Advanced Wound Repair Therapies

Advanced wound repair therapies

© Woodhead Publishing Limited, 2011

Related titles: Medical and healthcare textiles (ISBN 978-1-84569-224-7) Medical textiles remain one of the most dynamic areas of research in textiles. Medical and healthcare textiles is the fourth in a series of conferences held at the University of Bolton. Like its predecessors, it has attracted papers from some of the leading international centres of expertise in the field. Contributors cover a range of topics; these include emerging textile-based biomaterials, hygienic textiles, the use of textiles in infection control and as barrier materials, bandaging and pressure garments for managing chronic infections such as ulcers, the role of textiles in the management of burns and wounds, textile-based implantable devices such as tissue scaffolds and sutures, and intelligent textiles. Biomaterials for treating skin loss (ISBN 978-1-84569-363-3) The primary causes of wounds requiring skin replacement are severe burns and ulcers; materials must provide an effective temporary barrier, promote healing and minimise scarring. Massive improvements have been made to skin repair biomaterials in the last ten years with widespread adoption of new developments in the medical sector. This book provides a comprehensive review of biomaterials for treating skin loss. Chapters initially review the biomechanics and biological environment of skin. Further chapters discuss important methods and techniques for replacing and stabilising damaged skin, regulation and clinical trials. The final group of chapters discuss human origin and cultured dermal replacements and the principles of skin regeneration. Drug-device combination products (ISBN 978-1-84569-470-8) Drug delivery systems represent a vast area of research and development within biomaterials and medicine and the demand for sophisticated drug delivery devices continues to drive developments. Advanced drug delivery devices can offer significant advantages to conventional drugs, such as increased efficiency and convenience. Chapters in Part I discuss specific applications such as drug eluting stents and antimicrobial cements. Part II covers the development of drug device combination products with such topics as preclinical testing and regulation of products. This book provides a thorough analysis of the fundamentals, applications and new technologies of drug-device combination products for use throughout the human body. Details of these books and a complete list of Woodhead’s titles can be obtained by: • •

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Advanced wound repair therapies Edited by David Farrar

Oxford

Cambridge

Philadelphia

New Delhi

© Woodhead Publishing Limited, 2011

Published by Woodhead Publishing Limited, 80 High Street, Sawston, Cambridge CB22 3HJ, UK www.woodheadpublishing.com Woodhead Publishing, 1518 Walnut Street, Suite 1100, Philadelphia, PA 19102-3406, USA Woodhead Publishing India Private Limited, G-2, Vardaan House, 7/28 Ansari Road, Daryaganj, New Delhi – 110002, India www.woodheadpublishingindia.com First published 2011, Woodhead Publishing Limited © Woodhead Publishing Limited, 2011 The authors have asserted their moral rights. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publishers cannot assume responsibility for the validity of all materials. Neither the authors nor the publishers, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN 978-1-84569-700-6 (print) ISBN 978-0-85709-330-1 (online) The publisher’s policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp which is processed using acid-free and elemental chlorine-free practices. Furthermore, the publisher ensures that the text paper and cover board used have met acceptable environmental accreditation standards. Typeset by Toppan Best-set Premedia Limited Printed by TJI Digital, Padstow, Cornwall, UK

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Contents

Contributor contact details Introduction

xiii xix

Part I Introduction to chronic wounds

1

1

3

1.1 1.2 1.3 1.4 1.5 1.6 2

2.1 2.2 2.3 2.4 2.5 2.6 2.7

Dysfunctional wound healing in chronic wounds P. Stephens, Cardiff University, UK Normal skin wound healing Ageing skin and the onset of chronic, dysfunctional wound healing Dysfunctional healing of chronic skin wounds Conclusions Acknowledgements References The role of micro-organisms and biofilms in dysfunctional wound healing J. G. Thomas, H. Motlagh, S. B. Povey and S. L. Percival, West Virginia University, USA Introduction Microbiology and biofilms: not mutually exclusive Biofilms and the interactive cooperative community Biofilms in chronic wounds Biofilms as therapeutic or restorative microbiology/modeling Conclusion References

3 6 10 25 25 25

39

39 40 51 60 69 74 75 v

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3

Scarring and scarless wound healing B. J. Larson, A. Nauta, K. Kawai, M. T. Longaker and H. P. Lorenz, Stanford University School of Medicine, USA Introduction Wound healing process Fibroproliferative scarring Scarless fetal wound healing Adult versus fetal wound healing Treatment options for scars Future trends Conclusions References

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4

4.1 4.2 4.3 4.4 4.5 4.6 4.7 5 5.1 5.2 5.3 5.4 5.5

77

77 78 81 82 89 92 97 101 101

The discovery and development of new therapeutic treatments for the improvement of scarring 112 N. L. Occleston, A. D. Metcalfe, A. Boanas, N. Burgoyne, K. Nield, S. O’Kane and M. W. J. Ferguson, Renovo Group Plc, UK Introduction 112 Scar-free and scar-forming healing 113 In vitro and in vivo models to investigate the mechanisms of scarring and evaluate potential treatments 117 Translation from pre-clinical studies to clinical efficacy 122 Understanding the mechanisms of action of prophylactic scar improvement therapies 123 Conclusions 125 References 126 Monitoring chronic wounds and determining treatment 130 P. Plassmann, University of Glamorgan, UK Introduction 130 Wound size measurements 131 Wound colour measurements 139 Background material 149 References 150

Part II Biomaterial therapies for chronic wounds

153

6

155

6.1 6.2

Functional requirements of wound repair biomaterials R. M. Day, University College London, UK Introduction Wound pain and dressing materials

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6.3 6.4 6.5 6.6 6.7 6.8

Exudate management Prevention and control of infection Odour management Future trends Sources of further information and advice References

161 164 167 168 169 169

7

Tissue-biomaterial interactions S. Downes and A. A. Mishra, University of Manchester, UK Introduction: definitions Overview of tissue-biomaterial interactions Interactions at the biomaterial surface Tissue response to biomaterial Conclusion References

174

7.1 7.2 7.3 7.4 7.5 7.6

8

8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8

9

9.1 9.2 9.3 9.4 9.5 9.6

Polymeric materials for chronic wound and burn dressings A. Agarwal, J. F. McAnulty, M. J. Schurr, C. J. Murphy and N. L. Abbott, University of Wisconsin-Madison, USA Introduction Advanced moisture-retentive wound dressings Polymeric materials in moist wound healing dressings Infection control by polymeric wound dressings Conclusion Future trends Acknowledgements References

Dry wound healing concept using spray-on dressings for chronic wounds S. Jolly and S. Jolly, Clinogen Ltd, UK Introduction The key properties of an ideal wound dressing Using protein-based spray-on dressings in practice Case studies Conclusions References

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186

186 187 189 199 201 203 203 203

209 209 211 213 214 224 225

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Assessing the effectiveness of antimicrobial wound dressings in vitro J. Vaughan, R. Benson and K. Vaughan, Smith & Nephew Research Centre, UK Introduction Log reduction testing Zone of inhibition (ZOI) Bacterial barrier testing Other considerations Sources of further information and advice References

10.1 10.2 10.3 10.4 10.5 10.6 10.7 11

11.1 11.2 11.3 11.4 11.5 11.6 12

12.1 12.2 12.3 12.4 12.5 12.6 12.7 13

13.1 13.2 13.3

Adhesives and interfacial phenomena in wound healing B. J. Tighe and A. Mann, Aston University, UK Principles of adhesion, adhesivity and interfacial behaviour Bioadhesion: principles of adhesion applied to wound healing Adhesives in wound healing: materials overview Surgical adhesives and tissue sealants: structure and properties Conclusions References

227

227 229 234 237 241 244 245

247 247 253 257 273 280 281

Wound healing studies and interfacial phenomena: use and relevance of the corneal model 284 A. Mann and B. J. Tighe, Aston University, UK Wound dressing biomaterials: interfacial aspects of compatibility and wound response 284 The corneal model in wound healing and biomaterial studies 294 Interfacial phenomena in ocular surface contact lens studies 302 Wound fluid and the tear film collection 304 Biomaterials in mucosal wound healing 309 Conclusions 314 References 315 Sulphonated biomaterials as glycosaminoglycan mimics in wound healing B. J. Tighe and A. Mann, Aston University, UK Introduction Polymers and biomimesis Biomimetic models

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321 321 324 330

Contents 13.4 13.5 13.6 13.7

Sulphonated biomaterials in the context of biomimetic principles Sulphonated biomaterials and the chronic wound: possible modes of biomimetic behaviour Conclusions References

ix

335 341 351 354

Part III Molecular therapies for chronic wounds

359

14

Drug delivery dressings K. H. Matthews, Robert Gordon University, UK Introduction The role of drug delivery dressings in wound management Topically delivered therapeutic compounds Hydrocolloids Hydrogels Collagen Alginates Honey Future trends References

361

Molecular and gene therapies for wound repair E. Kiwanuka, F. Hackl, D. Nowinski and E. Eriksson, Harvard Medical School, USA Introduction Methods of gene delivery Gene therapy for wound healing Ethical issues Future trends References

395

Antimicrobial dressings M. IP, Chinese University of Hong Kong, Hong Kong Introduction Types of currently available dressings/formulations Types of ‘antimicrobials’ Future trends References

416

14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9 14.10

15

15.1 15.2 15.3 15.4 15.5 15.6

16 16.1 16.2 16.3 16.4 16.5

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361 362 363 373 374 377 380 383 385 387

395 400 407 410 411 412

416 418 439 442 443

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Avotermin: emerging evidence of efficacy for the improvement of scarring J. A. Bush, K. So, T. Mason, N. L. Occleston, S. O’Kane and M. W. J. Ferguson, Renovo Group Plc, UK There is a medical need for therapies that reduce scarring following surgery Current treatments for scar management are unsatisfactory New biological approaches are in development for the prophylactic improvement of scarring Conclusions and future trends References

17.1 17.2 17.3 17.4 17.5

450

450 451 452 458 459

Part IV Biologically derived and cell-based therapies for chronic wounds

461

18

Engineered tissues for wound repair N. J. Turner and S. F. Badylak, University of Pittsburgh, USA Introduction The wound microenvironment in wound repair Traditional approaches to wound repair Development of cellular therapies Development of acellular therapies Conclusion Acknowledgement References

463

Commercialization of engineered tissue products N. L. Parenteau, Parenteau BioConsultants LLC, USA Introduction Engineered templates and scaffolds Processed tissues Cell-based products Lessons from the first generation The second generation of advanced therapies Delivering value in advanced therapies Advanced therapies in the marketplace Conclusion References

495

18.1 18.2 18.3 18.4 18.5 18.6 18.7 18.8

19 19.1 19.2 19.3 19.4 19.5 19.6 19.7 19.8 19.9 19.10

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463 464 466 469 476 485 487 487

495 496 498 501 505 505 509 517 518 519

Contents 20

20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.9 20.10 21

21.1 21.2 21.3 21.4 21.5 21.6 21.7

xi

Biologically derived scaffolds K. Numata, RIKEN Institute, Japan and D. Kaplan, Tufts University, USA Introduction Polyhydroxyalkanoate (PHA)-derived scaffolds Silk-derived scaffolds Collagen-derived scaffolds Elastin-derived scaffolds Resilin-derived scaffolds Keratin-derived scaffolds Polysaccharide-derived scaffolds Conclusions and future trends References

524

Stem cell therapies for wound repair G. G. Gauglitz, Ludwig Maximilian University, Germany and M. G. Jeschke, University of Toronto and Sunnybrook Research Institute, Canada Introduction Frequently utilized sources of adult stem cells Clinical applications of stem cells to wound healing Conclusions Acknowledgement References Appendix: List of abbreviations

552

524 524 527 531 532 534 534 535 539 539

552 556 561 562 562 563 566

Part V Physical stimulation therapies for chronic wounds

569

22

571

22.1 22.2 22.3 22.4 22.5 22.6 22.7 22.8 22.9 22.10

Electrical stimulation for wound healing K. Balakatounis, Oxford Brookes University, UK Introduction Current of injury Physiological effects of electrical stimulation Antibacterial effects of electrical stimulation The effect of high voltage pulsed current (HVPC) on wound healing The effect of low intensity direct currents (LIDC) on wound healing Other types of electrical stimulation applied to wounds Discussion Conclusion References

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571 573 574 575 576 577 578 580 582 582

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Negative pressure wound therapy M. S. Miller, The Wound Healing Centers of Indiana, USA Introduction History of negative pressure wound therapy The science of negative pressure The pathophysiologic mechanisms of action of negative pressure The search for the perfect negative pressure technology Conclusions Acknowledgement References and further reading

587

Debridement methods of non-viable tissue in wounds D. J. Leaper, Imperial College London, UK and Cardiff University, UK, S. Meaume, Hôpital Charles Foix, France, J. Apelqvist, University Hospital of Skåne, Sweden, L. Teot, Hopital LaPeyronie, France and F. Gottrup, Copenhagen Wound Healing Centre, Denmark Introduction Background Complications of non-viable tissue in wounds and the need for debridement Presence of biofilm Organisation of debridement Timing and types of debridement Scoring the effectiveness of debridement Debridement in the diabetic foot Conclusions Sources of further information and advice References

606

Index

633

23.1 23.2 23.3 23.4 23.5 23.6 23.7 23.8 24

24.1 24.2 24.3 24.4 24.5 24.6 24.7 24.8 24.9 24.10 24.11

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587 588 593 594 598 602 602 603

606 607 609 613 614 614 623 624 627 627 628

Contributor contact details

(* = main contact)

Chapter 2

Editor D. Farrar Smith & Nephew Research Centre York Science Park Heslington York YO10 5DF UK E-mail: [email protected]

J. Thomas*, H. Motlagh and S. Percival Department of Pathology School of Medicine West Virginia University Health Sciences Center – North PO Box 9203 Morgantown, WV 26506-9203 USA E-mail: [email protected] [email protected] and

Chapter 1 P. Stephens Wound Biology Group Tissue Engineering and Reparative Dentistry School of Dentistry Cardiff University Heath Park Cardiff CF14 4XY UK

J. Thomas Honorary Professor School of Dental Medicine Cardiff University Cardiff, Wales, UK

E-mail: [email protected]

xiii © Woodhead Publishing Limited, 2011

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Contributor contact details

Chapter 3

Chapter 6

H. P. Lorenz Lucile Packard Children’s Hospital Stanford University School of Medicine Division of Plastic and Reconstructive Surgery Hagey Laboratory for Pediatric Regenerative Medicine MC 5148 257 Campus Drive Stanford, CA 94305-5148 USA

R. M. Day Division of Medicine University College London 5 University Street London WC1E 6JF UK

E-mail: [email protected]

Chapter 4 M. W. J. Ferguson Renovo Group Plc Core Technology Facility 48 Grafton Street Manchester M13 9XX UK E-mail: mark.ferguson@renovo. com

Chapter 5 P. Plassmann Department of Computing and Mathematical Science Faculty of Advanced Technology University of Glamorgan Pontypridd CF37 1DL UK

E-mail: [email protected]

Chapter 7 S. Downes* and A. Mishra School of Materials The University of Manchester PO Box 88 Manchester M60 1QD UK E-mail: sandra.downes@ manchester.ac.uk [email protected]

Chapter 8 A. Agarwal* and N. L. Abbott Department of Chemical and Biological Engineering University of Wisconsin-Madison 1415 Engineering Drive Madison, WI-53706 USA E-mail: [email protected] [email protected]

E-mail: [email protected]

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Contributor contact details

xv

Chapter 9

Chapter 12

Sujata Jolly* and Sapna Jolly Clinogen Ltd Robin Willis Way Windsor SL4 2PX UK

A. Mann* and B. J. Tighe Aston Biomaterials Research Unit Aston University Birmingham B4 7ET UK

E-mail: [email protected] [email protected]

E-mail: [email protected]

Chapter 14 Chapter 10 J. Vaughan, R. Benson* and K. Vaughan Smith & Nephew Research Centre Heslington York YO10 5DF UK E-mail: john.vaughan@ smith-nephew.com rachael.benson@ smith-nephew.com kiersten.vaughan@ smith-nephew.com

Chapters 11 + 13 B. J. Tighe and A. Mann* Aston Biomaterials Research Unit Aston University Birmingham B4 7ET UK E-mail: [email protected]

K. H. Matthews School of Pharmacy and Life Sciences Robert Gordon University Schoolhill Aberdeen AB10 1FR UK E-mail: [email protected]

Chapter 15 E. Eriksson Division of Plastic Surgery Brigham and Women’s Hospital 75 Francis Street Boston, MA 92115 USA E-mail: [email protected]

Chapter 16 M. Ip Department of Microbiology Chinese University of Hong Kong Prince of Wales Hospital Hong Kong E-mail: [email protected]

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Contributor contact details

Chapter 17

Chapter 18

J. A. Bush Renovo Group plc CoreTechnology Facility 48 Grafton Street Manchester M13 9XX UK

N. J. Turner and S. F. Badylak* McGowan Institute for Regenerative Medicine University of Pittsburgh 450 Technology Drive Pittsburgh, PA 15219-3130 USA

E-mail: [email protected] E-mail: [email protected] and

Chapter 19 Faculty of Medical and Human Sciences University of Manchester Oxford Road Manchester M13 9PT UK M. W. J. Ferguson* Renovo Group Plc Core Technology Facility 48 Grafton Street Manchester M13 9XX UK

N. L. Parenteau Parenteau BioConsultants, LLC 354 Money Hole Rd Fair Haven, VT 05743 USA E-mail: [email protected]

Chapter 20

E-mail: mark.ferguson@renovo. com

K. Numata RIKEN Institute 2-1 Hirosawa Wako-shi Saitama 351-0198 Japan

and

and

Faculty of Life Sciences University of Manchester Oxford Road Manchester M13 9PT UK

D. Kaplan* Department of Biomedical Engineering Tufts University 4 Colby Street Medford, MA 02155 USA E-mail: [email protected]

© Woodhead Publishing Limited, 2011

Contributor contact details

Chapter 21

Chapter 22

G. G. Gauglitz Department of Dermatology and Allergy Ludwig Maximilian University Frauenlobstrasse 9-11 80337 München Germany

K. Balakatounis 401 General Hospital Medical Corps Hellenic Army Greece

E-mail: [email protected]

Chapter 23

M. G Jeschke* Director Ross Tilley Burn Centre Sunnybrook Health Sciences Centre Department of Surgery Division of Plastic Surgery University of Toronto Canada

xvii

E-mail: [email protected]

M. S. Miller The Wound Healing Centers of Indiana Suite 1310 8244 US HWY 36 Avon, IN 46123 USA E-mail: [email protected]

Chapter 24

and Sunnybrook Research Institute Room D704 2075 Bayview Avenue Toronto, ON Canada M4N 3M5 E-mail: marc.jeschke@sunnybrook. ca

D. J. Leaper Cardiff University Heath Park Cardiff CF14 4XN UK E-mail: profdavidleaper@doctors. org.uk

© Woodhead Publishing Limited, 2011

Introduction

Most of us, for most of our lives, take wound healing and the remarkable regenerative capacity of our skin for granted. All of us experience cuts, grazes and minor burns throughout our lives – and perhaps our earliest experience of a ‘medical device’ is the use of a simple sticking plaster on a cut. Yet problems can, and do, occur in wound healing. Wounds can become infected, they may heal but with the formation of an unsightly scar or, worst of all, they may not heal at all. Alternatively, some of us may be unlucky enough to experience serious – sometimes even life threatening – burns. In cases such as these, a sticking plaster or simple gauze dressing will not suffice and we must turn to more advanced wound therapies. This is particularly true in the case of chronic wounds such as venous leg ulcers, pressure sores and diabetic foot ulcers. A chronic wound is defined as one that does not heal in an orderly way in a predictable amount of time the way that most acute wounds do. For example, a wound that has not healed in three months is often described as chronic. Chronic wounds are associated with ageing and underlying conditions such as diabetes. Demographic changes such as the ageing population and an increase in the incidence of diabetes mean that the number of chronic wounds is also increasing. Looking at the problem purely in terms of numbers, it is estimated that in the UK alone 70 000–190 000 people have a venous leg ulcer, 400 000 people per annum develop a pressure sore and there are 64 000 individuals with foot ulceration leading to 2600 amputations each year. The cost to the UK National Health Service of caring for patients with a chronic wound has been estimated at £2.2–3.1 billion per year. Figures in the USA are naturally even higher with estimates in the region of 2–5 million people suffering from chronic wounds at a cost of $20 billion to the US health system. And these are only the direct costs of treatment, they do not reflect the economic loss and, of course, the human cost in terms of pain, loss of self-esteem and impaired quality of life. Another problem on the increase is infection, which remains a major complication in both chronic and acute wounds. Issues such as antibiotic xix © Woodhead Publishing Limited, 2011

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Introduction

resistant ‘superbugs’ and hospital acquired infections have received much attention recently both in the scientific literature and the popular press and media. Fortunately, over the last two decades, there have been substantial advances materials and therapies for healing wounds. This progress continues and forms the subject of this book. Part I contains a background to the basic science of wound healing, in particular focusing on some of the problems that occur in healing. What is it that goes wrong in the case of a chronic wound and why won’t they heal without some kind of intervention? Why do some wounds heal with a scar? Why and how do some wounds get infected? Having addressed these questions, subsequent parts of the book go on to review some of the latest advances in wound healing therapies. Part II looks at developments in biomaterials for wound healing, as materials have always played an important role in treating wounds. Indeed, such treatment of wounds presents a number of challenges for material development in terms of providing the optimum physical environment for healing including the right moisture balance, protection from the external environment, protection from infection, management of wound exudate, optimum wound temperature, comfort for the patient and a host of other considerations. However, some of the biggest advances in wound healing therapies have come from going beyond this provision of the right physical environment to more actively influencing the biology of wound healing. In Part III we review molecular therapies for chronic wounds with approaches such as drug-delivery and anti-microbial dressings. Part IV deals with the even greater complexity of using biologically derived materials and cell-based therapies. This has been an area of intense activity over the last few years and has often seen wound healing products paving the way in terms of biologically derived scaffolds and tissue-engineered materials. However, as we shall see, there have been not only major technical challenges but also significant commercial challenges in bringing some of these more advanced therapies to market. Finally, in Part V, we look at the use of physical stimuli to promote wound healing. Here we include negative pressure therapy, which has emerged over the last few years as a completely new type of therapy but one that has rapidly grown in use and now represents one of the largest segments of the wound repair market, at least when viewed in monetary terms. Wound healing technologies sometimes appear to present something of a paradox. In the medical device technology arena, wound healing often seems to be something of a “poor relation” in comparison with the more glamorous areas of orthopaedics, spine, cardiovascular or drug delivery. Yet wound healing is where we have seen some of the first products introduced that lead the way in the application of new technologies. For example,

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Introduction

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antimicrobial wound dressings were, arguably, among the first combination products (before we even used the term), and the first tissue-engineered products to reach the market were aimed at wound healing. One of the most frequently cited early applications of nanotechnology in medicine has been the use of nanocrystalline silver wound dressings to prevent infection. Probably this lead in getting new technologies onto the market is connected with the somewhat easier regulatory pathway for products that contact the body but are not actually implanted within it. It is therefore hoped that this book will provide a snapshot of the state of development of advanced wound repair therapies as they are today and an insight into how this important field may develop in the future. D. Farrar Smith & Nephew Research Centre UK

© Woodhead Publishing Limited, 2011

1 Dysfunctional wound healing in chronic wounds P. S T E P H E N S, Cardiff University, UK

Abstract: This chapter discusses the normal wound healing process and how this is dysfunctional within chronic skin wounds in aged individuals. Skin ageing in relation to changes in cellular responses and composition of the extracellular matrix is described and introduced as a pre-cursor to aberrant wound repair. Theories for the development of these chronic wounds are suggested and the dysfunctional response is described in detail with respect to altered immune/inflammatory responses, re-epithelialisation, dermal repair and angiogenesis. Key words: chronic skin wound, ageing, dysfunctional cell response, altered extracellular matrix.

1.1

Normal skin wound healing

The wound healing process is a highly ordered and extremely complex process which, for the purpose of convenience, can be considered to consist of three successive but overlapping phases (for review see Clark (1996) and references therein and Table 1.1 for a general timescale for a normal wound healing process).

1.1.1 The vascular and inflammatory responses The loss of blood from a fresh wound has the effect of cleansing the wound of some of the invading foreign bodies and debris. Within seconds, vasoconstriction, platelet adhesion and aggregation, along with blood coagulation, result in the formation of a thrombus which encourages haemostasis. Coagulation proceeds via the two major enzymatic cascades and the resulting fibrin clot not only establishes haemostasis but also, in conjunction with fibronectin, provides a provisional matrix for the migration of many cell types into the wound space. The migration of initially neutrophils, and subsequently, monocytes into the injured tissue site is stimulated by a variety of chemotactic factors (e.g. fibrin degradation products, growth factors released from platelets). The neutrophils, via phagocytosis and by cellular killing, clear contaminating bacteria from the injured tissue site. On arriving 3 © Woodhead Publishing Limited, 2011

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Advanced wound repair therapies

Table 1.1 The time course of wound healing (for a simple, uncomplicated wound) – the individual processes of wound healing do not occur in isolation but overlap to varying degrees Time

Biological/cellular response

0 Hours 10–12 Hours 1 Day 3–7 Days

Vascular response initiated and blood coagulation begins. Initiation of new connective tissue formation. Fibrin network formed. Commencement of re-epithelialisation. Vascular response has peaked and started to subside. Peak of inflammatory response has been reached. Re-epithelialisation of superficial wounds completed (longer in more extensive wounds). Inflammatory response completed. Peak of formation of connective tissue. Maturation of collagen and remodelling of connective tissue.

12 Days 14 Days 6–16 Days >16 Days

at the wound site, the monocytes undergo a phenotypic metamorphosis to macrophages which phagocytose and digest pathogenic organisms and scavenge tissue debris. Macrophages continue to accumulate after neutrophil influx has ceased and release a number of growth and chemotactic factors that are necessary for the initiation and propagation of subsequent new tissue formation.

1.1.2 New tissue formation Re-epithelialisation Within hours after injury, movement of epidermal cells from the cut edges of the wound begins the process of re-epithelialisation. On completion of re-epithelialisation the keratinocytes revert to a normal phenotype and the basement membrane becomes reconstituted. The signals that drive this process involve the local presence of growth factors (e.g. Transforming Growth Factor-alpha (TGF-α), Transforming Growth Factor-beta (TGF-β), Keratinocyte Growth Factor (KGF) and Epidermal Growth Factor (EGF)), changes in keratinocyte exposure to extracellular matrix (ECM) molecules and changes in ECM tension. Fibroplasia and wound contraction After a lag of several days, fibroblasts migrate into the wound space, whence proliferation begins, stimulated by a number of fibroblast growth and chemotactic factors emanating from both the platelets and the macrophages and from the fibroblasts themselves. During fibroplasia, the fibroblasts change from having a migratory phenotype, to an ECM-producing

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Dysfunctional wound healing in chronic wounds

5

phenotype and finally to a myofibroblast phenotype when the cells have characteristics common to both fibroblasts and smooth muscle cells. The loose deposit of ECM initially produced by the fibroblasts is composed of large quantities of fibronectin and hyaluronic acid and is termed granulation tissue. These ECM components, along with tenascin, act as a scaffold for fibroblast migration throughout the wound space. Over the following few days the composition of the granulation tissue changes such that the collagen becomes the major component, with the fibronectin matrix acting as a scaffold for its fibrillogenesis. Early collagen deposition is highly disorganised but its subsequent organisation is primarily achieved by wound/ matrix contraction involving the action of myofibroblasts. Angiogenesis This process is vital for successful wound healing and its initiation occurs within days of injury. Stimulated by numerous angiogenic factors the endothelial cells from venules closest to the site of injury migrate through enzymatically fragmented basement membranes into the affected area. Endothelial cells within the parent vessel begin to proliferate (giving rise to capillary sprouts) and join the migrating population. When they meet the capillary, sprouts branch at their tips, join with the migrating cells and form capillary loops through which blood can flow. Activated endothelial cells then form further sprouts and loops giving rise to a capillary plexus.

1.1.3 Tissue remodelling The composition and structure of the continuously changing granulation tissue depends on both the time elapsed since injury and the distance from the wound margin. Initially the matrix contains components such as fibrin, fibronectin, vitronectin, types I and III collagen, tenascin and hyaluronic acid. This provides a suitable framework for the migration and influx of many cell types with fibronectin also possibly serving as a nidus for collagen fibrillogenesis. Over the following weeks, maturation of the matrix results in a great reduction of the fibronectin and hyaluronic acid content within the granulation tissue, but an increase in the amounts of collagen bundles and proteoglycans such as chondroitin sulphate and dermatan sulphate. Matrix metalloproteinases (MMPs) as well as the plasminogen activator/ plasmin system and hyaluronidase play an important role in this remodelling process as are tissue inhibitors of metalloproteinases (TIMPs). The result of all this tissue remodelling is mature scar tissue, which is, however, mechanically weaker than non-wounded tissue.

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Advanced wound repair therapies

1.2

Ageing skin and the onset of chronic, dysfunctional wound healing

Chronic skin wounds are more often than not found in the aged population and it is no coincidence that associated with the aged are changes in the structure and function of their skin. The typical signs of ageing such as wrinkling and sagging of the skin are all too permanent reminders of the encroachment of time or of too much time spent in the sun without the appropriate protection. So exactly what does ageing do to the largest organ of our bodies?

1.2.1 Skin ageing Ageing of the skin has been described in detail elsewhere (Stephens 2003); however, a brief overview will be given to set the scene for later sections concerning chronic wound healing (see also Table 1.2). Ageing is a basic biological process characteristic of all living organisms (Yaar & Gilchrest 2001). Inevitably it leads to reductions in maximal function and reserve capacity in all organ systems rendering the individual more susceptible to injury, disease and eventually death. Intrinsic or innate skin ageing (the skin becomes thin, pale and finely wrinkled) refers to the slow, but irreversible, degeneration of the skin’s structure and function (Uitto & Bernstein 1998). Extrinsic or ‘photo’ ageing (characterised by deep wrinkles, laxity and a leathery appearance, increased fragility, blister formation and poor wound healing) is the result of exposure to outdoor elements, primarily UV light (Gilchrest 1995). Both types of ageing are the result of distinct cellular and biological/biochemical alterations of the key cells and structures that constitute the skin. Ageing of the epidermis Aged skin demonstrates a reduced keratinocyte proliferative capacity (Yaar & Gilchrest 1999), an inability to properly terminally differentiate in order to form the stratum corneum and an inability to produce the approTable 1.2 The functional result of ageing in the skin Tissue

Functional change due to ageing

Epidermis Dermis Vasculature

Loss of barrier function, flattened dermo-epidermal junction. Decrease in skin thickness, increased skin stiffness. Reduction in number of papillary loop microvessels, reduced blood flow.

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priate cell signals in response to environmental stimuli (Yaar 1995). There are also decreased numbers of melanocytes (Gilchrest, Blog, & Szabo 1979), antigen-presenting lymphocytes and alterations in Langerhans cell activity within the epidermis as individuals age (Gilchrest et al. 1983; Thiers et al. 1984; Grewe 2001). Furthermore, the dermo-epidermal junction flattens with age due to retraction of the epidermal papillae and the microprojections of the basal cells into the dermis (Kurban & Bhawan 1990). As a result, cellular turnover within the epithelium is affected and this leads ultimately to the development of a dermo-epidermal junction and a skin structural unit which is less resistant to shear forces than younger skin is. Ageing of the dermis Cellular changes Skin thickness tends to decrease after the seventh decade (de Rigal et al. 1989) due to alterations in cellular biosynthetic/degradatory responses and direct alterations of the ageing ECM itself. This has been suggested to result from the accumulation of senescent cells within the aged dermis driven by organismal ageing (Dimri et al. 1995; Campisi 1996; Faragher & Kipling 1998). Furthermore, a decrease in the number and size of fibroblasts in aged skin has also been reported (Kligman & Lavker 1988). Within the dermis there are also reductions in the total number of papillary loop microvessels, decreased thickness of microvessel basement membranes and decreased numbers of perivascular cells (Braverman & Fonferko 1982). Once again this may be due to replicative senescence (Chang et al. 2002). With respect to the inflammatory and immune cell responses researchers have demonstrated decreases in both the number and size of mast cells in aged skin (Kligman & Lavker 1988). Furthermore, within aged individuals T lymphocyte numbers are reduced and their ability to proliferate in response to mitogens (e.g. interleukin (IL)-2 and -4) is impaired (Nagelkerken et al. 1991). Extracellular matrix changes The characteristic appearance of ageing dermis is due to distinct changes in its ECM composition including the collagens, elastin/elastic fibres, fibrillin and proteoglycans/glycosaminoglycans (see (Stephens 2003) and references therein). As well as alterations in the biosynthesis of ECM molecules there are also alterations in the cross-linking of these molecules with age (Bentley 1979). This is in part related to the fact that both collagen and elastin turnover slowly and are thus susceptible to age-related changes. The major alteration in collagen and elastin is the formation of intermolecular

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cross-links which can result in an over stiffening of the fibres when present in excess (Bailey 2001). Further stiffening of the fibres can then occur through a second process based on the reaction of glucose or its metabolites, giving rise to non-enzymic glycosylation-derived cross-links (glycation) and advance glycation end-products (AGEs). Concurrent with altered biosynthesis and cross-linking of the ECM there is also increased ECM turnover within ageing skin. This is brought about by an increased amount of a number of proteolytic enzymes including serine proteases and MMPs and a concomitant decrease in their inhibitors (e.g. TIMP levels (Parks 1999; Sardy 2009)). Such a tissue imbalance in aged skin may once again be due to the actions of senescent fibroblasts as they have been demonstrated within the ageing dermis (Dimri et al. 1995). Tissue turnover may also occur as a result of elevated levels of reactive oxygen and nitrogen species (ROS and RNS) and a decease in anti-oxidants leading to damage of a number of important cell components such as lipids, proteins and DNA (Waddington, Moseley, & Embery 2000). Studies suggest a correlation between the ageing process and the formation of ROS with increases in oxidative stress observed in some tissues (possibly due to alterations in mitochondrial function (Kwong & Sohal 2000; Podda & Grundmann-Kollmann 2001)). Tissue ageing is also associated with a reduction of both enzymatic and non-enzymatic antioxidants (Beckman & Ames 1998). Furthermore, the secondary effects of ROS on ECM degradation are that they up-regulate and activate MMPs and decrease TIMPs further shifting the balance within the tissue to that of a catabolic state.

1.2.2 Mechanisms of skin ageing So how does this ageing occur, what is the mechanism? A number of hypotheses have been proposed which attempt to answer this question and so some of these will now be briefly considered. Replicative senescence and telomere loss The first suggested mechanism is one which involves telomere loss leading from findings that the number of divisions for a normal cell such as a diploid fibroblast is finite (Hayflick 1965) (Fig. 1.1). The theory which has been proposed to explain this is the ‘telomere hypothesis’. Telomeres are made out of thousands of hexameric (TTAGGG) repeats (Griffith et al. 1999) and function to protect chromosomes against illegitimate fusions, to prevent the chromosome end from being recognised as a double strand break and to guide the pairing and movement of the chromosomes during mitosis and meiosis. However, during DNA replication the cells are faced with an ‘end replication problem’ (Olovnikov 1973). Due to the nature of the replication process, the far end of each telomere is not replicated and thus the telomeres shorten with

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NF (P6, 7.5PD)

CWF NF (P11, 17PD) (P11, 15.5PD)

CWF (P15, 23PD)

CWF (P12, 15PD)

0

CWF NF

NF CWF (P15, 21PD) (P20, 31PD)

0

10

20

30

40

50

60

(b)

200

300

NF CWF SEN NF SEN (P20, 31PD) (P27, 40PD) (P35, 53PD)

Time (days)

100

1.1 (a) Senescence associated beta-galactosidase activity (darker areas in right-hand image) in senescent chronic wound fibroblasts (CWF) but not patient-matched normal fibroblasts (NF); (b) replicative lifespan analysis demonstrating that CWF senesce (plateau out) well before patient-matched NF; (c) telomere analysis utilising STELA (Baird et al. 2003) demonstrating premature shortening of telomeres in CWF compared to patient-matched NF leading to premature senescence in this patient (adapted from Wall (Wall et al. 2008)). P = Passage, PD = Population doubling.

CWF (P6, 8.5PD)

(c)

(a)

NF (P29, 40PD) Population Doubling Level

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each round of cell division. Senescence occurs when a short, critical telomere length is achieved and the cell irreversibly exits the cell cycle. There are, however, exceptions to this rule in, for example, germ line cells, cancer cells and stem cells (Dhaene, Van Marck, & Parwaresch 2000), as these cells posses the enzyme telomerase which is able to synthesise telomeric sequence de novo and can therefore overcome telomere shortening. Free radical damage theory Another theory of ageing is the ‘free radical theory’ proposed by Harmann in 1956 (Harman 1956). That is, ROS and RNS are formed in aerobic organisms as a result of metabolic activity and that with time these cause irreversible damage to a number of important cell components. ROS encompass oxygen free radicals, including the superoxide radical (O2·−) and hydroxyl radical (·OH) species, as well as non-radical oxygen derivatives, such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl). Reactive nitrogen species include the nitric oxide radical (NO·) species and peroxynitrite (ONOO−) (Waddington, Moseley, & Embery 2000). To counteract these oxidative agents a number of cellular (enzymic) and extracellular (non-enzymic) antioxidants (e.g. tocopherol, ubiquinone, glutathione, ascorbate and urate) exist which help protect against the damaging effects of the ROS and RNS. Studies have suggested a correlation between the ageing process, the formation of ROS (for review see Sohal (2002)) and the reduction of both enzymatic and non-enzymatic antioxidants (Beckman & Ames 1998; Kohen & Gati 2000). Differential gene expression theory Another theory to explain the ageing phenomenon centres on the concept that as cells age, they alter their cellular activities due to programmed or epigenetic changes in gene expression (Campisi 1998; Uitto & Bernstein 1998). For example, with respect to ageing within the skin, distinct alterations in a variety of ECM and protease genes have been reported, which lead to markedly lower ECM biosynthesis and increased ECM turnover in aged individuals compared to foetal tissues (Uitto, Fazio, & Olsen 1989). Therefore, it may be the differential gene expression profiles of cells which ultimately determine how readily a tissue will age.

1.3

Dysfunctional healing of chronic skin wounds

1.3.1 Overview The repair of physical damage is an essential day-to-day function of the skin and as with other tissue functions, such as the mounting of an immune

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Dysfunctional wound healing in chronic wounds (a)

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(b)

(c)

1.2 Clinical images of (a) a venous leg ulcer, (b) a diabetic foot ulcer and (c) a pressure ulcer (Images courtesy of Professor Keith Harding, Cardiff University).

reaction against an infectious agent, it involves both cell proliferation and differentiation. Indeed, the ability of an organism to survive crucially depends on its ability to maintain integumental integrity (Ashcroft, Horan, & Ferguson 1995). Wound healing is therefore of paramount importance in mammalian homeostasis. When wounds fail to heal, they are classified as non-healing or chronic wounds. Such dysfunctional healing is generally associated with the aged, and is characterised by prolonged inflammation, defective wound ECM and failure of re-epithelialisation (Herrick et al. 1992; Ashcroft, Horan, & Ferguson 1995). Chronic wounds exist as one of three principle forms: venous ulcers, pressure ulcers and diabetic foot ulcers (Fig. 1.2; for a review see Falanga 2001). It is estimated that in the UK alone, chronic wounds affect approximately 3% of the population over the age of 65 years and cost healthcare providers over £1 billion annually (Harding, Morris, & Patel 2002). In addition to the financial cost, they can be both debilitating and painful for the sufferer and greatly reduce quality of life (Phillips et al. 1994; Krasner 1998; Persoon et al. 2004). Studies have demonstrated that incidence of lower limb ulceration increases steadily with increasing age after 65 years (Margolis et al. 2002). The commonest type of chronic wound is the venous leg ulcer, accounting for up to 60% of chronic wounds (Mekkes et al. 2003). Whilst pressure ulcers and diabetic foot ulcers have some underlying, tangible causative

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factors, there is no single unifying theory to explain why chronic venous wounds occur, although numerous theories and hypotheses have been proposed. It is generally accepted that the main contributory factor in the pathogenesis of venous leg ulcers is chronic venous insufficiency (CVI), resulting in venous stasis and microcapillary pathology (Baker et al. 1991). Some of the theories relating the pathophysiological effects of CVI to ulcer formation will now be outlined.

1.3.2 Theories for the development of chronic wounds A number of theories have been proposed to try to explain why chronic wounds form and ultimately persist. One theory is the fibrin cuff hypothesis (Browse & Burnand 1982), which proposes that the high ambulatory venous pressure within the calf muscle pump is transmitted, causing a widening of endothelial pores, allowing the escape of large molecules such as fibrinogen. Fibrin complexes then form and are not broken down due to inadequate fibrinolytic activity within the blood and tissue fluid. The fibrin complexes prevent the passage of oxygen and other nutrients, which normally sustain the cells of the dermis, the epidermis and the vasculature. This leads directly to cell death and ulceration. This theory was refined and expanded by Falanga and Eaglstein (1993) in their ‘trap’ hypothesis. They suggested that the macromolecules such as fibrinogen and α2-macroglobulin leaking into the dermis bind or ‘trap’ growth factors and matrix materials, hence making them unavailable for tissue repair and maintenance of tissue integrity. This hostile environment may also inhibit the de novo synthesis of ECM molecules by cells in the wound bed. Another theory, put forward is the proposed ‘leukocyte trapping’ hypothesis (Coleridge Smith et al. 1988; Scott, Coleridge, & Scurr 1991). This suggests that as CVI progresses there is a reduction in blood flow, resulting in adhesion of leucocytes to the endothelial cells of the vessels in turn leading to an increased number of white blood cells in the skin of patients with venous disease and resultant tissue damage and ulceration due to the build up of free radicals, toxic metabolites and proteolytic enzymes. Free radicals also form the basis of a theory proposed by Cheatle (1991) suggesting that the accumulation of iron sets up a reaction leading to the production of hydroxyl radicals causing tissue damage. Despite all these hypotheses it is still not clear exactly how the increased tissue pressure can result in the drastic cellular and matrix changes associated with chronic ulceration. However, a large number of studies are now starting to reveal the type and extent of the dysfunctional responses within these chronic wound environments (see below and Table 1.3 for an overview), which will certainly give the scientific/clinical community clues as to how these patients should be treated but also, possibly, into how these wounds can be avoided in the first place.

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Table 1.3 The key cellular events that are characteristic of a typical chronic, non-healing wound in aged skin Dysfunctional wound healing response

Change due to ageing

Immune response/ inflammation

Increased numbers of poorly activated macrophages, altered T cell numbers and type, increased B cells, increased levels of pro-inflammatory molecules (IFN-γ, TNF-α, IL-6 and -8), decreased levels of antiinflammatory molecules (IL-2 and -10), alterations in homeobox genes (Hox b13), increased MCP-1, decreased MIP-2, MIP-1α, MIP-1β and eotaxin, defective chemokine and chemokine receptor expression, COX-1 and -2 elevated, reduced phagocytic activity, increased oxidative stress. Decreased keratinocyte proliferation (as a population), failure to terminally differentiate correctly, decreased lipid synthetic capacity/barrier function, dysfunctional MMP production, altered integrin expression, failure to undergo EMT, dysfunctional Mast cells and DETCs, decreased number of melanocytes. Decreased fibroblast proliferation (senescence), altered ECM production/turnover (collagens, elastin/elastic fibres, fibrillin, proteoglycans/glycosaminoglycans), increased MMPs, PAs and other proteases, decreased TIMPs, increased ECM cross-linking, effects of AGEs on cell signalling, altered cytoskeleton, dysfunctional response to growth factors and cytokines, elevated oxidative stress/decreased anti-oxidants, dysfunctional production of growth factors and cytokines. Production of different ECM by endothelial cells, increased adhesiveness to leucocytes, increased responsiveness to TNFα, increased production of IL-1, decreased levels of key growth factors such as VEGF, FGF-2 and granulocyte/macrophage colony-stimulating factor, alterations in homeobox genes and integrin expression, decreased thickness of microvessel basement membranes, decreased numbers of perivascular cells.

Re-epithelialisation

Dermal repair

Angiogenesis

1.3.3 Chronic wounds and dysfunctional immune responses/inflammation Inflammatory cell populations The role of the inflammatory response in chronic dysfunctional wound repair in the aged is well established (for overviews see Eming, Krieg, & Davidson 2007; Menke et al. 2007). Whilst the generation of an inflamma-

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tory reaction is crucial to successful wound healing, it is clearly dysfunctionally regulated in wounds associated with the aged. Within human acute and chronic wounds an age-related increased in the number of mature macrophages has been reported (Ashcroft, Horan, & Ferguson 1998; Loots et al. 1998), which may seem counter-intuitive since macrophages are crucial for a successful wound healing response. Similar findings have been reported in mice with increased numbers of macrophages (but no change in neutrophils) in wounds made in aged versus young animals (Swift et al. 2001). However, a report by Moore (Moore, Ruge, & Harding 1997) suggests that despite their increased numbers the cells actually demonstrate a decreased activation and so may be dysfunctional in their responses. A delayed T cell infiltration into wounds in aged mice has also been demonstrated but despite this their number was ultimately greater in aged mice (Swift et al. 2001). Alterations in wound T cell content in humans have also been noted in other investigations (Ashcroft, Horan, & Ferguson 1998). Interestingly, although T cell depletion impairs healing (Peterson et al. 1987) wounds in athymic mice demonstrate increased breaking strength (Barbul et al. 1989). With respect to T lymphocytes there is a decreased ratio of CD4+ : CD8+ cells due to increasing numbers of CD8+ T lymphocytes within non-healing wounds (Loots et al. 1998). Furthermore, of the CD4+ cells that are present, a disproportionate number are of the pro-inflammatory Th1 type. Other studies have also demonstrated reduced staining for CD4+ and CD8+ cells within the ulcer bed (Galkowska, Olszewski, & Wojewodzka 2005). Despite there being very few B lymphocytes within wounds normally, numbers are generally increased within chronic wounds as are numbers of plasma cells (Loots et al. 1998). Persistent inflammation therefore seems to arise due to dysfunctional regulation of both cell numbers and cellular response, which in turn leads to an imbalance between pro- and anti-inflammatory molecules. Altered growth factor/cytokine/chemokine levels An imbalance between pro-inflammatory cytokines/growth factors and their inhibitors has been demonstrated within chronic wounds. Comparing chronic and acute wounds it has been demonstrated that overall levels of tumor necrosis factor (TNF)-α and IL-1β are increased in chronic wounds due to decreased levels of their respective inhibitors; p55 (the soluble TNF receptor protein) and the IL-1 receptor antagonist (Tarnuzzer & Schultz 1996; Wallace & Stacey 1998; Agren et al. 2000). Typical of some chronic non-healing wounds are increased levels of pro-inflammatory molecules (interferon-γ, TNF-α, IL-6 and -8) but decreased levels of anti-inflammatory molecules (IL-2 and -10) (Barone et al. 1998; Trengove, BielefeldtOhmann, & Stacey 2000; Agren et al. 2000). Indeed systemic administration of a

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monoclonal anti-TNF-α antibody in wounded ob/ob mice attenuated wound inflammation, improved re-epithelialisation and improved their wound healing (Goren et al. 2006). Recent studies suggest that this cytokine/growth factor imbalance could potentially be due to aberrant expression of the group of master control genes known as the homeobox genes, since over expression of, for example, Hoxb13 not only results in prolonged inflammation (demonstrated by a lack of neutrophil clearance), epidermal hyperplasia and blood vessel abnormalities but also in the upregulation of TNF-α (Mack & Maytin 2010). Whether such imbalances within chronic wounds are due solely to the ageing of tissues is unclear. However, it has been reported that production of monocyte-derived IL-1 and -6 is altered with ageing (Bradley et al. 1989; Roubenoff et al. 1998) as is macrophage production of vascular endothelial growth factor (VEGF) (Swift, Kleinman, & DiPietro 1999), which has important implications for blood vessel formation and the recruitment of blood-born wound healing cell types. Investigations of leukocyte chemoattractant levels suggest that there is increased monocyte chemoattractant protein (MCP)-1 in the wounds of aged mice but that macrophage inflammatory protein (MIP)-2, MIP-1α and MIP-1β and eotaxin are decreased (Swift et al. 2001). Although the exact role of chemokines in aged, dysfunctional healing remains to be fully elucidated, it has been demonstrated that chemokines such as IP-10 (a member of the alpha or cysteine-X amino acid-cysteine (CXC) chemokine family of chemotactic cytokines) may also be implicated in chronic, dysfunctional repair since studies in IP-10 transgenic mice have demonstrated an abnormal wound healing response characterised by a more intense inflammatory phase and a prolonged and disorganised granulation phase with impaired blood vessel formation (Luster et al. 1998). Furthermore, mice deficient for the XCX receptor CXCR2 show delayed healing (Devalaraja et al. 2000) and chronic wound fibroblasts have been reported to be deficient in the production of CXCL-1, 2, 3, 5 and 6 which may have a direct effect on inflammatory/immune cell attraction and retention within the chronic wound site (Wall et al. 2008). Interestingly, estrogen can reverse age-related impaired wound healing through down regulation of the inflammatory response (reduced neutrophil numbers, chemotaxis and expression of adhesion molecules) and increased matrix deposited at the wound site (Ashcroft et al. 1999). Recent studies report that this downregulation of inflammation by estrogen is through the concurrent downregulation of macrophage migration inhibitory factor (Ashcroft et al. 2003). Prostaglandins also have an important role in inflammation and their synthesis is mediated via cyclooxygenases (COX). In chronic wounds, constitutively expressed COX-1 is upregulated compared with normal skin and de novo expression of inducible COX-2 is also

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apparent (Abd-El-Aleem et al. 2001). Although COX-2 is not normally expressed in healthy cells, inflammatory mediators found in chronic wounds, such as lipopolysaccharide, IL-1α and IL-1β, may rapidly induce its expression (Endo et al. 1995; Konturek et al. 2001). Reaction products of COX-2 are responsible for many of the cytotoxic effects of inflammation (Seibert et al. 1994) and may therefore contribute to non-healing in chronic wounds. Phagocytosis is also a critical element of the inflammatory process; however, wound macrophages from aged mice exhibit a reduced phagocytic activity due to decreases in both the number of phagocytic cells and in the number of particles consumed by each cell (Swift et al. 2001). Decreases in macrophage phagocytic response may also be due to age-related changes in intracellular signalling as has been described for T cells (Patel & Miller 1992; Shi & Miller 1993; Chakravarti & Abraham 1999). Furthermore, the oxidative burst of both neutrophils and macrophages diminishes with ageing (Lipschitz & Udupa 1986; Davila et al. 1990; Alvarez & Santa 1996). The hypothesis that oxidative stress may be causative to the chronic wound phenotype has stimulated research in this area and an association between elevated ROS levels and chronic wounds has been made (Wenk et al. 2001; Allhorn et al. 2003; Yeoh-Ellerton & Stacey 2003; James et al. 2003). Elevated ROS production in the wound site may drive a deleterious sequence of events that result in a non-healing phenotype, as cellular functions including proliferation, migration, adhesion, matrix remodelling and apoptosis are all affected by ROS exposure (Wlaschek & Scharffetter-Kochanek 2005).

1.3.4 Chronic wounds and dysfunctional re-epithelialisation Ageing leads to an increase in the time taken to heal epidermal wounds. In a human-based study wound re-epithelialisation occurred more slowly in aged individuals (Holt et al. 1992), which in turn has been supported by investigations using aged mice (Swift, Kleinman, & DiPietro 1999). Agerelated decreases in the proliferative capacity of keratinocytes (Gilchrest 1983) and a failure to terminally differentiate correctly or respond to/ produce the appropriate cell signals (Rattan & Derventzi 1991) may contribute to this slow healing of minor injuries, ultimately giving rise to weaker scars and non-healing wounds (Gilchrest et al. 1983; Yaar & Gilchrest 2001). It is plausible that these alterations in epithelial renewal are attributable to the senescence of the keratinocyte populations since terminal differentiation of senescent keratinocytes occurs slower than for their growing counterparts (Norsgaard, Clark, & Rattan 1996). There are also problems in restoration of the barrier function of the stratum corneum after wounding in the aged, which is at least in part due to the decreased lipid synthetic capacity associated with ageing (Ghadially et al. 1995).

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Within chronic wounds re-epithelialisation is notably retarded, with evidence of ‘piling up’ of the keratinocytes at the edge of the wound and inhibition of cell migration. This may in part be due to aberrant regulation of MMPs (Saarialho-Kere 1998; Vaalamo, Leivo, & Saarialho-Kere 1999; Mirastschijski et al. 2002; Saarialho-Kere et al. 2002; Pirila et al. 2007) or plasminogen activator (Weckroth et al. 2004) at the wound edges or to altered cellular responses to the hypoxic wound environment (Xia et al. 2001). Interestingly, it has also been suggested that psychological stress, both acute and chronic, can impair cutaneous wound repair. Previously, this has been mechanistically ascribed to stress-induced elevations of cortisol (Choi et al. 2006; Christian et al. 2006); however, recent work suggests an alternative pathway namely stress-induced elevation of epinephrine levels resulting in activation of the keratinocyte beta2AR and the impairment of cell motility and wound re-epithelialisation (Sivamani et al. 2009). At the chronic ulcer edge the keratinocytes, apart from demonstrating a high degree of proliferation (elevated Ki67 labelling) and an activated phenotype (Keratin 16 positivity), show a reduced expression of LM-3A32 (the uncleaved, precursor of the alpha3 chain of laminin 5), a key molecule present on migrating epithelium (Usui et al. 2008). Crucial to this migration is the expression and production of cell surface molecules such as the integrins receptors. It is now apparent that such receptors have a key role to play in the lack of re-epithelialisation of chronic wounds since alphavbeta6 integrin is highly expressed in poorly healing human wounds (AlDahlawi et al. 2006) and mice that constitutively express the beta6-integrin in the epithelium frequently develop spontaneous, progressing fibrotic chronic ulcers associated with elevated levels of TGF-β1 (Hakkinen et al. 2004). Furthermore, there is a suggestion that during the re-epithelialisation process keratinocytes at wound margins undergo partial epithelial to mesenchymal transition (EMT) and that this is compromised with chronic wounds (Hudson et al. 2009). This is based on the fact that forty percent of Slug (a transcriptional regulator of EMT in development) null mice, but no wild type mice, developed non-healing cutaneous ulcers in response to chronic ultraviolet radiation. Other cell populations are also key to the dysfunctional epithelial response within chronic wounds. Whilst aberrant fibroblast and endothelial responses spring easily to mind, other cell types including mast cells and dendritic epidermal T cells (DETC) also appear to play a role. In chronic venous leg ulcers there was a significant increase in intact and degranulated mast cells in the surrounding skin and ulcer edge with mast cell granules and phantom cells (mast cells devoid of granules) predominantly observed in the epidermis (Abd-El-Aleem et al. 2005). With respect to DETC, in mice lacking these gamma delta T cells a delay in wound closure and a decrease in the proliferation of keratinocytes at the wound site have been observed,

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which may in turn be due to a decreased production of FGF-7 (Jameson et al. 2004). Following on from this, there is a developing link between failures in the immune system and the lack of re-epithelialisation within chronic wounds as the human cathelicidin anti-microbial protein hCAP18 (which has broad anti-microbial activity conferred by its C-terminal fragment LL-37) is found at only low levels with chronic wounds and its immunoreactivity is absent in ulcer edge epithelium (Heilborn et al. 2003).

1.3.5 Chronic wounds and dysfunctional dermal repair It has been suggested that impaired dermal healing in the aged dermis may be the result of replicative cellular senescence and its impact on tissue dysfunction. The reduction or even exhaustion of the proliferative capacity of the cells in the wound, would lead to a local accumulation of senescent cells which may then impact on the poor wound healing response. This is supported by investigations demonstrating impaired formation of granulation tissue in the aged due to a decrease in fibroblast number (Kligman & Lavker 1988), the fact that wounds in aged mice exhibit significantly delayed collagen synthesis (Swift, Kleinman, & DiPietro 1999) and that there are wellcharacterised changes in ECM composition/production by aged individuals/ cells (Ashcroft, Horan, & Ferguson 1995; Deie et al. 1997; Passi et al. 1997). The presence of senescent fibroblasts within the ageing dermis (Dimri et al. 1995) suggests that the tissue balance in aged skin is altered towards a more catabolic state. Indeed, tissue remodelling is known to be altered in aged patients and increases in MMP-2, PAI-1 and TIMP-2 production have previously been demonstrated in senescent versus normal fibroblasts (Zeng & Millis 1994; West et al. 1996; Ashcroft et al. 1997a; 1997b). Conversely, ageing matrix itself can affect fibroblast responses since AGEs/matrix glycation have been demonstrated to alter the signalling within fibroblasts inducing the misfolding of proteins (Loughlin & Artlett 2009) and also lead to direct affects on cellular morphology, attachment, proliferation, migration and ECM remodelling (Liao, Zakhaleva, & Chen 2009). Furthermore, such hyperglycemia has been reported to affect HIF1alpha stability and activation and that this impaired regulation of HIF1alpha has been reported to be involved in the development of chronic diabetic wounds (Botusan et al. 2008). Hyperglycemia has also been suggested to have a role to play in the dysfunctional response to Endothelin-1 by diabetic fibroblasts (Solini et al. 2007). Fibroblast responses The healing of dermal wounds is a complex multi-cellular process and a chronic, persistent wound is often the end result of a delay in this process in

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the aged. Whilst one obvious effect of senescence on the fibroblasts which subsequently infiltrate the wound would be their inability to proliferate, secondary effects include a decreased rate of movement (Schneider & Mitsui 1976; Kondo & Yonezawa 1992), increased latent time (Muggleton-Harris, Reisert, & Burghoff 1982), reduced responsiveness to stimulatory growth factors (Ashcroft, Horan, & Ferguson 1995) and a potential reduced overall ability of the population to remodel the ECM components (Bell, Ivarsson, & Merrill 1979; Almqvist et al. 2009). Workers have investigated the population of fibroblasts within the dermis of chronic wounds to determine if the impaired healing is related to cellular senescent changes and demonstrated decreased population doubling levels, proliferation and increased fibroblast senescence (Stanley et al. 1997; Vande et al. 1998; Mendez et al. 1998a; 1998b; Agren et al. 1999; Mendez et al. 1999; Vande et al. 2001; Vande Berg et al. 2005; Wall et al. 2008). The concept that replicative senescence in wound fibroblasts results in reduced proliferation and the failure of refractory chronic wounds to respond to treatment has therefore been proposed as an important factor in the tissue phenotype. These findings are, however, not universal and contrast with studies in three-dimensional collagen lattice systems and with the active cellular proliferation that is observed within these chronic wounds (Herrick et al. 1996; Stephens et al. 2003). Alterations in extracellular matrix With respect to ECM molecules chronic wound fibroblasts express higher levels of fibronectin mRNA and protein compared to normal fibroblasts in vitro (Mendez et al. 1998a). In situ, chronic wound tissue was found to contain fibronectin mRNA but this was not detectable in tissue from acute wounds and normal skin (Ongenae, Phillips, & Park 2000). However, researchers have demonstrated that intact fibronectin protein is not detected in chronic wound tissue and fluid (Rao et al. 1995; Herrick et al. 1992) but rather as a degraded form, along with α2-macroglobulin and α1-antitrypsin, the protein that normally protects fibronectin from proteolytic degradation (Rao et al. 1995; Grinnell & Zhu 1996). Abnormally high levels of the serine protease elastase are thought to be responsible for this (Rao et al. 1995; Grinnell & Zhu 1996; Herrick et al. 1997). Fibrin is elevated in chronic wounds and is localised to the fibrin cuff (Herrick et al. 1992). It is present mainly in an undegraded form (Claudy et al. 1991) due to the presence of plasminogen activator inhibitor-1 (PAI-1; see below; (Brakman et al. 1992)), which inactivates the serine proteases capable of degrading it. Collagen synthesis by chronic wound fibroblasts in vitro is thought to be reduced in both monolayer and in three-dimensional collagen gels (Herrick et al. 1996), although some researchers argue that there is no difference in synthesis, even in the presence of TGF-β (Hasan et al. 1997).

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Protease imbalance The above might suggest that the defective wound matrix within chronic wound lesions may be due to differences in ECM remodelling within the wound site, possibly mediated by alterations in local MMP activity (Herrick et al. 1996). To this end, researchers have demonstrated that total MMP activity is up to 30 times greater in chronic wound fluid than in acute wound fluid (Wysocki, Staianocoico, & Grinnell 1993; Weckroth et al. 1996; Trengove et al. 1999) with the MMPs detected in chronic wounds including MMP-1, -2, -3, -8, -9, -10, -11, -13 and -19 (Yager & Nwomeh 1999; Hieta et al. 2003)). Some researchers have reported that MMP-1 is the predominant interstitial collagenase in non-healing wounds (Weckroth et al. 1996) whereas others conclude that MMP-8 is more highly expressed (Nwomeh et al. 1999). A third collagenase, MMP-13 (collagenase-3), is also expressed to a lesser degree in chronic, but not acute, wounds (Vaalamo et al. 1997). The gelatinases MMP-2 and -9 are both significantly increased in chronic wound fluid (Wysocki, Staianocoico, & Grinnell 1993; Trengove et al. 1999). Increased MMP-9 is associated with expression of the active form of the serine protease urokinase plasminogen activator (uPA). A third serine protease whose activity contributes to the chronic wound phenotype is elastase, which degrades, amongst other things, fibronectin (Rao et al. 1995; Grinnell & Zhu 1996; Herrick et al. 1997). Levels of neutrophil elastase detected in chronic wound fluid are reported to be elevated up to 1300 times that of patient-matched plasma (James et al. 2003). It has also been demonstrated that significantly higher MMP-1 and MMP-3 levels can be induced by fibroblasts on exposure to chronic venous leg ulcer wound fluid (compared with acute wound fluid) and that TIMP-1 levels are significantly depleted (Subramaniam et al. 2008). Also that distinct deficits in nitric oxide (NO) production are linked with elevations in MMP-8 and -9 expression in diabetic human skin fibroblasts compared to normal (Burrow et al. 2007). Furthermore, aberrant expression of MMP inhibitors may also contribute to the non-healing phenotype of chronic wounds. Expression of TIMP-1 in acute wounds tightly regulates collagenase activity, but in chronic wounds TIMP-1 mRNA is sometimes undetected (Vaalamo et al. 1996), or present at levels inversely proportional to protease levels (Trengove et al. 1999). In addition, the TIMP-1 protein product is also reduced in chronic wounds (Nwomeh et al. 1999) as is TIMP-2 (Vaalamo, Leivo, & Saarialho-Kere 1999; Lobmann et al. 2002), which might contribute to excessive MMP-2 activity in the wound bed. However, other studies have reported that the overall activity of gelatinases MMP-9 and MMP-2 was not increased in chronic wounds compared to normally healing wound tissues (Mirastschijski et al. 2002) and that MMP levels are actually reduced (due to elevated TIMP levels) within chronic wound fibroblasts which explains, at least in vitro, why these cells

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demonstrate a decreased ability to reorganise their surrounding ECM environment (Cook et al. 2000). Wound fluid Prolonged exposure to chronic wound fluid itself can dramatically affect cell viability and motility which was observed to be related to altered cytoskeletal actin reorganisation (Raffetto et al. 2001; De Mattei et al. 2008). Also venous ulcer wound fluid directly inhibits the MAPK ERK pathway, suggesting that the venous ulcer wound environment has negative trophic factors that effect fibroblast proliferation and ulcer healing (Raffetto et al. 2006). Chronic venous leg ulcer fluid has also been shown to inhibit the growth of dermal fibroblasts by interfering with cell-cycle progression from G1 into S phase (Seah et al. 2005). Hence, continuous exposure of the resident wound fibroblasts to the chronic wound microenvironment may induce late cellular dysfunctions and lead to delayed wound healing. Alterations in growth factors/cytokines The presence and response to key wound healing growth factors/cytokines is also altered within chronic wounds. Researchers have reported that chronic wound fibroblasts isolated from patients with severe CVI exhibit decreased responsiveness to TGF-β, EGF, bFGF and Insulin-like Growth Factor compared with fibroblasts from patients with mild CVI without accompanying ulcer (Loot et al. 2002; Lal et al. 2003). In addition, decreased responsiveness of chronic wound fibroblasts to PDGF has also been reported (Vasquez et al. 2004), with mitogen-activated protein kinase pathways being key regulators in this process (Raffetto et al. 2006). TGF-β1-3 and type II TGF-β receptor could not be demonstrated within non-healing chronic venous ulcers (Cowin et al. 2001). Furthermore, decreased expression of beta ig-h3 (a TGF-betainduced gene involved in cell adhesion, migration, and proliferation) has been reported in chronic wounds and their fibroblasts (Cha et al. 2008). With respect to FGFs the presence of FGF inhibitory factors which possess heparinlike activity in fluids of chronic skin ulcers has been suggested to significantly contribute to the mechanism of the chronicity of these wounds (Landau et al. 2001). Furthermore, within chronic venous ulcers, there are decreased basal levels of fibroblast PDGF-alpha and -beta receptors which may explain the reduced proliferation of wound fibroblasts (Vasquez et al. 2004). Oxidative stress imbalance However, cellular ageing is not solely a product of replication-dependent telomere erosion. Cells may undergo premature senescence in response to a

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variety of external stimuli, for example oxidative stress (Toussaint, Medrano, & Von Zglinicki 2000; Wlaschek et al. 2003; Naka et al. 2004). Elevated levels of oxidative stress have been reported within chronic, inflamed wounds (Wenk et al. 2001; Allhorn et al. 2003; Yeoh-Ellerton & Stacey 2003; James et al. 2003) and it is plausible that stress-induced premature senescence, rather than ‘true’ replicative senescence, is the major factor affecting fibroblast regeneration potential in these non-healing wounds (Wlaschek & ScharffetterKochanek 2005). Furthermore, heat shock protein (HSP) 70 gives a cytoprotective effect against cellular stress but is attenuated in aged cells. Hence this impaired stress response in the aged may link with the development of chronic wounds in aged, which arise from repeated ischemia-reperfusion injury (Tandara et al. 2006). The cellular response to oxidative stress varies according to the level of exposure and whilst mild stress from low level reactive oxygen species (ROS) accumulation accelerates the rate of telomere erosion (Von Zglinicki 2000; 2002; Kawanishi & Oikawa 2004), high levels of stress from increasing concentration of ROS induce telomere-independent premature senescence (Wright & Shay 2001; 2002; Song, Lee, & Hwang 2005; Zhang 2007; Wall et al. 2008). Since chronic wound sites exhibit increased levels of oxidative stress, the ‘early onset’ senescence in chronic wound fibroblasts relative to patient-matched normal fibroblasts could be a consequence of increased ROS exposure. This in turn is compounded as it has been demonstrated that chronic wound fibroblasts produce increasingly elevated levels of ROS with time in culture (Wall et al. 2008) in accordance with elevated ROS generation by senescent fibroblast cell populations (Lee et al. 2002; Song, Lee, & Hwang 2005; Chiba et al. 2005; Zdanov, Remacle, & Toussaint 2006; Passos et al. 2007; Probin, Wang, & Zhou 2007). Therefore, high levels of oxidative stress in the wound site are probably responsible for depleting the chronic wound fibroblast capacity to withstand ROS accumulation leading to impaired chronic wound healing and cellular senescence/ageing, despite the presence of a variety of cellular and extracellular antioxidant entities (Finkel & Holbrook 2000; Sen 2003; Wlaschek & Scharffetter-Kochanek 2005). Indeed ROS overproduction can inactivate dermal enzymic antioxidants (superoxide dismutases (SODs), catalase, glutathione peroxidises, etc.) (Shukla, Rasik, & Patnaik 1997; Steiling et al. 1999), deplete non-enzymic antioxidant levels (Shukla, Rasik, & Patnaik 1997; James et al. 2001) and thus lead to DNA damage, telomere erosion, cellular senescence (Finkel & Holbrook 2000; Von Zglinicki 2000) and impaired cellular responses (Moseley et al. 2004). Chemokine deficiencies It has also been reported that chronic wound fibroblasts have an impaired ability to produce the correct stromal address code required to recruit the

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inflammatory cells necessary to drive an acute phase inflammatory response that could potentially overcome the non-resolving inflammation of chronic wounds (Wall et al. 2008). Such chronic wound fibroblasts demonstrated dysfunctional regulation of five different CXCL chemokines (CXCL-1, 2, 3, 5 and 6) all of which are closely related and are functionally similar: they all bind to CXCR2 (IL-8 receptor) at the neutrophil surface, initiating an acute inflammatory response upon injury (Baggiolini 2001). The importance of CXC chemokines in mediating chronic inflammatory responses has been well characterised using rheumatoid arthritis (RA) as a model (Bradfield et al. 2003; Buckley 2003a; 2003b; Burman et al. 2005a; 2005b); however, in this disease situation synovial fibroblasts from RA patients over-express these chemokines. Despite the clear difference between RA and nonhealing chronic wounds, the overriding hypothesis is that CXC chemokines play a crucial role in inflammatory resolution and that manipulation of this stromal address code could provide a key to the successful treatment of chronic wounds in the future.

1.3.6 Chronic wounds and dysfunctional angiogenesis In aged tissues the vascularity of the skin is diminished (Van de Kerkhof et al. 1994) which would have obvious effects on the wound repair process. Age-related declines in angiogenesis during repair have been reported (Reed et al. 1998) and delayed angiogenesis has been demonstrated in an aged murine wound model (Swift, Kleinman, & DiPietro 1999). It has been suggested that angiogenesis in wounds sustained within the ageing population is different to younger individuals, since endothelial cells from older individuals produce different ECM molecules (e.g. thrombospondin) compared to those isolated from younger individuals (Kramer et al. 1985). Furthermore, they demonstrated increased adhesiveness to leucocytes, increased responsiveness to TNF (which inhibits proliferation and phenotypically alters the cells (Gamble et al. 1985)) and increased production of IL-1 (resulting in decreased proliferation). These phenotypic changes may be due to the acquisition by the endothelial cell of senescent characteristics (Chang et al. 2002). Senescence has also been associated with atherosclerosis due to evidence of shortening telomeres with age, especially in blood vessels where the endothelium is under haemodynamic stress (Chang & Harley 1995; Okuda et al. 2000). Telomere shortening is possibly due to extensive cell division to replace cells lost due to this hemodynamic stress, which in turn could contribute to the formation of atherosclerotic plaques (Chang & Harley 1995; Okuda et al. 2000). As with the dysfunctional responses of all other cell/tissue types, a failure of angiogenesis is also observed within chronic wounds. Key to this failure is the altered levels of growth factors and cytokines within the wound

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environment. For example, in chronic dermal wounds there is decreased expression of VEGF; however, this can be ameliorated through the addition of antisense molecules to TGF-β (Riedel et al. 2007; 2008). Furthermore, it has been demonstrated that the aberrant expression of vascular endothelial growth inhibitor (an endothelial cell-specific cytokine and a potent inhibitor of endothelial cell proliferation and angiogenesis) is also linked to the outcome of healing within chronic wounds (Conway et al. 2007). It has also been reported that granulocyte/macrophage colony-stimulating factor has been successfully employed in the treatment of chronic skin ulcers through its effects on driving increased blood vessel density within the ulcer bed linked to the induction of VEGF (localised to monocytes/macrophages) but not the induction of placenta growth factor (Cianfarani et al. 2006). Interestingly, it has recently been hypothesised that within such chronic wounds such a dysfunctional control of VEGF could be attributed to microRNAs (≈22 nucleotide long, endogenously expressed non-coding RNAs that regulate the expression of gene products by inhibition of translation and/ or transcription) as the VEGF signalling pathway seems to be under repressor control by miRNAs (i.e. mature miRNA-dependent mechanisms impair angiogenesis in vivo) (Shilo et al. 2007). Furthermore, FGF-2-mediated angiogenesis through application of pulsed electromagnetic fields is able to accelerate wound healing under diabetic conditions (Callaghan et al. 2008). Long-term exposure to pro-inflammatory cytokines such as TNF-α or IL-1β (as observed within the chronic wound milieu) can induced the permanent transformation of endothelial cells into myofibroblasts (at least in vtiro) (Chaudhuri, Zhou, & Karasek 2007). Whilst such a transformation has not been directly reported in blood vessels within chronic wounds, it is interesting to speculate as to the influence of the chronic wound environment on the resident cell populations. However, it has been reported that in the dermis adjacent to an ulcer, the expression of IL-1α, IL-1β, IL-1Ra, EGF and PDGFa by endothelial cells was higher than the levels of expression in endothelial cells from the distant dermis and that expression of IL-6, TNFα and granulocyte/macrophage colony-stimulating was comparable to that in cells from intact dermis. This suggests that at the margin of the venous ulcers there is a preserved cytokine and growth factor secretory potential of dermal endothelial cells (Galkowska, Olszewski, & Wojewodzka 2005). Interestingly, control of endothelial function with the chronic wounds may be under the action of transcriptional regulators such as the HOX genes since over expression of Hoxb13 results in grossly abnormal dermal vessels possibly driven by upregulation of increased levels of both VEGF and TNF-α (Mack & Maytin 2010). Furthermore, it has been demonstrated that endothelial expression of αvβ3, α5β1 and urokinase-type plasminogen is regulated by Hox D3 during angiogenesis (Boudreau & Varner 2004) and

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indeed, the decreased levels of the αvβ3 integrin receptor demonstrated on chronic wound endothelial cells would concur with this (Herouy et al. 2000).

1.4

Conclusions

As time marches on we will all become personally aware of the processes associated with ageing through obvious manifestations within our skin. Whilst such alterations in the structure of this organ and the function of its cells are inevitable, it is unfortunately becoming more commonplace that such dysfunctional activities are leading to the development of chronic, non-healing wounds. Whilst we are still searching for a definitive answer as to exactly why such wounds arise in the first place, decades of research is now highlighting exactly what causes such wounds to further develop and persist within aged individuals. Understanding the chronic inflammatory response and the actions of senescent cell populations appear to be key in the development of treatments for these chronic conditions, as does understanding the role that bacteria play (the subject of a further chapter in this series). Indeed, such treatments are desperately needed as our population ages and we head towards a dramatic rise in the number of diabetic individuals within our society.

1.5

Acknowledgements

The author would like to thank Dr Ryan Moseley (Cardiff University) and Dr Ivan Wall (University College London) for their comments and proof reading and Professor Keith Harding (Cardiff University) for the provision of the clinical images.

1.6

References

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Swift, M. E., Kleinman, H. K., & DiPietro, L. A. (1999). “Impaired wound repair and delayed angiogenesis in aged mice”, Lab Invest, 79, 1479–87. Swift, M. E., Burns, A. L., Gray, K. L., & DiPietro, L. A. (2001). “Age-related alterations in the inflammatory response to dermal injury”, Trends Biotechnol, 117, 1027–35. Tandara, A. A., Kloeters, O., Kim, I., Mogford, J. E., & Mustoe, T. A. (2006). “Age effect on HSP70: decreased resistance to ischemic and oxidative stress in HDF”, J Surg Res, 132, 32–9. Tarnuzzer, R. W. & Schultz, G. S. (1996). “Biochemical analysis of acute and chronic wound environments”, Wound Repair Regen, 4, 321–5. Thiers, B. H., Maize, J. C., Spicer, S. S., & Cantor, A. B. (1984). “The effect of aging and chronic sun exposure on human Langerhans cell populations”, Trends Biotechnol, 82, 223–6. Toussaint, O., Medrano, E. E., & Von Zglinicki, T. (2000). “Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes”, Exp Gerontol, 35, 927–45. Trengove, N. J., Stacey, M. C., MaCauley, S., Bennett, N., Gibson, J., Burslem, F., Murphy, G., & Schultz, G. (1999). “Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors”, Wound Rep Regen, 7, 442– 52. Trengove, N. J., BielefeldtOhmann, H., & Stacey, M. C. (2000). “Mitogenic activity and cytokine levels in non-healing and healing chronic leg ulcers”, Wound Rep Regen, 8, 13–25. Uitto, J. & Bernstein, E. F. (1998). “Molecular mechanisms of cutaneous aging: connective tissue alterations in the dermis”, J Investig Dermatol Symp Proc, 3, 41–4. Uitto, J., Fazio, M. J., & Olsen, D. R. (1989). “Molecular mechanisms of cutaneous aging. Age-associated connective tissue alterations in the dermis”, J Am Acad Dermatol, 21, 614–22. Usui, M. L., Mansbridge, J. N., Carter, W. G., Fujita, M., & Olerud, J. E. (2008). “Keratinocyte migration, proliferation, and differentiation in chronic ulcers from patients with diabetes and normal wounds”, J Histochem Cytochem, 56, 687–96. Vaalamo, M., Weckroth, M., Puolakkainen, P., Kere, J., Saarinen, P., Lauharanta, J, & Saarialho-Kere, U. K. (1996). “Patterns of matrix metalloproteinase and TIMP-1 expression in chronic and normally healing human cutaneous wounds”, Brit J Dermatol, 135, 52–9. Vaalamo, M., Mattila, L., Johansson, N., Kariniemi, A. L., KarjalainenLindsberg, M. L., Kahari, V. M., & SaarialhoKere, U. (1997). “Distinct populations of stromal cells express collagenase-3 (MMP-13) and collagenase-1 (MMP-1) in chronic ulcers but not in normally healing wounds”, J Invest Dermatol, 109, 96–101. Vaalamo, M., Leivo, T., & Saarialho-Kere, U. (1999). “Differential expression of tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4) in normal and aberrant wound healing”, Hum Pathol, 30, 795–802. Van de Kerkhof, P. C., Van Bergen, B., Spruijt, K., & Kuiper, J. P. (1994). “Age-related changes in wound healing”, Clin Exp Dermatol, 19, 369–74. Vande Berg, J. S., Rose, M. A., Haywood-Reid, P. L., Rudolph, R., Payne, W. G., & Robson, M. C. (2005). “Cultured pressure ulcer fibroblasts show replicative senescence with elevated production of plasmin, plasminogen activator inhibitor-1, and transforming growth factor-beta1”, Wound Repair Regen, 13, 76–83.

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Vande, B. J., Rudolph, R., Hollan, C., & Haywood-Reid, P. L. (1998). “Fibroblast senescence in pressure ulcers”, Wound Repair Regen, 6, 38–49. Vande, B. J., Smith, P. D., Haywood-Reid, P. L., Munson, A. B., Soules, K. A., & Robson, M. C. (2001). “Dynamic forces in the cell cycle affecting fibroblasts in pressure ulcers”, Wound Repair Regen, 9, 19–27. Vasquez, R., Marien, B. J., Gram, C., Goodwin, D. G., Menzoian, J. O., & Raffetto, J. D. (2004). “Proliferative capacity of venous ulcer wound fibroblasts in the presence of platelet-derived growth factor”, Vasc Endovascular Surg, 38, 355–60. Von Zglinicki, T. (2000). “Role of oxidative stress in telomere length regulation and replicative senescence”, Ann N Y Acad Sci, 908, 99–110. Von Zglinicki, T. (2002). “Oxidative stress shortens telomeres”, Trends Biochem Sci, 27, 339–44. Waddington, R. J., Moseley, R., & Embery, G. (2000). “Reactive oxygen species: a potential role in the pathogenesis of periodontal diseases”, Oral Dis, 6, 138– 51. Wall, I. B., Moseley, R., Baird, D. M., Kipling, D., Giles, P., Laffafian, I., Price, P. E., Thomas, D. W., & Stephens, P. (2008). “Fibroblast dysfunction is a key factor in the non-healing of chronic venous leg ulcers”, J Invest Dermatol, 128, 2526– 40. Wallace, H. J. & Stacey, M. C. (1998). “Levels of tumor necrosis factor-alpha (TNFalpha) and soluble TNF receptors in chronic venous leg ulcers – correlations to healing status”, J Invest Dermatol, 110, 292–6. Weckroth, M., Vaheri, A., Lauharanta, J., Sorsa, T., & Konttinen, Y. T. (1996). “Matrix metalloproteinases, gelatinase and collagenase, in chronic leg ulcers”, J Invest Dermatol, 106, 1119–24. Weckroth, M., Vaheri, A., Virolainen, S., Saarialho-Kere, U., Jahkola, T., & Siren, V. (2004). “Epithelial tissue-type plasminogen activator expression, unlike that of urokinase, its receptor, and plasminogen activator inhibitor-1, is increased in chronic venous ulcers”, Br J Dermatol, 151, 1189–96. Wenk, J., Foitzik, A., Achterberg, V., Sabiwalsky, A., Dissemond, J., Meewes, C., Reitz, A., Brenneisen, P., Wlaschek, M., Meyer-Ingold, W., & Scharffetter-Kochanek, K. (2001). “Selective pick-up of increased iron by deferoxamine-coupled cellulose abrogates the iron-driven induction of matrix-degrading metalloproteinase 1 and lipid peroxidation in human dermal fibroblasts in vitro: a new dressing concept”, J Invest Dermatol, 116, 833–9. West, M. D., Shay, J. W., Wright, W. E., & Linskens, M. H. (1996). “Altered expression of plasminogen activator and plasminogen activator inhibitor during cellular senescence”, Exp Gerontol, 31, 175–93. Wlaschek, M. & Scharffetter-Kochanek, K. (2005). “Oxidative stress in chronic venous leg ulcers”, Wound Repair Regen, 13, 452–61. Wlaschek, M., Ma, W., Jansen-Durr, P., & Scharffetter-Kochanek, K. (2003). “Photoaging as a consequence of natural and therapeutic ultraviolet irradiation – studies on PUVA-induced senescence-like growth arrest of human dermal fibroblasts”, Exp Gerontol, 38, 1265–70. Wright, W. E. & Shay, J. W. (2001). “Cellular senescence as a tumor-protection mechanism: the essential role of counting”, Curr Opin Genet Dev, 11, 98–103. Wright, W. E. & Shay, J. W. (2002). “Historical claims and current interpretations of replicative aging”, Nat Biotechnol, 20, 682–8.

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Wysocki, A. B., Staianocoico, L., & Grinnell, F. (1993). “Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9”, J Invest Dermatol, 101, 64–8. Xia, Y. P., Zhao, Y., Tyrone, J. W., Chen, A., & Mustoe, T. A. (2001). “Differential activation of migration by hypoxia in keratinocytes isolated from donors of increasing age: implication for chronic wounds in the elderly”, J Invest Dermatol, 116, 50–6. Yaar, M. (1995). “Molecular mechanisms of skin aging”, Adv Dermatol, 10, 63–75. Yaar, M. & Gilchrest, B. A. 1999, “Aging of the skin,” in Fitzpatrick’s Dermatology in General Medicine, 5th edn, vol. 1 I. M. Freedberg, A. Z. Eisen, & K. Wolff, eds., McGraw-Hill, New York, pp. 1697–1706. Yaar, M. & Gilchrest, B. A. (2001). “Ageing and photoageing of keratinocytes and melanocytes”, Clin Exp Dermatol, 26, 583–91. Yager, D. R. & Nwomeh, B. C. (1999). “The proteolytic environment of chronic wounds”, Wound Rep Regen, 7, 433–41. Yeoh-Ellerton, S. & Stacey, M. C. (2003). “Iron and 8-isoprostane levels in acute and chronic wounds”, J Invest Dermatol, 121, 918–25. Zdanov, S., Remacle, J., & Toussaint, O. (2006). “Establishment of H2O2-induced premature senescence in human fibroblasts concomitant with increased cellular production of H2O2”, Ann N Y Acad Sci, 1067, 210–6. Zeng, G. & Millis, A. J. (1994). “Expression of 72-kDa gelatinase and TIMP-2 in early and late passage human fibroblasts”, Exp Cell Res, 213, 148–55. Zhang, H. (2007). “Molecular signaling and genetic pathways of senescence: Its role in tumorigenesis and aging”, J Cell Physiol, 210, 567–74.

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2 The role of micro-organisms and biofilms in dysfunctional wound healing J. G. T H O M A S, H. M O T L AG H, S. B. P O V E Y and S. L . P E R C I VA L, West Virginia University, USA

Abstract: The relationship of microbes and wounds has always been controversial; the unmasking of biofilms as the primary mechanism of microbial pathogenicity in chronic wounds has only heightened this controversy. Here, we attempted to give credence and data to support this theme-heightened hypothesis, utilizing: 1) “universal microbial principles” that are constant, independent of the site of infection; 2) integrating new terms and definitions reflective of updated microbial methods, detection, and modeling (pre-clinical trials); and 3) organized this education into four sections. Each section builds on the previous theme, with the final section revisiting the consequences of wound pathogenesis, focusing rather, on the benefits of a microbial biofilm (probiotics) and inhibition of planktonic, traditional wound pathogens. Key words: Biofilms or sessile communities, “critical colonization/climax community,” chronic or non-healing wounds, microbial staging or growth cycle: I–IV, probiotics in wound care, planktonic phenotype vs. biofilm phenotype: a biomarker, “ping-pong” infection, Anti-Koch, ratio: biofilm to planktonic phenotypes.

2.1

Introduction

We “live in a microbial world” and the diverse, dynamic, and flexible microbiota in Homo sapiens outnumber human cells 30 trillion to 100 trillion; its implication in skin and soft tissue infections (SSI), generally, and chronic wounds, specifically, in morbidity 70%, mortality 12%, and average cost increase (US $32,000) reflects an aging population which will only increase. This human “microbial world” exists both as free floating (planktonic – Pp) and sessile (biofilm – Pbf) phenotypes, but given the opportunity, 99.9% of organisms would preferably attach, form a multi-species biofilm, whose combined physical, chemical and biologic properties metamorph into a lava-like, hydrated polymer, encompassing physical laws of viscoelasticity. The 3-dimensional structure and Stages (I–IV) are influenced by eight features; the most important are stress and energy that drives the microbial community toward “critical colonization” or “climax community,” a pathogenic event in non-wound healing. Non-healing wounds advance through a 39 © Woodhead Publishing Limited, 2011

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bi-phasic pathway, early and late, with the former physiologic with endogenous microbes, the later exogenous origin, emphasizing the conversion from non-pathogenic to pathogenic via implantation of P.D. Marsh’s Ecologic Hypothesis (Anti-Koch), expanded upon by us to include an imbalance of planktonic microbes: biofilm phenotypes as a disease biomarker: >1. In contrast, studies by us using a triphasic wound model, employing eucaryotic cells, multi-species procaryotic biofilms, separated by a variablesize filter have shown that cell signaling via diffusion biofilms products, may be an active site for intervention, including wound dressings, where “pingpong” reinfection could be reduced. Neutralizing these signals by “critical colonization” or a stage III “climax community” with a beneficial probiotic (Lactobacillus reuteri), while maintaining a biofilm:plankotnic ratio, >1, reversing eucaryotic cell deterioration.

2.2

Microbiology and biofilms: not mutually exclusive

In this section the roll of planktonic and biofilm microorganisms will be discussed. The important aspect to consider is the ratio of planktonic (Pp) to biofilm phenotype (Pbf) which are present in chronic wounds and the potential for clinical assessment.

2.2.1 Disease, statistics and background Medical awareness is often driven by cost. The study of chronic wounds is no exception. Recent data says in the US 5.7 million patients acquire infections per year of which there is a cost of 20 billion dollars annually. In a more global sense for western cultures, wounds represent 1 to 2 percent prevalence of the population, specifically, in Germany there are 2 to 3 million patients per year with chronic wounds. In the UK there is approximately 3.55 per 1000 of the population (Vowden et al., 2009) who have chronic wounds. This problem will be be inflated by the increase in population (60.4 to 63.8 million, 5.6%) and percentage of residents 65 and over (9.5 to 13.0 million, 36%) by 2025 (Posnett and Franks, 2008). Another way to analyze the importance of cost in chronic wound infections is to combine the wounds in corresponding DRGs (diagnostic related groups) segregated by disease type in the US. There are six types of wound considered, including acute arterial ulcers, burns, diabetic foot ulcers, pressure ulcers, and venous leg ulcers. Chronic wounds represent a major feature and include foot and leg ulcers and pressure sores. Corresponding costs are significant when looking at DRGs. Diabetic foot pressure and venous leg

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ulcers are the most frequent wounds affected by the new categorization of Medicare-adjusted Severity DRG, or MS-DRG, respectively. Having the most reduced reimbursement, burns and arterial ulcers, are less frequently seen in the elderly groups. Interestingly from 1950 to 2005, total US population increased from 151 to 296 million with the elderly population growing 2.8 percent. In the year 2005, total charges for DRGs #139 (peripheral vascular disease) was greater than $1.5 million, yet Medicare reimbursed it by essentially 50 percent. For skin ulcers, DRG #271, the cost was $373 million (US) and Medicare paid $111 million. Similar trends of composition continue for wound debridement from injuries, DRG #440, $195 million, where total charges were only $63 million that was covered by Medicare.

2.2.2 A microbial world: anatomic barriers (artificial boundaries) versus microbial barriers “We live in a microbial world,” has been a statement recently maximized given a number of studies addressing the total microbial human composition. It is now estimated there are ten times the number of bacterial cells versus human cells in the body: 100 million or 108 million bacterial cells. A rudimentary and principal way of organizing this microbial population is to address anatomic locations, where the largest normal flora or reservoir exists (Table 2.1). Here, the four historical normal flora reservoirs of bacteria are listed recognizing the specific organ to organ system, i.e. the human source, the bioburden or central microbial population, the ratio of aerobe to anaerobe, and the diversity of microbial organisms recovered from each of those anatomic sites. In fact, this is a rather fundamental and rudimentary classification system, but one which helps organize initial classification, which will be under significant scrutiny as more and more is learned about the microbial population and the microbiota of the human beings. Of significant importance, more recently described, is the microbial barriers and the fact that microbes have small niches that extend and have significant diversity overlapping these four established barriers. Certainly there is cooperation and transmission between the four anatomic descriptions, but clearly within each there are significant barriers that are established by the microbial populations and their synergy antagonism and mutualism. In understanding the pathophysiology of wound microbial barriers, the reclassification by site may be as important as a traditional classification by anatomic reservoirs. Figure 2.1 expands the classification by the four established reservoirs and integrates not only the organization of diseases with more common flora, but describes them with an emphasis on organisms associated with disease. Hence, the anatomic location does have significance and can give

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Advanced wound repair therapies Table 2.1 This table describes the four reservoirs of Homo sapiens outlined in the ‘Infected patient’ (Figure 2.1) but emphasizes three additional features: organism bioburden, organism ratio (aerobic to anaerobic), and the diversity within the site described 4 normal flora reservoirs of bacteria an interactive continuum Human source

Bioburden

Ratio

Diversity

• • • •

1011 108 106 106

1000 : 1 100 : 1 10 : 1 1:1

200 200 700 50

GI Tract Urogenital Mouth Skin

When anaerobic infection might be suspected Brain abscess Cellulitis of jaw Cervical adenitis 1. Mouth • Streptococcus • Bacteriodes • Fusobacterium

Anaerobic pneumonitis Lung abscess Empyema Endocarditis Abscess Hepatic abscess Diverticulitis

4. Skin • Staphylococcus • Proprionibacteria • Diptheroids • Candida

Perotinitis Appendicitis Pelvic cellulitis Septic abortion Endometritis Post surgical vaginal cut infection Non‐gonococcal tubo ovarian abscess Skin infections Soft tissue infections Post amputation infections

Soil • Clostridium novie, septicum, perfringens • Clostridium tetani (gas gangrene)

Gas forming cellulitis

2.1 ‘The infected patient’ illustrates the four traditional anatomical barriers of Homo sapiens with the potential consequences of microbial colonization, their relationship to each other and the most common skin-associated infections.

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Septicemia

GI TRACT 2. Lower bowel & 3. Genital (vaginal) area • Bacteriodes fragilis • Anaerobic Strep • Clostridium novie, septicum, perfringens (gas gangrene)

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predilection and potential predication as to what the most common historical organisms are, recognizing the data is based, or has been up to now, on culture information from standard methodologies. Here, the classification systems also describe the populations in a rather broad sense and include aerobic organisms, which grow at a potential Eh of approximately +30 millivolts and anaerobic microbes, which favor an Eh or oxidation reduction potential of −180 millivolts, but in either case there was rather a broad range (Bowler, 1998). Table 2.2 describes the microbes associated in wound infections by aerotolerance. It, too, is historical, but builds upon the concept of microbial barriers, this time emphasized by oxidation reduction potential where there is a millivolt recognition and a more definitive characterization of the area and environment for which an organism will find biostability. There are basically three types in this scheme: type 1 – aerotolerant, type 2 – obligant anaerobes, and type 3 – strict anaerobes. There obviously is a significant overlap and many “traditional aerobic isolates,” are in fact obligant anaerobes and have the capacity to metabolically survive in either a relatively

Table 2.2 This table organizes organisms by aero-tolerance, recognizing the oxidation reduction potential (Eh) from an oxygen electrode at +420 millivolts to a hydrogen electrode at −410 millivolts Classification

Type I “Aerotolerant”

Type II “Obligate anaerobes”

Type III “Strict anaerobes”

Organism

Eh(mv)

Oxygen electrode @ pH 7 C. perfringens @ pH 6.0

+480 +250

B. vulgatus C. perfringens @ pH 7.8 Methylene blue becomes colorless @ pH 7.0 Resazurin

+140 +30 +11 0.0 −42

Anaerobic blood plates Mean upper value for abscess and infectious site

−150 −350

Treponemes Intestinal tract Crypts of mouth Hydrogen electrode @ pH 7

−280 −300 −410

Study orgainism

Candida spp. Pseudomonas spp. Enterococcus spp. Enterics (E. coli ) Staphylococcus spp. Streptococci spp.

Bacteriodies spp.

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oxygenated tissue or a relatively anaerobic tissue. Type 3 are strict anaerobes, which clearly have the capacity to only exist in a low Eh (oxidation reduction potential) and are often associated with late stage disease where the oxygen and Eh associated with it has been significantly reduced. NIH Human microbiome study The Human Microbiome study was initiated in 2007 (Thurnbaugh et al., 2007) to amplify the microbial flora utilizing molecular methods and complement the human genomic study. Its purpose was to better define the microbial tool, recognizing limitations of standard culture techniques and redefined again the anatomical location for skin by sebaceous moist and dry skin locations. The emphasis recognized that the majority of microorganisms inhabiting the skin were classified as viable, but non-culturable (VBNC). Twelve skin sites were cultured for the associated microbiota using molecular methods detecting 16 SRNA in the three micro environments. The samples were taken from healthy volunteers.

2.2.3 Definitions and pathophysiology of disease emphasising Eh/pH early and late colonization Wounds are generally described in a microbial sense as biphasic, a transition from an aerobic to an anaerobic environment as the wound process develops. Figure 2.2 integrates the clinical scenario with time based on producing an environment selecting for or amplifying an Eh that describes a favorable environment for organism growth. The simple schematic describes general health of the wound site from good to bad and integrates the transition from a planktonic environment to a biofilm environment; it also highlights the limitations of microbial recovery utilizing traditional culture methods. Sections I, II, and III (top to bottom), correlate to the aerotolerance, which defined the organisms relative to Eh, which is correlated with an Eh of the wound site. Here we see the characteristics of organisms in concert where environments select for cohabitation and synergy of selected microbes. Bacterial synergy is known to occur in chronic infected wounds, particularly between the anaerobic aerobic (positive Eh) and aerobic anaerobic (positive Eh, negative Eh bacteria). The aerobic-anaerobic synergy evident in chronic wounds is known to enhance degradation caused by bacterial enzymes of normally healthy tissue and also increased malodor in certain wound types. Essentially bacterial synergy or mutualism provides a competitive advantage to cohabiting bacteria within a protected biofilm environment, particularly within adverse tissue conditions. In essence, the wound environment and the selective pressures of pH/Eh and those eight

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Biphasic wound infection: a simple schematic (mixed aerobic: anaerobic organisms) Clinical O2 condition

I

II

III

H2

Poor

Tissue/patient responses

Good

Phase: Eh: Clinical: Representative organism: Variables:

aerobic anaerobic positive m.v. negative m.v. acute (peritonitis) late (abscess) E. coli Bacteroides fragilis organism, tissue integrity, host integrity, choice of antibiotics

2.2 ‘The biphasic wound’ illustrates the universal microbial principle that infections are routinely biphasic, ‘Early’ and ‘Late’ with the early most often associated with Gram positive organisms, the late with Gram negative.

features of biofilm selection define the microbial population which will be colonizing and essentially addressing critical colonization of the tissue.

2.2.4 Microbial location: planktonic (PP) organisms identified by clinical studies Over the years there have been innumerable studies describing the microbiology of wounds and the prevalence of selected phenotypes. These historical studies have given credence to the microbial etiology and potential consequences in a chronic wound, but have been wrought with problems particularly with interpretation of the data, the method of culture, and most recently the recognition of biofilms and their potential impact. Until the advent of molecular methods almost all studies were based on culture studies, which with their limitations gave confounding and often conflicting emphasis on both the microbes present in wounds and their significance in wound detail versus wound healing. Recognizing the growing importance of viable but non-culturable (VBNC) bacteria (Table 2.3) and the emergence of molecular methods

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Advanced wound repair therapies Table 2.3 This table highlights that we live in a ‘microbial world’ and if we evaluate the predominance of microbes in our body (a thousand prokaryotes to every single eukaryote) it is the environment and the globe around this that is predominantly an unrecognized microbial population Habitat

Cultured (%)

Seawater Freshwater Mesotrophic lakes Estuarine waters Activated sludge Sediments Soil

0.001–0.1 0.25 0.1–1 0.1–3 1–15 0.25 0.3

Note: Numbers based on direct cell counts. The majority of prokaryotes are non-culturable. Source: Daims, H. University of Vienna. Department of Microbial Ecology.

available to our diagnostic laboratories, non-culture techniques have grown in use in studies of chronic wounds. The molecular methods incorporated the use of 16-S ribosomal DNA-PCR incorporated with denaturing gradient gel electrophoresis (DGGE) (Fig. 2.3). DGGE is a recognized method that describes the diversity of a microbial population uninhibited by the limitations of bacterial culture. Its limits are obviously that it does not recognize organisms by Eh/pH nor does it recognize living from dead, but the bottom line is that there is another method to establish the diversity and complexity of a microbial pool often associated with biofilm structure (Hill et al., 2003). A number of studies have highlighted the use of 16-S RNA (Down et al., 2008). Davis et al. in the “Analysis of the microflora of healing in nonhealing chronic venous leg ulcers” using 16-S ribosomal DNA-PCR and denaturing gradient gel electrophoresis (DGGE), highlighted the complexity of the microbial community, the limitations of culturing and the use of this method to distinguish the microbial pool from healed versus nonhealed tissue (Davis et al., 2008; Dowd et al., 2008; Down et al., 2008). Studies by Dowd et al. (2008) indicated the complexity of the wound bed and the lack of correlation, culture to non-culture techniques. The unique point is that molecular methods are not limited by techniques necessarily established in large laboratories and allow for global signature of organisms associated with wounds. Table 2.4 compares the disparity between non-culture and traditional methods (Bowler and Davies, 1999; Wolcott and Dowd, 2008; Wolcott et al., 2009).

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DGGE profiles of bacterial DNA from healed and non-healed tissues Healers

Non-Healers

30%

S 48 12 53 19 24 26 28 49 2 5 6 7 14 15 17 18 25 29 S a a a b

A B

a

a a

c

a

a

a

b

b

b

a

a

a b

b

b c b

b

B

a d

b

C

A

a b

a

b

• Each band is thought to represent a bacterial species

a

C

b

a

a

• Each band labelled A, B, C, or D was excised and sequenced

c c

b

D

D a

b c

60%

2.3 DGGE (density gradient gel electrophoresis) is a method for defining the population diversity in microbial pool that employs two universal primers for replication of the nucleic acids (16S RNA) followed by gel electrophoresis separation in an acid and urea environment.

The NIH Microbiome study may be the benchmark to clarify the organism disparities; here using molecular methods and multiple cultures, the core microbiota is being catalogued, it is unmasking organisms previously undetected in skin, and illuminates the VBNC problem. Further, Fig. 2.4 highlights differences in methods of reporting; Fig. 2.4 utilizes data from USA clinical laboratories, reporting organisms defined by susceptibility testing and sorted by: 1) patient and 2) results (Sun et al., 2008). To help clarify significance, one of the historical aspects has involved quantification. A plethora of studies have described the use of quantification of the potential bench marks of certain colony counts of certain organisms to potential pathogenicity. This has always been fraught with problems, but underscored the difficulty in assessing this mixed microbial population. Historically, 104 CFUs/ml (colony forming units) have been used as a baseline to encourage laboratories to identify individual organisms rather than reporting general versus species. A number of studies have indicated that that bench mark was wrought with problems, however, and more recently CFUs/cm3 has gained credibility.

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Peptostreptococcus spp. Propionibacterium spp. Clostridium spp.

S. aureus S. epidermidis Coag-neg Staph spp. Streptococcus spp. Enterococcus faecalis Enterococcus spp. Corynebacterium spp. E. coli Proteus mirabilis Proteus spp. Klebsiella pneumoniae P. aeruginosa

Martin JM, et al. J Investigative Dermatology. 2010:130:38–48.

Bacteroides spp. Prevotella spp. Finegoldia spp.

Anaerobic

Aerobic

Traditional culture

S. aureus S. dysgalactiae Streptococcus spp. Enterococcus spp. Acinetobacter spp. Hemophilus spp. Rhodopseudomonas spp. Sphingomonas spp. Citrobacter spp. Enterobacter cloacae Proteus spp. Morganella morganii Pseudomonas spp. Serratia spp. Stenotrophomonas spp.

Aerobic

Non-culture techniques

Comparison of wound organism detection by methods and aero-tolerance

Anerococcus spp. Anaerococcus vaginalis P. asaccharolyticus Peptococcus spp. Peptoniphilus spp. Dialister spp. Veillonella atypia Clostridium spp. Finegoldia magna Bacteroides fragilis

Anaerobic

Table 2.4 This table outlines the difficulties of organism detection, depending on the culturable methods utilized and the emphasis on aerobic and anaerobic

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Ten most common wound organisms (patients) Jan 2000‐Aug 2010 (TSN 2010) Enterobacter cloacae Proteus mirabilis Staphylococcus epidermidis Enterococcus faecalis Klebsiella pneumoniae Enterococcus species Pseudomonas aeruginosa Escherichia coli Staphylococcus species, (coagulase negative) Staphylococcus aureus 0

20,000 40,000 60,000 80,000 100,000 120,000 140,000 160,000

Ten most common wound isolates (results) Jan 2000‐Aug 2010 Enterococcus faecalis Enterococcus species Enterobacter cloacae Proteus mirabilis Staphylococcus epidermidis Klebsiella pneumoniae Pseudomonas aeruginosa Staphylococcus species, (coagulase negative) Escherichia coli Staphylococcus aureus 0

500,000

1,000,000

1,500,000

2,000,000

2,500,000

2.4 Wound isolates tabulated by the USA, The Surveillance Network (TSN) where organism detections are entered into a repository from over 500 participating institutions on a daily basis.

2.2.5 Clinical implications: the biofilm/plantonic phenotype ratio As the complexity and dynamic environment of wound cultures has been clarified, it has also become apparent that limitations utilizing standing microbial principles of quantification and/or species named were also inadequate. We have felt for some time that the importance of biofilms was underappreciated, but that the importance of a biofilm in itself was limited, given that in the milieu of a wound both planktonic and biophenotypes are prevalent. In fact, it is our hypothesis, that the ratio of biofilm to planktonic phenotype is perhaps the most unrecognized important feature in the assessing of either delayed healing or the improvement and transition from non-healing to healing. We propose that the microbial lifecycle of a wound in fact involves three phenotypes, none of which are mutually exclusive. This involves

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Advanced wound repair therapies Percival/Thomas: Dual Hypothesis Pathogenesis INTER-RELATIONSHIP CRITICAL COLONIZATION (Anti-Koch) HYPOTHESIS I

ENDOGENOUS SKIN FLORA TISSUE Eucaryote DEPTH

EXOGENOUS PATHOGENS

EARLY Critical Colonization

HYPOTHESIS II

Procaryote

BIOFILM RATIO LATE

ENVIRONMENT pH/Eh/STRESS 8 SELECTIVE PRESSURES

5–7 Day

1.

2.

BI-PHASIC CHRONIC WOUND ENVIRONMENT

ORGANISM SELECTION / BF STRUCTURE

2.5 ‘The Percival/Thomas Dual Hypothesis’ is an amplification of Figure 2.2 that describes the biphasic nature of most microbial infections, Gram positive to Gram negative, integrating the tissue environment (Percival and Cutting 2009).

the interface of a planktonic strain and a biofilm strain, but recognizes the biofilm strain may be attached or unattached to abiotic or biotic surfaces. These all have an influence on the proportion of these different phenotypes that magnify the success or the failure of wound management. Our hypothesis is shown in Fig. 2.5. Figure 2.6 shows a presentation of our biofilm: planktonic ratio hypothesis, which has been supported by data found by us in recent studies (Section 2.5). We feel that the ratio is a biomarker and can be simply applied using Congo red plates. The concept is not new and rather is an expansion of an age old microbial principle that says an infection (ID) is equal to the numbers (N) times the virulence (V) divided by the immunology (I) of the site (ID = (N × V)/I). This is our means of amplifying the well-recognized principles of balance required in normal tissue involving the immune response.

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Congo Red+ / Biofilm (CFUs / ml / cm2)

COLONIZATION RESISTANCE 1000

RATIO: CFUs

BAD >5 >1 and <5 COLONIZATION

BAD BioBurden

PHENOTYPE Biofilm Planktonic

GOOD <1

GOOD

Congo Red > Blood Agar Plate Colonies

100 100

Congo Red-/ Planktonic (CFUs / ml / cm2)

1000

2.6 “Biofilm: planktonic ratio.” This figure illustrates the importance of the Percival/Thomas Hypothesis and the transition in the late stage of a biphasic wound infection shown in Figure 2.5.

2.3

Biofilms and the interactive cooperative community

This is an overview of the role of biofilms as well as the mechanisms which are employed for communication amongst the combination of bacteria.

2.3.1 Definitions The term “biofilm” was originally coined by engineers, who were particularly interested in the impact of slime bacteria and the efficiency of water filtration. In fact, the original work published was most often found in sanitation and water engineering journals and appeared in nonmedical journals because medicine and biofilms had not had an interface in the early 1980s. Today much has changed and the definitions associated with biofilms have reflected the participants and their background in the growing importance of biofilms in medicine and dentistry. The evolution has involved first the clarification of its structure, the importance of its function, and most recently the impact in diseases, particularly the recognition in chronic disease evidence. From a microbiological sense, a biofilm is both anti-Pasteur, anti-Koch, and anti-Darwin. The biofilm favors “group level selection” rather than “individual selection” and the key comparative points are emphasized into one. Here, the anti-Koch, anti-Pasteur concept is emphasized by addressing

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competitive behaviour versus cooperation, limited diversity versus population heterogeneity, decreased virulence versus increased virulence, and resource consumption versus economical use of limited resources (HallStoodley et al., 2004). The initial definition of a biofilm includes the following features: • • • • •

Attachment in cascade fashion, Gram positive to Gram negative 3-dimensional architecture glue like outer skin: EPS (extra-polymeric substances) physical features defined by rheology and hydrated polymer loss of antibiotic susceptibility.

A biofilm is a group of microorganisms attached to a surface surrounded by an EPS (extra polymeric substance). It was recalcitrant to traditional planktonic antimicrobials. Interestingly, given the opportunity, 99.9 percent of organisms would attach rather than free flow. The biofilm phenotype or lifestyle is the one preferred by all microorganisms evaluated. Whether these are involved in non-diseased states, i.e. environmental or medical/ dental disease, or industrial, the theme is the same. The defined structure and three-dimensional organization of biofilms has continuously evolved with new evidence-based research. Today it is recognized more in a developmental sense, defined as a primitive type of developmental biology in which special organization of the cells within the matrix optimizes the utilization of the nutritional resources available. It can be further defined as an immobilized enzyme system in which the milieu and the enzyme activities are constantly changing and evolving to an appropriate steady state. The steady state can be radically altered by applying physical forces such as high shear, a very important feature in medical diseases. Four definitive factors integrate to build a biofilm and the consequences of its structure, where “structure equals function”; these properties can be sorted by: 1) structural, 2) physiologic gradients, 3) chemical and physical, and lastly, 4) biologic. The components of the biofilm are often reflective of the environment, but in generality the following observations can be made: up to 97 percent can be fluid, reflecting the environment; microbial cells; multiple species may represent 2 to 5 percent; polysaccharide 1 to 2 percent, usually neutral or polyanic; proteins less than 1 to 2 percent; DNA and RNA less than 1 percent; ions variable. Host factors, such as fibrin, red blood cells (RBCs) and white blood cells (WBCs) all reflective of the environment of the biofilm.

2.3.2 Biofilm structure where “structure equals function” The 3-D architecture of the biofilm is reflected in its four stages of growth and embodies the statement, “structure equals function.” The four stages

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Table 2.5 This table highlights the staging of a biofilm and the universal cyclic growth of microbes between Lag, Log, Stationary, and Death, respectively Stages I, II, III, and IV Planktonic PP/biofilm PBF staging Staging

Description

Characteristics

0 I A, B II III IV A, B

Free floating Early Immature/moderate Mature/highly complex Late/apoptosis or necrosis

Individual cells Loose, open Increased biomass Increased biodiversity Free-floating

(I–IV) of the microbial lifecycle in a biofilm integrate the universal microbial growth pattern of: I – Lag, II – Log, III – Stationary, and IV – Death or dispersal. It also highlights the relative times associated with each of the stages, the most famous of which is the “mushroom-like” appearance, often described for Pseudomonas biofilms; as shown in Table 2.5, each stage has a unique description (Penhallow, 2005). Stages I and IV are the most unique in that they clearly represent reversible processes. In the Lag phase, conditioning of the surface either abiotic or biotic is critical; “pioneering” bacteria will be selected based on the preconditioning of the surface to which they adhere. That is a reversible process followed by an irreversible process when the pioneering bacteria recruit and actively aggregate in a specially defined arrangement as one shifts from the Stage of I to II, the log growth of this community. Simultaneously Stage IV has a reversible step which defines the transmission and transfer from the biofilm phenotype to the planktonic phenotype. There may be death of these components, or there may be a recycling from the biofilm to the planktonic scheme, which is represented in the figure by the upper half or the light-colored background. Please note: All microbial communities, in fact, involve a cyclical nature of both the biofilm phenotype and the planktonic phenotype. Figure 2.7 defines this interface in a more integrated manner recognizing the planktonic (Pp), or Stage “0”, is not the preferred phenotype, but that Stage I, II, III, and IV all represent growth in a cyclic nature of the biofilm (Pbf) interface with its environment and the planktonic phenotype (Pp) (Kirketerp-Moller et al., 2008). Staging, description, and characteristics of the individual cells are described in Table 2.1, which highlights the characterization and organization of the biofilm or its planktonic component in the previously described sections. The corresponding SEM depicting the growth of biofilms in

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Advanced wound repair therapies MICROBIAL STAGE CYCLE

SESSILE ATTACHED

PLANKTONIC FREE-FLOATING

STAGE

I-A I-B LAG

II LOG

UNIVERSAL GROWTH CURVE

STOP HERE

LAG

Attachment Pioneer Bacteria Conditioning Seconds

III STATIONARY

Conversion or Transformation Social Behavior Diversity Recruitment

Minutes

Growth Biomass Gradients

Maturity Competition

Surface

Hours

IV-A IV-B DEATH

PROMOTE HERE

Detachment Eroding and sloughing (metastis) Recycle

Days

John Thomas, Ph.D, West Virginia University

Intra Oral Sessile (PBF) ( ) and planktonic (PP) ( ) life forms are not mutually exclusive, but biofilms are the preferred growth vehicle

2.7 The four cyclical stages (I–IV) of biofilm development, incorporating the universal microbial principles of microbial growth: I – Lag, II – Log, III – Stationary, and IV – Death.

poloxamer is shown in Fig. 2.8. This highlights key characteristics of each of the featured stages of biofilm characterization emphasizing key words such as “channelling,” “domain,” and “aggregates.” Stage III is where the biofilm has reached a “climaxed community” and achieved “microbial homeostasis.” We also refer to this stage as the ‘biopot’ stage, characterized by the flexibility of a kelp bed, for example, able to move with the changing tides and currents of water surrounding it. Figure 2.9 is perhaps the most relevant in discussing medical wounds and further evaluation of biofilms and the contribution they may play in dysfunctional healing; whereas, Fig. 2.7 represented a general discussion of biofilm in its interrelationship with its planktonic counterpart. The reality is that biofilms in medicine, often in wounds, are multi-species organizations and that the overall presentation is greater than the individual parts and within each organism niche there are different communities that have different cycles and different presentations within the biofilm. We often talk about monospecies biofilm, co-biofilm, and complex biofilms. Catheter

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Biofilm staging of wound specimens

I

II

III

IV

2.8 ‘Electron micrographs depicting the four stages of biofilm phenotype’ as characterized in Figure 2.7.

Biofilm development: mono to complex Monospecies biofilm Co-biofilm Medical ICU CR-BSI Biofilm lifecycle Line sepsis IMD

Complex-biofilm Dental Periodontal

III III

Biomass

III II II I

IV II

I

I

I

Sa, Ca, Sm

Microbes:

IV

IV

Gram positive

IV

II

MRSA, Pa, Lactobacillus Time Gram negative

III

P. ging, F. nucleatum

Anaerobic

Extrapolymeric Substance:

2.9 The growth of a biofilm in numbers of organisms is shown here over time, and focuses on the disease entities generally found with a monospecies or a complex biofilms.

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related sepsis, or line sepsis, and indwelling medical devices often are associated with monospecies biofilms. Complex biofilms could be associated with dental as in periodontal disease, or in wounds given the length of time of colonization in the environment and proximity to large reservoirs of microorganisms. Often the early colonizers are aerobic, shifting to a Gram negative anaerobic flora as time goes on and as the evolution of the concept biphasic early to late is manifested in the actual environment. It is important to recognize the key contribution of the EPS (extra polymeric substance). It is a major regulatory feature of biofilms and controls a great deal of the environment within the biofilm. Clinical diagnosis has also evolved, but is often controversial compared to the original criteria of Donnaly and Costerton. Singh et al. (2002) utilized criteria that defined the biofilm as: 1) surface associated, 2) composed of microbial aggregates, 3) localized infections that were intractable to antibiotic treatment despite the demonstrated susceptibility of the corresponding planktonic bacteria. Stoodley and Stoodley, in 2009, decided that diagnostic criteria for biofilm infections should include: 1) 2) 3) 4) 5) 6)

pathogenic bacteria associated with a surface, direct examination of infected tissue demonstrated aggregated procaryotic cells in cell clusters encased in a matrix, infection was confirmed to a particular location in the host, infection location recalcitrant to antibiotic treatment despite demonstrated susceptibility to planktonic bacteria, culture negative results in spite of clinically documented high suspicion of infection confirmed by culture independent methods, ineffective host clearance evidenced by the location of bacterial cells and bacterial clusters in discrete areas in the host tissues associated with host inflammatory cells.

This 3-D architecture is complex, but in research there are at least eight factors, which are involved in the architecture in ultimate configuration of the mature biofilm. These are listed in Fig. 2.10. The eight features are shown; of those, four are particularly germane and key in its organization. First, it is a species that is chosen in the colonization and in the selection of the other bugs associated with it. Second, is the substratum, the surface to which colonization occurs following conditioning. Third, is the nutrient and energy source, what is available for the population and who can use it most efficiently in the structure of “I” versus “We”. Fourth, the mechanical factors in sheer forces, which are critical in environments, distinguished by vortex turbulence or parallel sheer. These can be measured in Reynolds units and are incredibly important for ventilator-associated pneumonia and endotracheal colonization versus a more static environment of a wound surface.

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BIOFILM DEVELOPMENT EXAMPLE-VAP 8-Factors that are involved in biofilm ARCHITECTURE Species colonization, community makeup * (Anti-infective) hostile forces The physico-chemical environment

Genotypic factors * Cyclic stage

THE BIOFILM COMMUNITY STRUCTURE AND EVOLUTION Mechanical factors and shear forces

Substratum

Nutrient energy,resource * Intrinsic

2.10 The eight factors that define the 3-D architecture of a biofilm structure are listed with the four most important in wound infections highlighted by gray composition.

2.3.3 Chemical, physical and environmental properties of biofilms Biofilms have unique properties that make them recalcitrant to antimicrobial therapy, and resistant to physical forces which enable them to persist and survive in hostile environments. First, biofilms in fact act as a hydrogel or extremely hydrated polymer gel. As a hydrogel the physical properties of the biofilm are described by viscoelasticity in which they can exhibit elasticity associated with solids, and viscous liquid-like properties simultaneously. The consequence is that in seconds biofilms absorb increased sheer by behaving elastistically and in long periods the sheer is dissipated through viscous flow or streamlined to reduce drag of the biofilm 3-D structure. This viscoelasticity can be best described by features of rheology. In fact, rheology was a description first heralded by the Greek philosopher Heraclitus (540 to 580 BC) of Ephesus. He stated that everything flows and nothing abides; everything gives way and nothing stays fixed. In fact, these properties can be best demonstrated by lava flow and there is a relationship between them the sheer stress, elastic stress, and viscous stress. This allows for very exacting methodology of forces needed to address a biofilm and the consequence of these forces in the ultimate 3-D architecture. These properties are considered attributable to the EPS. That is, the EPS, which maximizes the characteristics of viscoelasticity in the individual property of rheology.

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Advanced wound repair therapies SURFACE O2 gradient

+ 420

14

Nutrition sugar

Environment oxygen

Saccharolytic

Aerobic Anaerobic Aciduric

Eh redox agents

Sugar substitutes

Ph control agents

Eh Scale

Micro-aerophilic

pH Scale

Asaccharolytic Anaerobic

A

Domain B

– 410

Abiotic Enamel

H2 Domain

D C

Domain

Domain

Substratum POCKET

Cells Biotic Tissue

1

2.11 A complement to Figure 2.10 this diagrammatic representation highlights the importance from top to bottom of the biofilm and its gradients described by environmental pressures Eh (oxygen reduction potential) and pH.

The environmental properties described usually in physiology, gradients, and metabolism are highlighted in Fig. 2.11, which shows the interface of both Eh and pH in the environment of the biofilm formation and describes then the lattice and barriers with which microbes will search and find stability. A wound environment, in fact any environment, espouses the principle of Galileo. Organisms will search for the environment that favors their nutritional success and the cooperation is defined by the “I” (Robert Koch) versus “We” concept (anti-Koch). The most interesting and unique finding of biofilms is the reality that their actions and activities mimic activities in a developmental sense of another multi cellular organization, tumors. In fact, it has been our hypothesis that communities having linked microbes masquerading as a biofilm, are really procaryotic tumors. It is our further hypothesis that in fact biofilms would be better called “BioTumors” which highlights and unmasks the relationships of biofilms in tumors and also describes the interface with planktonic bacteria: biofilms in fact have more characteristics in common with tumor cells of a eucaryotic

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nature than with planktonic bacteria, their legacy. The fact is that there has been a significant amount of information discussed with unique biofilm form of organism, Candida albicans, which is a eukaryotic organism and this will be described later. Key features that are shared are highlighted by the cyclical nature of the biofilm, its community structure, its division of labor, heterogeneity, and its metastases, which is associated with the planktonic form.

2.3.4 Microbial components Obviously one of the most important features of the biofilm and community is individuals that compose that community. The microbes under the influences of the previous description are organized in a special arrangement to give the community the greatest efficiency to survive in the environment provided. There are, however, some universal principles again following the microbial world as described in Section 2.2.1, where it was pointed out that the initial “colonizers” are initially Gram positive cocci and the late colonizers are generally Gram negative and rod-shaped. This co-adhesion and organization is influenced by all of the previous discussions of the eight features, the nutrition, and the stress environment particularly. The key is this multilayered capacity is a major feature in providing dilution of potential toxic materials including antimicrobials. Hence, in addition to the barrier provided by the EPS, dilution of the anti-infectives is a major feature in protecting the independent individuals within the biofilm community. It is estimated that a dilution factor of 1 : 1000 is not unrealistic, recognizing therefore that antimicrobials and their achieved peak serum value may have no relationship to the value obtained within the biofilm. It is also important to recognize that a mixed species biofilm is significantly more stable, robust, and cohesive than a monospecies biofilm. This cohesion is facilitated by greater cross-talk and co-aggregation amongst the features it defines, where biodiversity equals stability which also equals survival.

2.3.5 Clinical implications: electron micrographs/key definitions Defining structure from a variety of sources, recognizing that biofilms are universal, whether they are seen in industry, medicine, dentistry, or the environment is key, because there is a crossover of information between the selective and divergent eco-systems, but the rules are the same. Figure 2.12 illustrates the biofilm as a “signature” of wound Stage (III) and the individual’s microbiota. It is influenced mostly by pH, Eh, and stress and for wounds, this is usually “static” compared to ventilator-assisted pneumonia (VAP) and the environment in mechanical ventilation (MV) in

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Advanced wound repair therapies Chronic wound

2.12 Electron micrograph from chronic wounds highlighting selected structures of complex Stage IV biofilms in chronic wounds.

the ICU. The 3-D structure depicted highlights the very rough topography with various morphotypes covered by the EPS as a “balloon.”

2.4

Biofilms in chronic wounds

The role of biofilms in chronic wounds will be further discussed and analyzed. There will also be discussion on understanding the various roles biofilms can play in such wounds: good, bad, and unknown.

2.4.1 Chronic wounds As in any complex process there is a plethora of definitions for the six wounds classified by the Delphi Panel, with the most controversial and yet open for scientific improvement, being those dealing with chronic wounds. In the simplest sense, chronic wounds are those where the normal wound process is considered disturbed and prolonged. The extent and depth of the definitions depend upon the complexity of the wound and the potential factors involved in the non-biological schemes that set up the environment that may favor colonization and the continuum in the wound infection process. Factors affecting healing and important in defining the role of microbes include the type of wound, site of damage, depth, and underlying pathology

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Factors that delay wound healing • Microbial numbers/pathogenicity/virulence/ synergy • Granulation tissue hemorrhagic/fragile • Inflammatory mediators • Inactivated state of neutrophils • Bacterial and human toxins • Tissue hypoxia • Metabolic wastes • Reduce fibroblast number/collagen production

2.13 This figure focuses on the cellular environment, the patient’s underlying risk factors, and the management heretofore used in dealing with the chronic wound.

(Cutting et al., 2008). Established factors that delay wound healing are listed in Fig. 2.13. These again point out the concept of the immunologic factors and the barrier factors of tissue itself in its relationship to the microbial pooling, colonization, and potential status (Guo and DelPietro, 2010). As highlighted earlier, features that impact on the organization of the biofilm and are most critical include the conditioning film on the wound site, the structure of the wound bed and the stress or transport mechanisms of either laminar or turbulent flow surrounding that wound site. The environment is very dynamic and hence speaks volumes to the necessity for continued evaluation of that wound site, all processes having some influence on the microbial ecology.

2.4.2 The wound continuum The wound infection continuum can be complex or simple based on the depth with which one wishes to describe the process. In its simplest form it can be described as going from contamination to systemic infection with reversible and irreversible steps in between highlighted by the term critical colonization. This theme addresses the more established microbial assessment focusing on the bioburden. Remember that the classic statement of infection equals numbers × virulence over immunity I = V × N/I; this theme addresses the bioburden in a more clinical as well as microbial content, particularly relating to metabolic load imposed by the microorganisms in the environment of the wound. This concept also integrates the subclinical and clinical presentations and brings into focus the balance between the immunologic component of the wound site, the microbial components. It highlights the biofilm as a major feature in this “climax community” or “critical colonization” (Stage I–II) site from which major decisions are influenced within the

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wound continuum. Here some of the consequences of bacterial growth including extra cellular enzymes and toxins released are emphasized, as well as the production of an extra polymeric substance (EPS) ultimately associated with the biofilm and the community of sociomicrobiology. The microbial contribution is obviously significant. It is a reflection of the organisms present, which is a reflection of the environment of the wound bed itself. A combination of aerobic, microaerophilic, and anaerobic organisms all integrates in the sociomicrobiology favoring the production of a protected survival oriented biofilm.

2.4.3 “Critical colonization”/“climax community” (Stage III) “Critical colonization” is a term used to focus on those time and/or population dynamics that favor a branch point towards cellular destruction and delayed repair versus the benefits of healing. “Critical colonization” has been used as a bench mark and has in it the assessment of a population having made part of the decision to treat response for failure in the next healing step of wound management (Cutting et al., 2008). There are a number of features of critical colonization which have been described as, in a general sense, associated with numbers of multiplying planktonic and biofilm bacteria. The cells reach a critical level where the potential for the wound to become infected increases significantly (Davis et al., 2008). This critical level is considered by many to be approximately 105 colony forming units (CFUs) per gram of tissue often described in a planktonic sense. Critical colonization refers to bacteria replication without a significant host reaction in conjunction with this replication. It is the inability of the wound to maintain a balance between an increasing microbial bioburden and with down regulation of bacterial replication by the immune system and/or therapeutic management used at the same time. “Critical colonization” and infection are two very similar yet distinct concepts and distinguishing between the two can be difficult. Hence, we have addressed this by the utilization and the reference of planktonic to biofilm establishing a biomarker that enhances and describes further the microbial and bioburden contribution to the wound process. There is no question that the bioburden, as established by colony forming units, has value in discerning the differences between infection sites. However, the concept of colony forming units is fraught with problems given that there may be both aerobic and anaerobic contributions as well as yeast contributions. The colony count may be of the featured individual microbe versus the entire pool or eco system of microbiota involved. Furthermore, this is found to be influenced by the site of culture, the type of the wound bed, i.e. burn versus acute, and by the influence of the methodology for detection; standard culture, viable bacteria versus non-culturable

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techniques, i.e. molecular methodology. What is the impact of the VBNC (viable but non-culturable) microbes? There is growing awareness that the unrecognized population of microbes may in fact be very important in defining the outcome of the infected site (James et al., 2008; Kaplan, 2010).

2.4.4 Dual hypothesis of Percival and Thomas Hypothesis I and Hypothesis II (Dual Hypothesis of Percival and Thomas) first established in 2006, addressed, for the first time, the importance of biofilms and the contribution of various members within the multispecies community (Fig. 2.14) (Percival and Rogers, 2005; Percival and Cutting, 2009). Although it was based on dental plaque/biofilm, its concept was recognized to have universal application in that clearly there was a transition between microbes that were colonized with endogenous/normal flora verses those that were overgrown due to major ecological pressures with an increased exogenous bioburden of more significant or established pathogens. P.D. Marsh’s Ecologic Hypothesis (Marsh, 2003) described a disease threshold based on bioburden and major ecological pressures and involved features of periodontal disease which mimics the features of a wound site given the emphasis and the process driven by changes in Eh/pH (i.e., “a wound is a toothless periodontal pocket” (Bjarnshol et al., 2008)).

Percival/Thomas II: Pathophysiology Pathogenesis INTER-RELATIONSHIP

Normal Flora ratio P BF Number of organisms I

II Nonspecies specific

Critical colonization Colonization

IV Phenotype ratio BF P Species specific III

>2 CFUs >5 MBEC

Pathogenesis

2.14 A detailed description highlighting four features that in a cascade fashion will determine the outcome of the infected wound and are a more detailed description of the Percival/Thomas Hypothesis II ‘Early’ and ‘Late’ (Figure 2.5).

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We expanded the Ecologic Hypothesis to focus on not only the bioburden, but the ratio of the phenotypes biofilm to planktonic as a major feature of critical colonization in wounds. There was a direct analogy. We entitled it the ‘critical colonization threshold’, but featured not the microbial pool alone as a base, but rather the presence of the more survival-directed recalcitrant population in a biofilm community versus a planktonic community, noting that they are not mutually exclusive. Environments will favor organism selection for a specific phenotype. We also recognized the importance of Candida albicans and it was the driving influence of the biofilm capability of Candida species that influenced the direction of the hypothesis and the subsequent elaboration of a Hypothesis I to expand to a Hypothesis II. A conceptual presentation of the Percival/ Thomas dual hypothesis is featured in Fig. 2.14; it integrates in the early phase, and the features that most readily select for a phenotype or drive an equation that favors a biofilm structure rather than a planktonic for survival of the organism. These features, well described earlier in this chapter, include the depth of the tissue which is a direct reflection of the environment, the pH, the stress, and further incorporate the eight features described earlier in Section 2.2, which are the hallmarks of a biofilm formation. This early stage drives the equation towards the irreversible status of colonization, which ultimately leads to critical colonization or anti-Koch with integration of Hypothesis I to Hypothesis II. Hypothesis I is the creation of an unbalanced microbial ecology, the organisms most often associated with disease related to high virulence and the corresponding reduction in the normal colonization process (endogenous). Hypothesis II recognizes the completion of this transition during critical colonization by the numerical superiority of the biofilm phenotype for the selected exogenous organisms. Hypothesis II is an elaboration of the Ecologic Hypothesis (Marsh, 2003), but clearly uses as its bench mark the biofilm inhabitance in the ratio of significant pathogens greater than the planktonic isolates; it is this switch to a more recalcitrant survival oriented community which drives critical colonization (Stage I–II) to poor wound response and delayed healing (Climax community, Stage III) and the ratio of microbial phenotype (Singh et al., 2002). The relationship between the microbial progression and biofilm formation in wounds and the ultimate significance of the ratio is highlighted in Table 2.6, which correlates the biofilm stage, the formation, the disease process, biphasic classification, and the relationship of planktonic to biofilm phenotype. Although this is a rather complicated table that really integrates the importance of the stage of the biofilm, the growth of the microbial community in an environment that is protected as a biofilm, the corresponding clinical consequences, i.e. structure equals function, with the ultimate recognition that there is a process usually defined as early and late dependent upon the pool of organisms involved and the classification of the disease

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Disease progression Contamination Colonization “Critical” colonization and infection Transmission metastasis

Biofilm formation

G +ve’s (Aerobes) G +ve’s G −ve’s (Aerobes) G +ve’s G −ve’s Aerobes Anaerobes Candida albicans Adhesion Progression “Critical” ratios BF:PL phenotype Detachment

CLINICAL

MICROBIOLOGIC

John G. Thomas Ph.D. and Staff, 2009.

IV A B

IAB II III

Biofilm stage

Relationship between microbial progression and biofilm formation in wounds

Host control Host resistance Microbial imbalance Microbial control

“Late”

“Early”

Disease classification “biPhasic”

Table 2.6 This complex table integrates the microbial consequences and the cellular response during the early and late development of a wound with colonized microbiota

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process. In the conversion of Stage II to Stage III of the biofilm, the preponderance of the biofilm phenotype is associated with microbial imbalance during this critical colonization. It also again emphasizes that universal principle of microbiology that early is associated with Gram positive anaerobes and late is associated with Gram negative anaerobes.

2.4.5 Clinical implications: application of hypothesis and utilization of biofilm to planktonic ratio We have made significant progress in establishing the creditability of the concept in the use of the ratio in wound classification. Is the ratio a marker for disease progress? Initial studies have been undertaken where we have studied in 40 wound isolates the ratio of the biofilm to planktonic presentation by employing the MIC of the isolates in either phenotype. Hence, the presentation of the ratio was defined by an MIC for the planktonic organism or a BEC (biofilm elimination concentration) for the biofilm phenotype. This numerical value was made as a ratio to help further clarify the Hypothesis I and Hypothesis II theme. Data presented at a number of international meetings has given the critical ratio quantitative value. A ratio of BEC to MIC of greater than 5 established a biofilm dominance and a categorization of unresponsive or UR. A ratio of the BEC to MIC of greater than 1, but less than 5, established an indeterminate or “I” value where the ratio was not an indicator of disease process. In a ratio of biofilm BEC and planktonic MIC of less than 1 indicated planktonic dominant responsive or were often associated with wound repair (Fig. 2.15, Fig. 2.16). The major significance was the fact that certain organisms had a predilection for biofilm ratio superiority, but it was unpredictable in the assays determined that certain organisms, although less likely to favor a biofilm phenotype, were nonetheless clearly capable in the right environment of producing that ratio and the feature of the ratio was critical in assessing the outcome of the wound.

Bioreactor and the wound environment: collateral damage Wound management often employs a dressing or some kind of barrier to potential recolonization from external sources. This environment in fact favors the formation of continual potential biofilm phenotype superiority given that there is a “ping-pong effect” and that the underside of the gauze may act as a considerable reservoir for either the planktonic or biofilm isolates, hence the continuation and the insertion of Hypothesis I to Hypothesis II is uninterrupted. Maintenance of the wound only addresses half of

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μg/ml

COLONIZATION RESISTANCE RATIO: Resistance

1000

BAD

MBEC/ Biofilm

ANTI-INFECTIVE >5 >1 and <5

Colony Type

COLONIZATION RESISTANCE

RATIO MBEC MIC

GOOD

ANTI-INFECTIVE <1 0.1

MBEC > MIC 0.01

256

μg/ml

MIC/Planktonic

2.15 This is the complement to Fig. 2.6, but the ratio of biofilm: planktonic phenotypes are based on MBEC (minimal biofilm elimination concentration).

Specimen Source/Organisms Respiratory

Blood

15 Ratio

15

Ratio

10

32

10 5

5

SA

SL 001 SM 001 CA 001

00 1

00 2

S C

00 1

EF

00 3 L

00 4

EC

EC

KP

EC

EC

L

00

00 1 F C

00 2

0

00 1

0

2

5

00 2

5

00 7

10

Wound 93

EC

10

Ratio

15

KP

Ratio

Urine 15

PA

M R

PA 003

00 SA 1 00 VR 1 E 00 1 C N S 00 G 1 B 00 1 C N S 00 2

0 0

2.16 Amplification of the theory as outlined in Fig. 2.14. This ratio, or biomarker, has had significant impact in patient management given that the ratio of a value of greater than 5 had been associated with wounds with least ability to recover based on standard therapy.

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Advanced wound repair therapies Integrating biofilm (BF) stage and planktonic(P) transmission “ping-pong” pathogenesis ABIOTIC

Eh O2

pH

Dressing BF

Planktonic

Nutrient supply & stress

11

Planktonic

BF 2

H2 Wound bed BIOTIC

2.17 A diagrammatic representation of our findings that the matrix of dressings is an ideal biofilm environment and that the wound bed and the dressing act as reservoirs that ‘ping-pong’ via the planktonic phenotype between them.

Biofilm reactors: chronic wounds /gauze The covered wound bed is a static biofilm reactor influenced by Eh and pH via perfusion, enhancing up regulation to recalcitrant biofilm phenotype (PBF) and changing ratio from planktonic phenotype (PP) in chronic wounds Planktonic predominate ≤1 (Susceptible)

PBF PP

≥1 Biofilm predominate (Recalcitrant)

2.18 This diagram highlights the significance of the ratio and the importance of the preponderance of microbial phenotypes, planktonic or biofilm.

the ping-pong problem and is described in Fig. 2.17. A covered wound bed is a static biofilm reactor influenced by Eh/pH via profusion, enhancing up-regulation to recalcitrant biofilm phenotype. Hence, the benefit of materials within the gauze may reduce this capability (Fig. 2.18). Lastly, and very important within this bioreactor environment, there is also a favorable exchange of genes between the organisms themselves, which will enhance the potential for genetic resistance focused on antibiotic markers. Although evidence is slow to accumulate, there is no question that the proximity of the gene pool within the matrix and this 3-dimensional organization enhances the potential of antibiotic resistance in gene

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migration within the communities of the biofilm, hence the bioreactor is not only a place that favors the production of an environment more associated with critical colonization and lack of healing, it is in this same environment that the potential resistant component will be transferred from species to species not only then enabling the environment to be particularly recalcitrant to management, but to be a continual reservoir for antibiotic resistance to other sites within the patient’s care.

2.5

Biofilms as therapeutic or restorative microbiology/modeling

With increasing interest in probiotics this section will focus on employing biofilms as a therapeutic method in regards to non-healing wounds and protection. It will also incorporate the use of a Triphasic Wound Model PLUS, which focuses on features described earlier about the environment of the wound and the utilization of a biofilm organism, in this case Lactobacillus reuteri.

2.5.1 Background We have had considerable experience in developing and engineering a model or simulator closely replicating the environment of ventilator associated pneumonia. This initially focused on adult ventilator associated pneumonia with the engineering of the ventilator endotrach lung model (VEL), which incorporated the significant features in a dynamic environment for luminal growth of a biofilm in an endotrach. The model incorporated a ventilator and the features of X – the forced dynamics, Y – the positioning of the endotrach in a pathologic correct position, and Z – the utilization of a bi-lobe Michigan lung, which together with the forces of X – the 840 VE ventilator, created the dynamic environment associated with ventilation in the ICU. The model stimulator for pre-clinical evaluations enabled us to describe and study the biofilm environment using quantitative microscopic (CLSM) and non-culture (PCR) techniques.

2.5.2 Triphasic Wound Model PLUS Our wound model utilized key features from our VAP studies and incorporated the eight recognized forces that determine a biofilm, but integrated and engineered a unique combination of eucaryotic and procaryotic cells. The model is shown in Fig. 2.19. Eucaryotic cells were fiberblastic cells NIH3T3 grown overnight, whereas the procaryotic cells were grown in a Poloxamer F-127 (a reverse gel) and incorporated four biofilm-established

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Advanced wound repair therapies Triphasic Wound Model A. EUCARYOTE

Triphasic wound simulation

Liquid media Fibroblast cells NIH3T3

B. Growth interface (48-hour incubation)

PROCARYOTE BIOFILM 1. Ca 2. MRSA (F127) 3. PA Poloxamer (containing biofilm) 4. SA Filter

Mono BF or Co BF or Complex BF

Lr

48 Hours incubation Separation Diffused material Assay for cell death Variable Lr - Strain 1 Lr - Strain 2

Molecular structure imaging Variable

Time

Ph 5.5 Ph 8.5

Eh + 30 Eh –180

2.19 A graphic representation of our pre-clinical model called the ‘Triphasic PLUS Wound Model’ that integrates the environment of the wound, the challenge of the biofilm microorganisms, and allows for their integration in a flexible environment.

microbes including Staph aureus, MRSA, P. aeruginosa, and Candida albicans. The procaryotic pool could vary from 1 to 4 organisms and simulate a monospecies to a complex biofilm depending on the combination of organisms chosen. The F-127 Poloxamer is a well known medium for inducing biofilm formation and its structure. The organisms, either eucaryotic or procaryotic, were grown independently for 48 hours and then combined in a U-prep spin column separated by a Whitmin #3 filter paper to evaluate diffusible products from the biofilms and their influence upon the eucaryotic monolayer via quorum sensing (QS). The combined eucaryotic/procaryotic filter paper Triphasic environment could be influenced by pH/Eh anaerobic incubation and incubated at 37 degrees for a variety of time frames. Ultimately, the two cell types could be separated and the impact of the biofilm on the cells analyzed by a variety of markers of cell death and toxicity. The staging of the biofilm (I–IV) in the procaryotic poloxamer tube assayed by confocal laser scanning microscopy (CLSM) and the use of COMSTAT to quantify with live/dead stains, the staging of the biofilm, and its stage impact upon the eukaryotic cells. The results were quite stimulating; they confirmed the concept of an engineered Triphasic Wound Model with flexibility while integration features that

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Average cell death counted × 104 10 9 8 Results 7 6 5 4 3 Average cell death based on the number of organisms × 104 2 1 0 8

7 6 5 4 3 2 1 0

One Organism

Two Organisms

Three Organisms

Organism Only Organism & L. reuteri

Staph aureus (Sa)

Candida Pseu. Sa/Pa Sa/Ca Pa/Ca Sa/Pa/Ca albicans Aeruginosa (Ca) (Pa)

Average cell death counted × 104 9 8 7 6 5 4 3 2 1 0 Organism & L. reuteri

Staph aureus (Sa) Candida albicans (Ca) Pseu. Aeruginosa (Pa) Sa/Pa Sa/Ca Pa/Ca Sa/Pa/Ca

Organism Only

2.20 Initial results of a single oral Probiotic Lactobacillus reuteri used in preliminary studies to measure the impact of a probiotic on the biofilm as assayed in the Triphasic PLUS Wound Model elaborated in Figure 2.19.

emphasized biofilm growth, with targeted cells, yet flexible start (pH, Eh) depending upon the wound type and stage evaluated. Hence, the Percival/ Thomas Hypothesis could be studied. Preliminary results indicated that the model was able to assay various contributing facts with outcomes measured both visually and by eucaryotic assays that included viability, SEM, toxicity, and cyto-chemistry strains. The results emphasized the more complex the biofilm the more impact on the growth of the eucaryotic cells, confirming observations that a more complex, older biofilm enhanced by cross-talk and aggregation is a feature of non-wound healing (Fig. 2.20).

2.5.3 Intervention with probiotics The Triphasic Wound Model was engineered to ultimately allow for evaluation of various interventions and the placement of various containing dressings in the filter separating the eucaryotic and procaryotic fields (Hill et al., 2003; Yan et al., 2008). Conceptually it was always our intention to ultimately use the model once its value was proven to measure various

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minimal interventions and to potentially allow for the empowerment of patients through therapeutic microbiology or the use of probiotics. There has been considerable interest in the use of probiotics in oral care and it has most recently had some success in the evaluation of ventilator associated pneumonia. Probiotics in wound care have always been evaluated as a lesser option, but our model gave us the chance to assess particularly the use of Lactobacillus reuteri (BioGaia), which had proven very beneficial in oral care. Conceptually, the use of probiotics would be maximized, which would, in our Hypothesis I and II driven model, interfere with the conversion in “critical colonization” of the biofilm to a more recalcitrant ratio, where the biofilm of pathogenic organisms outnumbered the ratio of planktonic organisms. Here, our concept was to suggest that a ratio beneficial to biofilm of a probiotic may, in fact, interfere with the continued of demise of the cellular background and reverse the non-healing process. The Triphasic Wound Model PLUS gave us the opportunity for evaluating this concept (Martin et al., 2010 and Percival et al., 2008). We approached this strategy with two studies. We initially utilized Lactobacillus reuteri Prodentis of which there are two strains; we chose initially to use one strain (ATCC 55730), which had a proven value in out-competing via a better biofilm than oral care with previous isolates. The Lactobacillus was added at the time of the biofilm formation in the procaryotic mix and the effects of this beneficial biofilm, combination analyzed by the visualization of cell growth was interesting; the use of the poloxamer-probiotics mix clearly reversed the damage of cells associated with the biofilm grown in the poloxamer environment alone. The second phase utilized two different Lactobacillus strains, L. casei and L. plantarum, one, a lytic causing organism, the other immune modulating (Fig. 2.21, Fig. 2.22). Although considerable work is still necessary the Triphasic Wound Model PLUS is an in vitro simulator which, under controlled conditions allows for the assessment of the wound environment maximizing Eh/pH and those conditions which might favor the growth of a biofilm as established by a ratio in our hypothesis (Percival and Bowler, 2004; Percival et al., 2010). It also allows for the intervention a variety of methods including various coated diffusible dressings and a combination of potential probiotics, which would herald the potential use of therapeutic or “restorative microbiology” in reversing the delay in healing of a chronic wound (Potera, 2010; Rennie et al., 2003).

2.5.4 Development The development of the engineered Triphasic Wound Model PLUS allows for significant studies under controlled conditions (in vitro) with the use of

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90 80 70 60 50 40 30 20 10 0

None Casei Plantarum 24 hour 48 hour 72 hour Incubation Time

% Bacterial Death

% Bacterial Death

Staphylococcus aureus

73

80 70 60 50 40 30 20 10 0

None Casei Plantarum 24 hour 48 hour 72 hour Incubation Time

Candida albicans

% Bacterial Death

7 6 5 4

None Casei Plantarum

3 2 1 0

24 hour 48 hour 72 hour Incubation Time

2.21 The effect of Lactobacillus casei and Lactobacillus plantarum on planktonic Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. L. plantarum was consistently more effective than L. casei in reducing colonization of the planktonic S. aureus and P. aeruginosai, while neither probiotic was successful in reducing colonization of planktonic C. albicans. This figure complements Figure 2.20, but employs two strains of lactobacillus, each focusing on the major features of 3 species biofilm inhibition: 1) antibiofilm activity, and 2) immune modulation of the host defences.

molecular imaging and longitudinal fluorescent studies in mice, a transition from in vitro to in vivo can occur. With the use of the Xenogen IVIS luminal imaging system, we can perform real-time in vivo imaging to monitor and record cellular and genetic activity within a mouse using bioluminescent recorders. With the recent use of biofilm micromarkers such as Pseudomonas aeruginosa and Staph aureus real-time biofilm development within living animals can be performed. The Triphasic Wound Model PLUS provides screening that would allow us to then integrate the data from the initial in vitro results and incorporate optimum combinations of probiotics particularly in a real-time animal model and assess not only the consequences of the immediate tissue reaction, but the potential consequences of a stalled healing with systemic

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35 3 2.5 2 1.5 1 0.5 0

SaLc

Pseudomonas aeruginosa

SaLp

Bacteria and Probiotic Inoculation

# Viable 3T3 Mouse Fibroblast Cells (¥ 105)

# Viable 3T3 Mouse Fibroblast Cells (¥ 105)

Staphylococcus aureus 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

PaLc

PaLp

Bacteria and Probiotic Inoculation

# Viable 3T3 Mouse Fibroblast Cells (¥ 105)

Candida albicans 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 CaLc

CaLp

Bacteria and Probiotic Inoculation

2.22 The effect of Lactobacillus casei and Lactobacillus planterum on the reduction of diffusible products from monospecies biofilms of Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. There is a significant association between probiotic administered and the reduction of diffusible products from monospecies biofilms as measured by mouse fibroblast cell viability (p < 0.001). The columns on the left represent 3.0 μm pore sized inserts, while the columns on the right represent 0.4 μm pore sized inserts. The variability in viable fibroblast cells based on pore size indicates that communication between the diffusible products of the probiotic and the bacteria biofilm happens pre- and not post-filtration. Here, the diffusible products of the mono-species biofilm of lactobacillus are inhibited or reduced relative to death of targeted mouse fibroblasts.

involvement simultaneously. This expands our knowledge of the use of therapeutic intervention and replacement or therapeutic microbiology.

2.6

Conclusion

Chronic wounds are global, causing significant international morbidity and mortality. Illustrating the pathogenesis via biofilms should unmask targets for intervention, not the least of which could be probiotics. Here, we highlighted: 1) the importance of the human microbiome studies and cataloging

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of unrecognized skin species, 2) the significance of a four phase cascade in tissue colonization to pathogenesis, 3) the importance of the biofilm: planktonic ratio as a biomarker of wound tissue deterioration, 4) the importance of in vitro modeling and 5) the potential benefit of wound colonization with a beneficial probiotic, single or mixed pools. It is important to remember that a wound is a diverse procaryotic and eucaryotic mixture, generally in a closed environment when dynamics of interaction are stressed based on Eh and pH, and communication amongst all cells is key to management.

2.7

References

Bjarnshol T, Kirketerp-Moller K, Jensen P, et al. Why chronic wounds will not heal: a novel hypothesis. Wound Repair and Regeneration. 2008;16(1):2–10. Bowler PG. The anaerobic and aerobic microbiology of wounds: a review. Wounds. 1998;10:170–78. Bowler PG, Davies BJ. The microbiology of infected and noninfected leg ulcers. Intl J Derm. 1999;38:101–06. Cutting KF, White RJ, Mahoney P, Harding KG. Clinical Identification of Wound Infection: Delphi Approach. In identifying Criteria for Wound Infection. EWMA Position Document. London: MEP Ltd. 2008. Daims H. Vienna. Personal Communication, USC. Los Angeles, CA. Biofilm Meeting. 2006. Davis S, Ricotti C, Cazzaniga A, et al. Microscopic and physiologic evidence for biofilm-associated wound colonization in vivo. Wound Repair Regen. 2008;16(1):23–29. Dowd S, Yan S, Secor P, et al. Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing. BMC Microbiology. 2008; 81:15. Down SE, Sun Y, Secor, et al. Survey of bacterial diversity in chronic wounds using Pyrosequenceing, DGGE, and full ribosomal shotgun sequencing. BMC Microbio. 2008;8:43–58. Guo S, DelPietro LA. Factors affecting wound healing. J Dent Res. 2010;89: 219–29. Hall-Stoodley L, Costerton WJ, Stoodley P. Bacterial biofilms: from the natural environment to infectious diseases. Nature Reviews: Microbiology. 2004;2:59–108. Hill KE, Davies CE, Wilson MJ, et al. Molecular analysis of the Microbiota in chronic let ulcerations. J Med Microbiol. 2003;52(4):365–69. James GA, Swogger E, Walcott R, et al. Biofilms in chronic wounds. Wound Rep Reg. 2008;16:37–44. Kaplan JB. Biofilm dispersal: Mechanisms, clinical implications, and potential therapeutic uses. J Dent Res. 2010;89:205–18. Kirketerp-Moller K, Jensen PO, Fazli M, et al. Distribution, organization, and ecology of bacteria in chronic wounds. J. Clin. Microbiol. 2008;46:2717–22. Lewis K. Persistor cells and the paradox of chronic infections. Microbe. 2010;5:429–37. Marsh PD. Are Dental diseases examples of ecologic catastrophies? Microbiology. 2003;149:279–94.

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Martin JM, Zenilman JM, Lazarus GS. Molecular Microbiology: New dimensions for cutaneaos biology and wound healing. J Investigative Derm. 2010;130:38–48. Penhallow K. A review of studies that examine the impact of infection on normal wound healing process. J of Wound Care. 2005;14:123–26. Percival SL, Bowler PG. Biofilms and their potential role in wound healing. Wounds. 2004;16:234– 40. Percival SL, Rogers AA. The significance and role of biofilms in chronic wounds. In McBain et al. Biofilms Persistence and Ubiquity. The Biofilm Club 7th Meeting. Gregynog Hall, Powys. 2005;171–78. Percival SL, Cutting KF. Biofilms: possible strategies for suppression in chronic wounds. Nursing Standard. 2009;23:64 –72. Percival SL, Thomas JG, Williams DW. Biofilms and microbial imbalances in chronic wounds: Anti-Koch. Int Wound J. 2010;7:169–75. Percival S, Bowler P, Woods E. Assessing the effect of an antimicrobial wound dressing on biofilms. Wound Repair & Regeneration. 2008;16(1):52–57. Posnett J, Franks PJ. The burden of chronic wounds in the UK. Nursing Times. 2008;104(3):44–45. Potera C. Current topics; biofilms are key challenge in treating chronic wounds. Microbe. 2010;5:414. Rennie RP, Jones RN, Mutnick AH. Occurrence and antimicrobial susceptibility patterns of pathogens isolated from skin and soft tissue infections: report from the SENTRY Antimicrobial Surveillance Program (United States and Canada, 2000). Diag Micro Inf Dis. 2003;45:287–93. Singh Pk, Parsek MR, Greenberg EP, Welsh MJ. A component of innate immunity prevents bacterial biofilm development. Nature. 2002;6888:552–55. Stoodley L, Stoodley P. Microreview: evolving concepts in biofilm infections. Cell Microbio. 2009;11(7):1034– 43. Sun Y, Dowd SE, Wolcott RD, et al. In vitro multispecies Lubbock chronic wound biofilm model. Wound Rep Reg. 2008;16:805–13. Thurnbaugh et al. International Human Microbiome Project. NIH. 2007. Vowden K, Vowden P, Posnett J. The resource costs of wound care in Bradford and Airedale primary care trust in the UK. J of Wound Care. 2009;18(3):93–4, 96–8, 100. Wolcott RD, Dowd SE. A rapid molecular method for characterizing bacterial bioburden in chronic wounds. Journal of Wound Care. 2008;17:513–16. Wolcott RD, Gontcharova V, Sun Y, Dowd SE. Evaluation of the bacterial diversity among and within individual venous leg ulcers using bacterial tag-encoded FLX and Titanium amplicon pyrosequencing and metagenomic approach. BMC Microbiol. 2009;9:226–37. Yan S, Dowd S, Smith E, Rhoads D, Wolcott R. In vitro multispecies Lubbock chronic wound biofilm model. Wound Repair & Regeneration. 2008;16(6):805–13.

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3 Scarring and scarless wound healing B. J. L A R S O N, A. N AU TA, K. K AWA I, M. T. L O N G A K E R, and H. P. L O R E N Z, Stanford University School of Medicine, USA

Abstract: In contrast to adult wound healing, the early-gestation fetus has the remarkable ability to heal skin wounds without scar formation. Over the past several decades, an intensive research effort has greatly increased our understanding of scarless fetal wound healing. This chapter discusses the differences that exist between the extracellular matrix (ECM), inflammatory response, cellular mediators, stem cell function, and gene expression profiles of fetal and postnatal wounds. This chapter then reviews the currently available treatment options for scars and concludes with a discussion of future trends in scar treatment. Key words: wound healing, fetal wound repair, scarless wound healing, skin development, review.

3.1

Introduction

A scar is a macroscopic disturbance in the normal tissue architecture that involves regulated collagen deposition in response to tissue injury. Scarring is the normal outcome in the wound healing process, and occurs in the setting of both surgical and traumatic wounds. The mechanism of scar formation involves inflammation, fibroplasia, formation of granulation tissue, and scar maturation. The skin is the most frequently injured tissue in the body, and dermal scarring can result in loss of function, movement restriction, and disfigurement. Scar formation is a major medical problem that can have devastating consequences. The adverse physiologic and psychologic effects of scars are broad, and there is currently no reliable way to prevent or treat scarring. In contrast to adult wound healing, the early-gestation fetus has the remarkable ability to heal skin wounds without scar formation. This observation was first reported more than three decades ago (Rowlatt, 1979) and has subsequently been confirmed in both animal models and human fetuses (Adzick and Longaker, 1992). Since that time, an intensive research effort

Financial disclosure and products: We have no commercial associations or financial disclosures that might pose or create a conflict of interest with information presented in this chapter.

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Table 3.1 Adult vs. fetal wound healing

Collagen content

Hyaluronic acid ECM modulators Adhesion proteins

Platelets Inflammatory cells Interleukins

TGF-β Gene expression Progenitor cells

Adult wound healing

Fetal wound healing

Predominance of Type I collagen provides strength and rigidity, but impedes cellular migration and regeneration Low levels of hyaluronic acid inhibit cellular movement Low MMP to TIMP ratio favors accumulation of collagen Diminished upregulation of adhesion proteins results in slower fibroblast migration Release PDGF, TGF-β1 and TGF-β2 Many

Predominance of Type III collagen is optimal for cellular migration and proliferation

Rapid increase in proinflammatory cytokines, such as IL-6 and IL-8 High levels of TGF-β1 and TGF-β2 Delayed upregulation of genes involved in cell growth and proliferation Skin progenitor cells found at inadequate numbers to mediate scarless repair

High levels of hyaluronic acid facilitate cellular movement and trap water High MMP to TIMP ratio favors ECM turnover and remodeling Rapid upregulation stimulates cell attachment and migration

Decreased degranulation and aggregation Few Increased expression of the anti-inflammatory cytokine IL-10, which decreases production of IL-6 and IL-8 Low levels of TGF-β1 and TGF-β2. Increased TGF-β3 Rapid upregulation of genes involved in cell growth and proliferation Skin progenitor cells migrate to sites of injury and mediate scarless repair

has focused on unraveling the mechanisms underlying scarless fetal wound repair (Table 3.1). We now know that significant differences exist between the extracellular matrix (ECM), inflammatory response, cellular mediators, stem cell function, and gene expression profiles of fetal and postnatal wounds. These differences may have important implications in the clinical treatment of scars.

3.2

Wound healing process

Wound repair in the normal adult is a tightly regulated process that follows three temporally overalapping phases: inflammation, tissue formation, and

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tissue remodeling. In response to tissue injury, inflammatory cells hone to the wound bed. The proliferation of fibroblasts follows the acute inflammatory response. Fibroblasts are responsible for synthesizing various tissue components, including collagen and fibrin. During the acute inflammatory phase, circulating progenitor cells migrate to injured tissue. Cells rapidly proliferate, which results in the formation of new blood vessels and epithelium. Fibroblasts then differentiate into myofibroblasts, the cells responsible for collagen deposition and wound contraction. Excess accumulation of an unorganized extracellular matrix results in scar formation. Although scar remodeling occurs for months to years after the initial injury, complete restoration of the normal extracellular matrix architecture is never achieved (Colwell et al., 2005b). Mature scars restore only 70% of the tensile strength of normal skin and pre-scar function is never completely recovered (Madden and Peacock, 1971). Thus, wound healing is a fibroproliferative response that results in incomplete regeneration of the original tissue.

3.2.1 Inflammation Cutaneous injury often results in a disruption of the underlying vascular network. To prevent extravasation of blood and plasma, platelets aggregate to form a fibrin-rich clot (Clark, 1996a). In addition to providing hemostasis, platelets release growth factors and pro-inflammatory cytokines, such as transforming growth factor β1 (TGF-β1) and platelet derived growth factor (PDGF) (Moulin et al., 1998). Injured epithelial cells also release chemotactic molecules and vasoactive mediators which recruit inflammatory cells to sites of injury (Clark, 1996a). Neutrophils respond to chemotactic signals and are the first inflammatory cells recruited to wounded tissue. Neutrophils release degradative enzymes, phagocytose foreign material, and secrete proinflammatory cytokines (Hubner et al., 1996). In the absence of infection, the neutrophilic infiltration subsides within a few days after injury. In the presence of infection, a persistent neutrophil-rich inflammatory response results, which leads to poor wound healing and excess fibrosis (Muller-Eberhard, 1992). Following the acute inflammatory phase, monocytes are recruited to the wound site in response to a variety of intravascular and extravascular chemotactic signals. Circulating monocytes transmigrate (diapadese) across the blood vessel endothelium and become tissue macrophages. As monocytes transform into tissue macrophages, proinflammatory cytokines are released. These cytokines are critical for normal wound healing, despite their proinflammatory properties (Leibovich and Ross, 1975). Monocytes and activated macrophages promote wound debridement by attaching to the extracellular matrix (ECM), which induces ECM phagocytosis. However,

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similar to neutrophils, a persistent macrophage response may lead to excess scar formation (Juliano and Haskill, 1993).

3.2.2 Tissue formation Within hours after injury, the process of reepithelialization begins. Epidermal cells proliferate and migrate through a fibroblast-derived provisional matrix (Winter, 1962). In areas of hair growth, stem cells found in the infundibular hair bulge contribute to the reepithelialization of denuded skin (Cotsarelis et al., 1990, Ito et al., 2005). Neoangiogenesis depends on local and circulating endothelial cells. Components of the ECM, as well as a number of cytokines, have been implicated in neoangiogenesis (Folkman, 1996). Additionally, low oxygen tension, lactic acid, plasminogen activator, and collagenase have all been shown to play a role (Singer and Clark, 1999). Activation of apoptoic pathways ultimately stops the process of neoangiogenesis (Ilan et al., 1998). Granulation tissue is a vascular rich neostroma that comprises macrophages, fibroblasts, and loose connective tissue. Granulation tissue begins to form approximately four days after injury and replaces the initial fibrinrich clot (Xu and Clark, 1996). The extracellular matrix that is created serves as a scaffold for neovascularization and forms a protective barrier against invading pathogens (Hunt, 1980, Roberts and Sporn, 1996). Fibroblasts create and modify the granulation tissue, which is initially composed predominantly of Type III collagen. Type I collagen, a stronger structural protein, replaces the weaker Type III collagen, and becomes the dominant isoform in scar tissue. When collagen density reaches a critical level, fibroblast proliferation and collagen synthesis cease (Grinnel, 1994). Dysregulation of this negative feedback loop results in pathologic scar formation and fibrotic disorders, such as keloids, morphea, and scleroderma (Singer and Clark, 1999).

3.2.3 Tissue remodeling Wound contraction and tissue remodeling occur during the second week post-injury, and represent the final phases of the wound healing process. During this period, fibroblasts undergo a phenotypic transformation to become myofibroblasts, which are cells with increased smooth muscle actin expression (Desmonliere and Gabbiani, 1996). Several growth factors, such as TGF-β and PDGF, regulate wound contraction (Montesano and Orci, 1998). Matrix metalloproteinase (MMPs), enzymes responsible for degrading excess connective tissue and extracellular matrix proteins, orchestrate tissue remodeling (Parks, 1999). Tissue remodeling results in a decrease in the number of fibroblasts and a progressive decline in the vascularity of the

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wound. During this phase, collagen matures, scar tissue becomes paler and the wound gains tensile strength. Fibroblasts secrete mature type I collagen, which forms along the lines of tension in the wound, in place of immature and randomly deposited type III collagen. Collagen remodeling may be disturbed by a number of factors, leading to raised scar formation (Johnstone and Farley, 2005).

3.3

Fibroproliferative scarring

Fibroproliferative scarring represents an amplification of the wound healing process, characterized by overdeposition of connective tissue. Although the degree of scar formation is often unpredictable, there are several factors that influence the severity of scarring. These factors include genetics, tissue site, sex, race, age, the magnitude of injury, and wound contamination. Generally speaking, areas of thicker dermis scar more than areas of thinner dermis. Premenopausal women often scar more than both postmenopausal women due to estrogen’s negative impact on wound healing. In general, patients with darkly pigmented skin are more prone to fibroproliferative scarring. Large and contaminated wounds also tend to result in increased scar formation (Ferguson and O’Kane, 2004, Ashcroft et al., 1997a, 1997b, 1997c). Scar formation results from excess accumulation of unorganized extracellular matrix. In cutaneous wounds, hypertrophic scars and keloids are the two major pathologic scar types.

3.3.1 Hypertrophic scars Hypertrophic scars are more common than keloids. Hypertrophic scars are defined as raised, erythematous, pruritic lesions that do not extend beyond the boundaries of the original wound (Peacock et al., 1970). Hypertrophic scars are often initially brownish-red in color, but can become pale with age. These lesions tend to be less nodular than keloids, rarely raising more than 4 mm above the skin surface (Niessen et al., 1999). Hypertrophic scars usually begin to form six to eight weeks after injury and reach a plateau by six months. Unlike keloids, hypertrophic scars often occur over extensor surfaces, such as the elbows and knees. When located over joints, debilitating contractures may result. Regression may be observed without intervention and the extent of scarring is directly related to the depth and location of injury (Haverstock, 2001). Histologically, hypertrophic scars are characterized by collagen bundles that are fine, well-organized, and parallel to the epidermis. Unlike with keloids, myofibroblasts are present, and alpha smooth muscle actin is expressed in a nodular formation. Mucin is absent, and hyaluronic acid is a major component of the papillary dermis.

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3.3.2 Keloids Keloid scars are also raised, erythematous and pruritic, but they extend far beyond the original wound margins (Peacock et al., 1970). Keloids form over a period of two to three months and almost never spontaneously regress. These growths can be extremely irritating, but the clinical manifestations are highly variable between patients. Keloids can be particularly disfiguring due to their nodular appearance, size, and color, which tends to be dark and erythematous (Shaffer et al., 2002, English and Shenefelt, 1999, Atiyeh et al., 2005, Niessen et al., 1999). These scars can cause pain, burning, and itching. The most common areas affected by keloids are the upper body sebaceous areas. Keloids do not commonly involve joints and contracture is rare (Tuan and Nichter, 1998). Interestingly, keloids only occur in humans and no keloid animal model exists. Histologically, keloids are characterized by thick, large, closely packed bundles of disorganized collagen. Alpha smooth muscle actin is oriented around the vessel wall, and myofibroblasts are absent. Mucin is deposited focally in the dermis, and hyaluronic expression is confined to the thickened, granular/spinous layer.

3.3.3 Pathogenesis of fibroproliferative scarring Fibroproliferative scarring results from an imbalance between ECM synthesis and degradation. During the acute inflammatory phase, increased levels of IL-8 lead to the release of pro-fibrotic growth factors, such as TGF-β, insulin-like growth factor-1 and PDGF (Iocono et al., 2000). Of these factors, the TGF-β family has a major, defined role in tissue fibrosis (Border and Noble, 1994). Interestingly, TGF-β1 and TGF-β2 have been reported to increase fibrosis, while TGF-β3 has been shown to decrease fibrosis. TGF-β1 and -β2 have also been found to potently stimulate fibroblast contraction (Smith et al., 1999). In fibroproliferative states, collagen degradation does not proceed normally, which leads to impaired tissue remodeling. Furthermore, the collagen in fibroproliferative states is often randomly oriented and inadequately cross-linked (Robson, 2003).

3.4

Scarless fetal wound healing

3.4.1 Fetal cutaneous wound healing Scarless wound healing has been observed in the fetuses of mice, rats, pigs, monkeys and humans (Colwell et al., 2005a). In fetal mammals, the ability to repair wounds scarlessly is age-dependent (Cass et al., 1997, Somasundaram and Prathap, 1970). In other words, fetal skin heals scarlessly prior to a certain gestational age, after which point typical scar formation occurs. In

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humans, scarring of wounds begins at approximately 24 weeks of gestation, while in mice scarring of wounds begins on embryonic day 18.5 (average gestation period for mice is 20 days) (Lorenz et al., 1993, Colwell et al., 2006, Lorenz et al., 1995). This transition point is also modulated by wound size. As wound size increases in fetal lambs, the ability to heal scarlessly is lost at an earlier point in gestation (Cass et al., 1997). In response to tissue injury, the fetal dermis has the ability to regenerate a non-disrupted collagen matrix that is identical to that of the original tissue (Longaker et al., 1990, Whitby and Ferguson, 1991, Beanes et al., 2002). In addition, dermal structures such as sebaceous glands and hair follicles form normally after fetal injury (Fig. 3.1) (Rendl et al., 2005). Although the exact

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3.1 Scarless healing of E16 fetal mouse wounds (H&E Stain). Black arrows indicate India ink tattoo made at the time of wounding in order to demonstrate scarless wound location. Healed wounds (a) and (c) at 72 h (100×). The epidermal appendage (developing hair follicles) pattern shows numerous appendages directly in the healed wound. Magnified views of the same wounds (b) and (d) showing epidermal appendages (open arrows) within the wound site (200×). No inflammatory infiltrate is present. Reproduced with permission from Lippincott Williams & Wilkins. Original publication: Beanes SR, Hu FY, Soo C, et al. Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition. Plast Reconstr Surg. 2002;109:160–170.

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mechanisms of scarless fetal wound healing is still unknown, it is thought to be due to differences between the ECM, inflammatory response, cellular mediators, and differential gene expression in fetal and postnatal wounds (Beanes et al., 2001b, 2001a, Lorenz et al., 2001, Soo et al., 2000, Hildebrand et al., 1994, Frantz et al., 1993, Oshima et al., 2001, Wagers et al., 2002, Shizuru et al., 2005).

3.4.2 Fetal skin The structure and histology of fetal skin changes dramatically with development. In the human embryo, a primitive epidermis first appears at gestational day 20. During weeks 4 to 8 of gestation, the primitive epidermis develops into a two-layered periderm and basal cell layer. The fetal epidermis begins to stratify at week 9, and keratinization begins at week 14. By week 16, the fetal epidermis has many of the components of the adult epidermis: a basal cell layer, an intermediate layer, hair follicles, sweat glands, and follicular keratinization. After 24 weeks gestation, fetal skin development is characterized by rapid growth and maturation, and at birth the neonatal skin is histologically indistinguishable from adult skin (Fig. 3.2) (Lane, 1986, Lorenz et al., 1992a, Coolen et al., 2010). The extracellular matrix plays an important role in cell adhesion, differentiation, and proliferation. The ECM is a dynamic layer of collagen, glycosaminoglycans, proteoglycans, and adhesion proteins that undergoes a series of changes before reaching its adult phenotype (Beanes et al., 2001b,

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3.2 Histology of human fetal skin at 14 and 20 weeks gestation and adult skin. At 14 weeks, the epidermis consists of a basal layer, an intermediate cell layer and a periderm. At 20 weeks, the number of intermediate cell layers has increased and developing hair follicles are visible. Adult skin has a less cellular dermis, and a multilayered epidermis with basal, spinous, granular and cornified layers. Scale bars 100 μm. Reproduced with permission from Springer. Original publication: Coolen NA, Schouten KC, Middelkoop E, et al. Comparison between human fetal and adult skin. Arch Dermatol Res, 2009.

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2001a, Lorenz et al., 2001, Hildebrand et al., 1994). The fetal ECM is optimized to facilitate cellular migration and proliferation, which may have important implications in wound healing. Thus, the fetal ECM may provide an environment that is conducive towards scarless wound repair.

Collagen content There are phenotypic differences between the collagen content and crosslinking patterns in fetal and postnatal wounds. In fetal wounds, Type III collagen is rapidly deposited in a fine reticular network that is indistinguishable from uninjured skin (Fig. 3.3) (Beanes et al., 2002). Postnatally, the ratio of Type I to Type III collagen in wounds increases (Merkel et al., 1988). The predominance of Type I collagen in postnatal wounds provides regenerating tissue with more strength and rigidity, but may impede cellular migration and regeneration. Early scar formation in late gestation fetal wounds demonstrates larger collagen fibers with greater interfiber space (Fig. 3.4) (Beanes et al., 2002).

Hyaluronic acid Hyaluronic acid (HA) is a glycosaminoglycan that is one of the chief components of the ECM. The net negative charge of HA traps and impedes water molecules, which allows resistance to deformation and facilitates cellular movement (Clark, 1996a). In general, fetal skin contains more HA than adult skin, and fetal fibroblasts express more HA receptors (Alaish et al., 1994, Longaker et al., 1989). In scarless fetal wounds, the HA content of the ECM increases more rapidly than in adult wounds. Fetal wounds also have fewer proinflammatory cytokines, such as IL-1 and TNF-alpha, that act to downregulate HA expression (Kennedy et al., 2000). Since fetal skin contains more HA than adult skin, several investigators have proposed a role of HA in scarless healing (Mast et al., 1991, 1993).

Proteoglycan extracellular matrix (ECM) modulators Proteoglycan ECM modulators regulate collagen synthesis, organization, and degradation. Levels of decorin, a modulator of collagen fibrillogenesis, are upregulated in adult wounds (Beanes et al., 2001b). Conversely, levels of fibromodulin, another modulator of collagen fibrillogenesis, are lower in adult wounds (Soo et al., 2000). Fibromodulin inactivates TGF-beta, a key cytokine involved in wound healing, and has been shown to have an

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3.3 (opposite) Scarless healing of E16 fetal rat wounds (confocal microscopy). Collagen fibers are stained with sirius red and appear white. (a) Healed wound harvested at 72h (200×). The epidermis is thickened at the wound site (arrow). The collagen fiber is reticular and unchanged from the surrounding dermis. (b) Healed wound harvested at 72h under a higher magnification (1000×). The collagen fibers are thin and closely approximating each other with little interfiber space. The fibers are arranged in a wispy reticular pattern. (c) Non-wounded E19 skin at the same magnification as (b) (1000×). The dermal collagen fiber pattern is identical to (b). Reproduced with permission from Lippincott Williams & Wilkins. Original publication: Beanes SR, Hu FY, Soo C, et al. Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition. Plast Reconstr Surg. 2002;109:160–170.

antiscarring effect during wound repair (Soo et al., 2000). Lysyl oxidase expression is increased in adult wounds, and this modulator has been implicated in the pathogenesis of some fibrotic diseases (Beanes et al., 2001a). Matrix metalloproteinases (MMPs) and tissue-derived inhibitors (TIMPs) are involved in ECM turnover. Scarless fetal wounds have a higher ratio of MMP to TIMP expression, which favors remodeling over accumulation of collagen (Dang et al., 2003). Adhesion proteins Scarless fetal wounds have an enhanced ability to upregulate ECM adhesion proteins, such as tenascin and fibronectin (Whitby and Ferguson, 1992, Cass et al., 1995, Whitby et al., 1991). These adhesion proteins mediate cellular attachment to the ECM and attract fibroblasts, keritinocytes, and endothelial cells to sites of injury (McCallion et al., 1996). During development, tenascin facilitates cell movement and fibronectin facilitates cell anchoring. Early gestation fetal rabbit wounds express fibronectin 4 hours after wounding, while fibronectin expression is not seen until 12 hours postwounding in the adult (Longaker et al., 1991). Similarly, tenascin appeared at 1 hour in the wounded fetus, 12 hours in the wounded neonate, and at 24 hours post-wounding in the adult mouse (Whitby et al., 1991). The rapid deposition of tenascin in fetal wounds may stimulate early cell migration, while the large amounts of fibronectin may stimulate cell attachment and wound repair. These factors may promote rapid deposition of an organized matrix that has less scarring.

3.4.3 Scarless repair is intrinsic to fetal skin The capacity for scarless repair was initially attributed to the sterile intrauterine environment. Amniotic fluid is rich in hyaluronic acid and growth

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3.4 (opposite) Early scar formation after the transition point in E18 fetal rat wounds (confocal microscopy). Collagen fibers are stained with sirius red and appear white. (a) Healed wound at 72 h (200×). The wound dermal collagen pattern (open arrows) is different from the surrounding nonwounded dermis (shaded arrow). The fibers are less densely compacted. No epidermal appendages are present. Neovascularization is shown with the white arrows. (b) Healed wound at 72h at a higher magnification (1000×). The collagen fibers are thicker, but with greater interfiber spaces compared to nonwounded dermis. (c) Nonwounded skin at E21 days gestational age (1000×). When compared to wound collagen fibers (b), nonwounded dermal collagen fibers are thinner with less interfiber space. Reproduced with permission from Lippincott Williams & Wilkins. Original publication: Beanes SR, Hu FY, Soo C, et al. Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition. Plast Reconstr Surg. 2002;109:160–170.

factors but devoid of bacteria and inflammatory stimulators. Therefore, this environment was proposed to be an environment conducive to scarless wound repair. However, early studies demonstrated that the intrauterine environment is neither necessary nor sufficient for scarless repair. Fetal marsupials develop in a maternal pouch outside the uterus but are still able to heal wounds scarlessly (Armstrong and Ferguson, 1995). Experiments have also shown that adult sheep skin transplanted onto the backs of fetal sheep, bathed in the amniotic fluid of the intrauterine environment, heal incisional wounds with scar while adjacent wounds in fetal skin heal scarlessly (Longaker et al., 1994). Moreover, human fetal skin heals without scar when it is transplanted into the subcutaneous tissue of adult athymic mice (Lorenz et al., 1992b).

3.5

Adult versus fetal wound healing

3.5.1 Differences in cell type and function Platelets The absence of an acute inflammatory infiltrate in scarless wounds may be partly explained by decreased fetal platelet degranulation and aggregation. Although there is no difference in size, organization, or granule content by transition electron microscopy in fetal compared to adult platelets, fetal platelets produce less platelet-derived growth factor (PDGF), TGF-ß1, and TGF-ß2 than their adult counterparts (Olutoye et al., 1996). The proinflammatory signals that are released from the platelet plug are responsible for mediating the early phase of the wound healing process. Although exposure of fetal platelets to collagen in vitro stimulates growth factor release, the

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capacity for fetal platelet aggregation is age-dependent (Olutoye et al., 1997). Early gestation fetal platelets do not aggregate, whereas late gestation fetal platelets aggregate just as effectively as adult platelets. The transition point for this platelet response corresponds to the transition point from scarless regeneration to formation of scar. Thus, the reduced function of early gestation fetal platelets may be one mechanism of scarless repair. Neutrophils Neutrophils neutralize and engulf bacteria. Cytokines TGF-ß1 and PDGF recruit neutrophils to the site of injury. Neutrophils then release selfstimulating cytokines and chemoattractants for fibroblasts and macrophages (Singer and Clark, 1999). Fewer neutrophils are present in the fetal wound, and researchers have demonstrated an age-dependent defect in the ability of fetal neutrophils to phagocytose pathogenic bacteria in fetal sheep (Jennings et al., 1991). Fetal wounds heal rapidly with minimal inflammation. This observation has stimulated interest in the role of cellular inflammatory mediators, cytokines, and growth factors in fetal wound healing. In the postnatal animal, disruption of tissue integrity stimulates platelet activation, cytokine production, and chemotaxis of macrophages and neutrophils (Singer and Clark, 1999). However, scarless wounds are characterized by a relative lack of inflammation (McCallion et al., 1996). Furthermore, introduction of inflammation into normally scarless wounds produces dose-dependent increases in wound macrophages, neutrophils, collagen deposition, and scarring (Frantz et al., 1993). Conversely, reduction of inflammation in postnatal wounds also reduces scarring. This suggests an important role of inflammation in scar formation. Fibroblasts Synthesis and remodeling of the ECM by fibroblasts is essential for wound healing. Adult and fetal fibroblasts reside locally in the injured tissue or are recruited to the site of injury by soluble chemoattractants released by macrophages and neutrophils (Clark, 1996b). Fetal wounds have fewer inflammatory cells and less cytokine expression, yet heal more rapidly than adult wounds. This may be partly explained by intrinsic differences between adult and fetal fibroblasts. Fetal and adult fibroblasts display differences in synthetic function of collagen, HA, and other ECM components. In vitro, fetal fibroblasts synthesize more type III and IV collagens than their adult counterparts, correlating with an increase in prolyl hydrolase activity, the rate-limiting step

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in collagen synthesis (Thomas et al., 1988, Lorenz and Adzick, 1993). Collagen synthesis is delayed in the adult wound while fibroblasts proliferate. In contrast, fetal fibroblasts simultaneously proliferate and synthesize collagen (Clark, 1996b). Fetal fibroblasts have more surface receptors for HA, which also serves to enhance fibroblast migration (Chen et al., 1989). Additionally, TGF-ß, which inhibits migration of confluent fibroblasts in vitro, is decreased in the fetal wound (Ellis and Schor, 1996). Differences in myofibroblasts have also been reported. Myofibroblasts, which are contractile fibroblasts detected by the presence of alpha-smooth muscle actin, appear in the adult wound one week after wounding. The content of myofibroblasts is greatest during the 2nd and 3rd week and decreases with time (Clark, 1996b), whereas wounds made early in gestation have virtually no myofibroblasts. In contrast, scarring fetal and postnatal wounds have progressively more active myofibroblasts, which correlates with contraction and degree of scarring (Estes et al., 1994). Overall, the fetal fibroblast has an intrinsic ability to synthesize a dermal ECM that is superior to the adult fibroblast in terms of its ability to generate an organized dermis at sites of injury.

3.5.2 Differences in cytokine profiles Transforming growth factor-beta (TGF-ß) Since shortly after its discovery more than 20 years ago, TGF-ß was shown to be involved in wound healing. The TGF-ß isoforms are involved in all steps of the wound repair process, and have divergent effects on scar formation and wound healing. The expression of TGF-ß1 and TGF-ß2 is increased in adult wounds, while it is unchanged in fetal wounds (Nath et al., 1994). Scarless wounds in fetal mice have less TGF-ß1 than neonatal or adult wounds (Krummel et al., 1988). Insertion of PVA sponges containing TGF-ß1 into fetal wounds leads to scar formation (Krummel et al., 1988). Similarly, treatment of adult rat wounds with neutralizing antibodies to TGF-ß1 and TGF-ß2 reduces scar formation (Nath et al., 1994, Shah et al., 1994). The relative proportion of TGF-ß isoforms, and not the absolute amount of any one isoform, may determine the wound phenotype. In scarless fetal wounds, TGF-ß3 expression is increased while TGF-ß1 expression is unchanged. Conversely, TGF-ß1 expression is increased and TGF-ß3 expression is decreased in scarring fetal wounds (Hsu et al., 2001). Treatment of adult wounds with exogenous TGF-ß3 reduces scar formation (Shah et al., 1995). This suggests the ratio of TGF-ß3 to TGF-ß1 may determine whether tissue regenerates with or without scar.

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Interleukins Interleukins are cytokines important in chemotaxis and activation of inflammatory cell mediators. IL-6 stimulates monocyte chemotaxis and macrophage activation, while IL-8 attracts neutrophils and stimulates neovascularization (Liechty et al., 2000a). Wounding stimulates a rapid increase in IL-6 and IL-8, which persists at 72 hours in the adult but disappears by twelve hours in the fetus (Liechty et al., 2000a, 1998). Both IL-6 and IL-8 expression are significantly lower in early fetal fibroblasts compared to adult fibroblasts. The addition of IL-6 to fetal wounds produces scar in wounds that would otherwise heal without scar. IL-10 is anti-inflammatory through decreased production of IL-6 and IL-8. Fetal skin grafts harvested from early gestation IL-10 knock-out mice grafted onto syngeneic adult mice heal with significant inflammation and scar (Liechty et al., 2000b). In an initial study, adult mouse wounds were treated with an IL-10 over-expression adenoviral vector. Inflammation was reduced, and wound healed without scar (Gordon et al., 2001). The strategy of overexpressing IL-10 could be used to decrease scar formation in adult human wounds. Differences in gene expression profiles Genomic microarray analysis has shown that the gene expression profiles of scarless fetal wounds and scarring postnatal wounds are different (Colwell et al., 2008). Scarless fetal wound healing is characterized by a rapid upregulation of groups of genes involved in cell growth and proliferation, which likely contributes to rapid wound closure. These genes are important for DNA transcription, DNA repair, cell cycle regulation, protein homeostasis, and intracellular signaling. At early time points, the proportion of genes with increased expression is significantly higher in fetal wounds. However, by 24 hours, the postnatal wound transcriptome has more genes with increased expression compared with fetal wounds (Colwell et al., 2008). The rapidly changing gene expression profile in fetal wounds likely reflects the rapid healing rate in the fetus.

3.6

Treatment options for scars

Currently available therapeutic interventions do not adequately address the issue of scar formation. Research has not adequately demonstrated efficacy or safety for many treatments secondary to either small treatment groups or a lack of well-designed controls. However, treatment algorithms for the management of keloids and hypertrophic scars have been published and these can be used to guide clinical practice (Figs 3.5 & 3.6).

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(a) Exclusive diagnosis of similar diseases and differential diagnosis of hypertrophic scars and keloids Hypertrophic scars (HSs)

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3.5 Treatment algorithm for hypertrophic scars (HSs). Reproduced with permission from Wolters Kluwer Health. Original publication: Ogawa, R. The most current algorithms for the treatment and prevention of hypertrophic scars and keloids. Plast Reconstr Surg. 2010; 125(2):557–568.

3.6.1 Surgery Scars can often behave in an unpredictable way, and remodeling can occur for more than a year following injury. During this period, scars can flatten, soften, lose their dark pigmentation, and contractures can lessen. Therefore,

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3.6 Treatment algorithm for keloids. Reproduced with permission from Wolters Kluwer Health. Original publication: Ogawa, R. The most current algorithms for the treatment and prevention of hypertrophic scars and keloids. Plast Reconstr Surg. 2010; 125(2):557–568.

surgery is usually considered only after one year of observation and after other, less invasive therapies have failed. There are many options for surgical treatment of scarring, including excision with direct closure, local skin flap coverage, or more extensive vascular flap coverage.

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3.6.2 Cryotherapy Cryotherapy has been used as an adjunct to surgery in the treatment of keloids and hypertrophic scars. Cryotherapy is thought to destroy scar tissue and decrease collagen synthesis. This therapy has shown promise, the largest study showing 79.5% response rate with 80% reduction in scar volume (Har-Shai et al., 2003, Zouboulis et al., 1993). Cryotherapy side effects include hypopigmentation and depressed, atrophic scar formation, and long term efficacy has not yet been established (Rusciani et al., 2006).

3.6.3 Compression therapy Pressure dressings have been in use for decades (Tolhurst, 1977); however, their efficacy in either prophylaxis or in the treatment of scars has not been demonstrated (Reish and Eriksson, 2008). Pressure earrings have been used at sites of earlobe keloid excisions but have not been shown to eliminate recurrence. These therapies are likely to be most efficacious in polytherapy, but further investigation is warranted.

3.6.4 Corticosteroids Corticosteroids, used topically and via intralesional injection, are commonly used to treat symptomatic scars. Triamcinolone is the most common agent used, and the mechanism of action is most likely due to reduction of the inflammatory response. As a result, collagen degradation increases as its synthesis decreases. Fibroblast proliferation decreases, and keratinocytes decrease expression of TGF-β1 and TGFβ2 (Perez et al., 2001, Wu et al., 2006, Rosenberg et al., 1991, Stojadinovic et al., 2007, Manuskiatti and Fitzpatrick, 2002). Experimental studies, though limited by lack of appropriate controls and poor study design, report 50 to 100% efficacy (Manuskiatti and Fitzpatrick, 2002, Tang, 1992, Darzi et al., 1992, Reish and Eriksson, 2008). Side effects of corticosteroids include delayed healing, hypopigmentation, dermal atrophy, and scar widening. These effects are neither benign nor rare, occurring in 63% of patients (Manuskiatti and Fitzpatrick, 2002, Maguire, 1965). The most appropriate use of corticosteroids is thought to be in polytherapy, as the concurrent use of other agents reduces the steroid dose required to produce therapeutic effect.

3.6.5 Lasers Pulsed dye laser therapy is thought to reduce vascularity by producing photothermolysis of the microvasculature, resulting in thrombosis and ischemia and decreased collagen content (Reiken et al., 1997). In

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comparison to corticosteroids, laser therapy produces fewer adverse effects (hyperpigmentation in 1 to 24% and transient purpura in some). Laser therapy has been shown to reduce scar erythema in limited studies (Alster and Williams, 1995, Reiken et al., 1997, Alster, 1994). However, more research with well-designed controls is required to confirm its efficacy.

3.6.6 Topical silicone gel Topical silicone gel sheets hydrate the wound bed and are thought to inhibit collagen deposition and downregulate TGF-β2. This is an attractive therapy for both prophylaxis and treatment of excessive scarring because silicone gel is noninvasive and associated with few adverse effects. However, studies on its efficacy show conflicting results (Ahn et al., 1991, Quinn, 1987, Carney et al., 1994).

3.6.7 Radiation Radiation is thought to decrease fibroblast proliferation and neovascular bud formation, decreasing collagen content at the wound bed. This therapy is often used perioperatively and has been shown to be most effective for recurrent keloids if a single dose is given within 24 hours of excision. When used in this manner, as an adjunct to surgery, radiation therapy decreases recurrence rates from between 45 and 100% to between 16 and 27% (Ragoowansi et al., 2003, Ship et al., 1993, Berman and Bieley, 1996, Kovalic and Perez, 1989). Although radiation therapy has been shown to be useful in eliminating keloid recurrence, specific dosing regimens have not been established. One group reports efficacy with 300 to 400 Gy applied in three to four fractions or 600 Gy for three fractions (Reish and Eriksson, 2008).

3.6.8 5-Fluorouracil (5-FU) 5-Fluorouracil is thought to inhibit fibroblast proliferation and TGF-β1induced collagen synthesis (Mallick et al., 1985, Blumenkranz et al., 1982, Wendling et al., 2003). Although this therapy has shown good results in combination with corticosteroids alone or with corticosteroids and laser therapy, its use alone has shown little efficacy (Manuskiatti and Fitzpatrick, 2002, Fitzpatrick, 1999). Further studies are required to establish 5-FU as a mainstream scar therapy.

3.6.9 Bleomycin Bleomycin is an antibiotic known to produce antibacterial, antitumor, and antiviral activity. As a scar-reducing therapy, this agent is thought to inhibit

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lysyl-oxidase or TGF-β1, decreasing collagen synthesis (Hendricks et al., 1993, Lee et al., 1991). Intralesional injection of bleomycin has been effective in reducing the appearance of keloids and hypertrophic scars (Saray and Gulec, 2005, Naeini et al., 2006, Espana et al., 2001). However, the studies are limited due to lack of well-designed controls. Adverse effects of bleomycin injection include hyperpigmentation in 75% of patients and dermal atrophy surrounding the injection site in 10 to 30% of patients (Bodokh and Brun, 1996).

3.7

Future trends

3.7.1 Growth factors and cytokines As discussed, the tight regulation of growth factors, cytokines, and chemokines, as well as a wound environment supportive of activity, is essential to proper wound repair. While acute wounds progress through the temporally overlapping stages of wound repair, chronic wounds become arrested in the inflammatory process, creating a proteolytic environment upregulating proinflammatory cytokines and chemokines. This environment impedes the action of other growth factors and cytokines, preventing the re-establishment of the skin’s barrier function. Most of the research on growth factor and cytokine therapy has focused on the use of these agents to enhance wound healing in chronic ulcers. One major impediment in the topical delivery of growth factors has been in the creation of sustained release factors resistant to degradation in the chronic wound’s proteolytic environment. It is thought that debridement is essential prior to administration of topical growth factors, as this process restores growth factor receptor expression absent at the nonhealing edges of chronic ulcers (Brem et al., 2007, 2003). The growth factors currently receiving the most research attention for clinical therapeutics are VEGF, bFGF, and GM-CSF. Currently, patients are being treated with PDGF-BB, bFGF, and GM-CSF, although only PDGF-BB is the only growth factor that has passed FDA regulations in the United States (Barrientos et al., 2008). The constituents of the PDGF growth factor family are produced by platelets, macrophages, vascular endothelium, fibroblasts, and keratinocytes (Bennett et al., 2003). PDGF has been implicated at every stage of wound healing, and PDGF levels are markedly decreased in chronic wounds (Robson, 1997). A recombinant human variant of PDGF-BB known as Becaplermin has been approved by the FDA for the treatment of diabetic wounds and pressure ulcers. This drug has been determined, through randomized controlled clinical trials, to be both safe and efficacious (Margolis et al., 2000, 2004). VEGF is a large family of growth factors active in angiogenesis. VEGF-A has been shown in several in vitro studies to be essential to proper wound

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healing by promoting endothelial cell migration and proliferation (Suzuma et al., 1998, Senger et al., 1996, Pepper et al., 1992, Goto et al., 1993). Chronic ulcers contain large areas of ischemia, making VEGF-A an attractive therapeutic agent. Although in vivo studies show that the administration of VEGF-A can improve angiogenesis in diabetic ischemic limbs and diabetic wounds (Walder et al., 1996, Bauters et al., 1994, 1995, Takeshita et al., 1994a, 1994b, Galiano et al., 2004), its exogenous administration is associated with vascular leakage and the formation of disorganized blood vessels and dysfunctional lymphatic vessels (Nagy et al., 2002, Carmeliet, 2000). VEGF-C is another member of the VEGF family that has been linked to improved wound healing in treatment of diabetic ulcers. In vivo studies show increased angiogenesis and lymphangiogenesis, as well as accelerated healing when VEGF-C was administered by an adenoviral vector to type I diabetic mice (Saaristo et al., 2006). Living cell therapy has been approved by the FDA as a novel approach to wound healing. Fibroblasts and keratinocytes produce at least 17 growth factors, and it is postulated that the application of living cell therapies induces their release, stimulating the patient’s own cells to participate in wound healing (Brem et al., 2003, Phillips et al., 2002, Falanga et al., 2002). Juvista, the first pharmaceutical scar reducing product, recently completed Phase I and II trials in the UK. According to the company’s website, this recombinant TGF-β3 polypeptide improves scar appearance with intralesional injection. Seventy percent of the wounds treated exhibited a positive response, and the drug was found to be safe in the population tested, which was over 1500 patients (see www.renovo.com). A second product under development by the same company is Juvidex, a mannose-6-phosphate topical formulation, an estradiol derivative known to inhibit TGF-β1 and TGF-β2. Research shows that although estradiol upregulates TGF-β levels in unwounded skin, it actually downregulates TGF-β1 levels in wounded skin, facilitating repair (Stevenson et al., 2008, Ferguson and O’Kane, 2004).

3.7.2 Small molecule compounds Although there are no current small molecule compounds commercially available, the development of small molecule probes of regenerative pathways are currently under investigation. These compounds might offer some advantages over other methods of preventing or treating scar formation, including the ability to disrupt pathways with precise temporal control. Through comparisons of zebrafish and other regenerative healing models, groups such as Mathew et al. strive – through in vivo chemical genetics – to identify the molecular differences between mammals and lower vertebrates.

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Identifying these differences through throughput genetic screening would allow the induction of regenerative healing with the introduction of small molecule clinical therapeutics (Mathew et al., 2007). Many pathway interventions have been proposed to prevent or reduce scar formation. Fibromodulin, hyaluronic acid, and hepatocyte growth factor have been identified as factors that, if increased in expression, could induce regenerative healing (Stoff et al., 2007, Iocono et al., 1998, Ha et al., 2003). Other approaches have focused on the inhibition of MMP (e.g. GM6001) (Witte et al., 1998), pro-collagen C-proteinase (Fish et al., 2007), angiotensin peptides (Rodgers et al., 2003), and dipeptidyl peptidase IV enzymes (Thielitz et al., 2008). Adenovirus-p21 overexpression has also been associated with scar reduction (Gu et al., 2005).

3.7.3 Stem cells At this point, tissue regeneration in adult human tissues has not been accomplished by single molecule-specific therapy. Regenerative repair may require cell-based therapy in which multiple cascades of signaling pathways are affected. Stem cell therapy is promising due to its ability to differentiate cells into various cell types. Initially, embryonic stem cells (ES cells) were thought to be the best candidates for inducing regenerative repair at sites of tissue injury. However, there are several obstacles associated with the transplantation of embryonic stem cells. By far, the largest obstacle is the immune system. To overcome this barrier, authors have suggested either the transient use of immunosuppressive agents with gradual withdrawal or encapsulation of ES cells with biodegradable polymer membrane prior to transplantation (Heng et al., 2005, Orive et al., 2003). In both cases, transplanted cells would ultimately be eliminated by the host immune system, but only after regenerative repair was well underway. The other major obstacle to ES cell transplantation is the risk of teratoma formation. Although in theory this risk would be ameliorated using the aforementioned methods, the risk to the patient is too large to make ES cell transplantation a current, viable therapeutic option. Mesenchymal stem cells (MSCs) are thought to be an ideal source for stem cells used for scar-reducing therapies, based on their ability to expand in vivo and differentiate into multiple tissue types (Lee et al., 2004, Zuk et al., 2002). These cells could be isolated from an adult, then manipulated and transplanted back into the same patient. Therefore, the use of MSCs avoids the immune system obstacles associated with ES cell therapy. Human studies show that MSCs are immune privileged and can be used without tissue typing or HLA matching (Hematti, 2008, Ryan et al., 2005). Moreover, MSCs isolated from both the bone marrow and adipose tissue are

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thought to have favorable safety profiles (Fang et al., 2007, Garcia-Olmo et al., 2005, 2009). Two human investigations of MSCs in wound healing demonstrate the ability of these cells to improve wound healing. The first was a small trial performed by Falanga et al. in which MSCs combined with fibrin spray was applied topically to acute surgical wounds and chronic lower extremity ulcers. Data from this study show accelerated healing of wounds in the treatment group in comparison to controls. The speed of wound healing was directly proportional to the number of stem cells applied (Falanga et al., 2007). Yoshikawa et al. performed a larger study involving the application of bone marrow derived MSCs with dermal replacement – with or without autologous skin grafts – to various nonhealing wounds. Results showed accelerated healing in wounds treated with MSCs (Yoshikawa et al., 2008). Further research is needed to characterize MSCs and their niche. As purification techniques improve, the role of MSCs in wound healing will gain clarity. Recent research has identified another reservoir of stem cells in the skin’s epidermis. A number of stem cell niches are present in the epidermis, and the interfollicular epidermal stem cells and hair follicle bulge region stem cells can supply each other when damaged. At this point, epidermal stem cell therapeutic potential is largely theoretical, but research is likely to develop rapidly, as skin grafting for burn victims provides abundant clinical opportunities for research. Finally, induced pluripotent stem cells (iPS cells) have gained recent popularity as a potential cell-based therapy to induce regenerative repair. In a landmark paper, Takahashi and Yamanaka describe the process of reverting differentiated tissue cells back to a pluripotent state through transduction with specific transcription factors (Oct4, Sox2, Klf4, and c-Myc). These experiments have been successful in both murine and human cells, and both types of iPS cells are expected to behave like ES cells (Takahashi et al., 2007, Takahashi and Yamanaka, 2006, Maherali et al., 2007, Wernig et al., 2007). The use of iPS cells for tissue regeneration would allow for the use of autologous cells to create patient-specific cell lines, which could then be used for regenerative therapy. These therapies have the advantage of avoiding the immune rejection and ethical concerns typically associated with ES cell therapy. However, due to the use of viral vectors, concern regarding insertion mutagenesis and uncontrolled genome modification mandate further research into the development of other methods of reprogramming (Pera and Hasegawa, 2008, Zhou et al., 2009, Stadtfeld et al., 2008, Okita et al., 2008). To date, all reprogramming factors have been shown to be oncogenic when overexpressed. Therefore, before the introduction of iPS therapy in clinical practice, rigorous investigation into therapeutic safety must be performed.

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Scarring and scarless wound healing are complex and highly regulated events that are influenced by a multitude of different cells and mediators, including cytokines, signaling molecules, and receptors. Over the past several decades, an intensive research effort has greatly increased our understanding of scarless fetal wound healing and the more primitive cells orchestrating the process, but the precise mechanism behind this complex event remains unknown. Based on our current knowledge of scarless wound healing, we have only been able to develop marginally effective scar treatments. A more complete understanding of the mechanisms underlying scarless wound healing will likely lead to the development of improved anti-scar therapeutics.

3.9

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4 The discovery and development of new therapeutic treatments for the improvement of scarring N. L. O C C L E S T O N, A. D. M E T C A L F E, A. B OA N A S, N. B U R G OY N E, K. N I E L D, S. O’ K A N E and M. W. J. F E R G U S O N, Renovo Group Plc, UK

Abstract: Scarring is a major cause of physical and psychological morbidity and, whilst a variety of model systems exist that have increased our understanding of the pathways and processes underlying scar formation, they have typically not translated to the development of effective therapeutic approaches for scar management. In this chapter we describe the basic science underlying both scar-free and scar-forming healing, as well as the utility and translation of pre-clinical model systems to humans. Finally we outline our pioneering approach to the discovery and development of therapeutic approaches for the prophylactic improvement of scarring in man. Key words: scarring, skin, wound healing, TGFβ3, avotermin.

4.1

Introduction

Wound healing in both children and adults is essentially a repair process, which normally results in scarring at the wound site. Scar tissue is not identical to the tissue which it replaces and is usually of inferior functional quality. Scarring is a major cause of physical and psychological morbidity (1–8) and, whilst a variety of model systems exist that have increased our understanding of the pathways and processes underlying scar formation, they have typically not translated to the development of effective therapeutic approaches for scar management. Despite a number of potential treatment regimens, no single therapy is accepted universally as the standard of care (9–11). Scar improvement still remains an area of clear medical need. In this chapter we describe the basic science underlying both scar-free and scar-forming healing, as well as the utility and translation of pre-clinical model systems to humans. Finally, we outline our pioneering approach to the discovery and development of therapeutic approaches for the prophylactic improvement of scarring in man. 112 © Woodhead Publishing Limited, 2011

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There is a spectrum of wound healing that ranges from the ability to completely regenerate tissue in amphibians, through scar-free healing in embryos of different mammalian species, to scar-forming healing in children and adults. From an evolutionary perspective, the scarring response results in rapid replacement of missing tissue and, although sub-optimal in terms of appearance and function, results in a reduction in the likelihood of infection and an increased likelihood of organism survival following injury. The characteristics of scar-free healing after incisional wounding have been shown in various studies (12). Such healing has been demonstrated in the early embryos of a range of mammalian species including mice, rats, rabbits, sheep, pigs, marsupials and monkeys (13). They include minimal inflammation and complete restoration of normal skin structure, with normal collagen deposition and regularly distributed hair follicles, capillaries and glands. Comparison of the architecture of regenerated skin in embryos with that of adults demonstrates that it is the organisation of collagen that is largely responsible for scar formation. Whereas the dermis of embryonic skin is restored to the normal ‘basket weave’ architecture of collagen, in adult scars the collagen is abnormally organised in parallel bundles of fine fibres that are distinct from the normal skin (Fig. 4.1). It is of particular note that there appear to be no major differences between the composition of the dermal tissue of scar-free and scar-forming healing. This indicates that primarily scarring is a failure of the regeneration of the normal skin structure rather than a biochemical problem relating to an abnormal composition of the scar tissue (12). The mechanisms underlying scar-free and scar-forming healing have been studied at the molecular, biochemical and cellular level. Whilst there are a number of differences that have been identified between healing in the embryo and the adult, many of these are not mechanistically causative. In illustration, the mammalian embryo is surrounded by the sterile aqueous environment of the amniotic fluid, whereas adult wounds are exposed to air and a range of potentially contaminating agents including bacteria and foreign bodies. Originally, it was thought that the sterile aqueous environment provided by the amniotic fluid was important in scar-free healing. However, studies on marsupials such as the opossum, which complete development in their mother’s pouch, proved otherwise (14). In this model, incisional wounds were made in young pouch opossums and at an equivalent embryonic time to mouse embryos in an amniotic environment. Pouch opossums, like embryonic mammals, were found to heal without scarring despite developing outside of a sterile amniotic environment. Following injury to the embryo, the inflammatory response (by virtue of a less than mature immune system) is less marked, and differs in terms of the types

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e

rr

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4.1 (opposite) Scarring results from an abnormal deposition and organisation of collagen cutaneous scar in a non-Caucasian subject at 12 months following a 1 cm full thickness incision to the inner aspect of the upper arm (a). Histological staining of the excised scar with Van Geison’s stain demonstrating collagen (blue/green) in the normal skin compared to scar tissue and a normal undulating epidermis with rete ridges in the normal skin compared to a flattened epidermis overlying the scar (b). Picrosirus Red staining of ascar viewed using polarised light (c), illustrating the normal ‘basket-weave’ organisation of collagen in the normal skin resulting in organised light scattering (birefringence) compared to the abnormal organisation of collagen fibres within the scar resulting in a lack of birefringence. Arrowheads indicate the border of normal skin and scar tissue. Scale bars in (b) and (c) are 500 μm. In (b) and (c) rr = rete ridges; e = epithelium.

and number of inflammatory cells that enter the wound (15). Finally whilst the profiles and quantities of growth factors and cytokines associated with scar-free healing are often different to those in adult scar-forming healing (16–19), there are only a few of these factors that present themselves as potential therapeutic targets (12, 13, 20). Data from our studies and those in the literature demonstrate that the scarring response represents a continuous spectrum of phenotypes in organisms ranging from scar-free through to scar-forming healing (Fig. 4.2). Both scar-free and scar-forming healing can occur in the same animal, e.g. an axolotl can regenerate an amputated limb but heals an incisional wound on the flank with a scar; if part of the liver is removed in mammals by hepatectomy the liver regenerates, whilst stab wounds made to the liver heal with scarring; MRL/MpJ and other strains of adult mice including athymic nude-nu mice regenerate ear wounds, where the absence of T-lymphocytes in wounded ears provides a microenvironment conducive to regeneration of mesenchymal tissues, which is in contrast to wounds made on the dorsum of MRL/MpJ mice that heal with a scar; penetrating wounds to the cheek of humans heal with scarring of the external cutaneous surface but the oral mucosal surface heals with no discernable scar (12, 21–26). The likelihood is that tissue repair and regeneration are not that dissimilar and, in fact, share many common mechanisms that differ very subtly. Furthermore, the fact that all mammalian embryos exhibit a regenerative capacity demonstrates that even adult mammals contain the genetic program for regeneration. Notably, the growth factor profile of embryonic healing wounds is very different from that of an adult wound. Scar-free embryonic wounds contain reduced levels of Transforming Growth Factor-beta 1 (TGF-β1), Transforming Growth Factor-beta 2 (TGF-β2) and Platelet Derived

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Regenerative healing

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Species

Exemplar model / body site

Axolotl

Limbs

Mouse (MRL and other strains)

Ear

Mouse, rat, pig, sheep, marsupial, monkey

Embryo

Human

Liver, gums, oral mucosa, small cutaneous wounds (all adult)

Mouse, rat pig, human

Prophylactic cutaneous scar reduction therapeutics (in adult) e.g., TGFb 3

Axolotl

Incision (on flank)

Mouse Embryo (TGFb 3 knockout) mouse (MRL strain) Incision (on dorsum) Human (children and adults)

Cutaneous wounds Hypertrophic scars Keloids

4.2 Scar-free to scar-forming healing in vertebrates represents a continuous spectrum of responses.

Growth Factor (PDGF; released in the adult from degranulating platelets and inflammatory cells) and elevated levels of Transforming Growth Factorbeta 3 (TGF-β3). Experimental manipulation of the growth factor profile of adult wounds by exogenous addition of TGF-β3 or neutralisation of TGF-β1, TGF-β2, PDGF, etc., results in markedly reduced or absent scarring (12). The above findings and observations are critically important as they demonstrate that organisms retain the ability to heal via a regenerative as well as a scar-forming process, which gives a biological basis for therapeutic modulation of the healing response in adults to reduce scar formation.

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In vitro and in vivo models to investigate the mechanisms of scarring and evaluate potential treatments

The healing and scarring response consists of a robust series of complex, dynamic and interacting cellular, and molecular processes including haemostasis, the inflammatory response, granulation tissue formation and remodelling. The functions of the cell types involved in these processes are also regulated by a wide range of extracellular stimuli including growth factors/ cytokines as well as interaction with the extracellular matrix (27), which elicit effects by cell surface receptors and a range of intracellular signalling cascades that result in changes in gene and protein expression. Taken together these events contribute to complex dynamic microenvironments within the injured tissue during healing and scar maturation with which resident and infiltrating cells interact. A number of in vitro models have been utilised to investigate the various aspects of the healing and scarring response at the molecular and cellular level and include different cell types (e.g., neutrophils, macrophages, lymphocytes, keratinocytes, melanocytes, fibroblasts, fibrocytes and endothelial cells) and different molecular and cellular processes (e.g., signal transduction, gene expression, proliferation, migration, growth factor production, extracellular matrix production and remodelling) (28–32). Whilst in vitro models represent applicable systems to study individual components, these systems do not always accurately model the vastly more complex interactive in vivo situation. Typically we employ in vitro systems to further evaluate and refine findings generated from in vivo models. A number of species including mice, rats and pigs have been used as potential models of scarring in preclinical studies (Table 4.1). These studies include the use of excisional and incisional wounds in rodents and pigs, typically with macroscopic and/or microscopic endpoints for scarring, as well as the use of transgenic mice and more recently the Red Duroc pig, which exhibits some of the features of hypertrophic scarring seen in humans (33–35). Most studies have not systematically compared the molecular, cellular and tissue responses in these models to those in man. More importantly, most studies have not investigated the translation of therapeutic modulation in pre-clinical models to that in man. Addressing these issues is key to not only discovering and developing potential therapeutics for humans, but also for investigating and understanding their mechanisms of action. We have addressed these issues in a series of extensive longitudinal studies utilising a range of endpoints and technologies both in pre-clinical models and humans. Our studies in mice, rats and pigs have demonstrated that scars are stable and mature at ≥70 days post-wounding in mice/rats and ≥6 months in pigs compared to 6 to 12 months in man. Comparison of the

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Gene deletions/additions can elicit lethal effects and may provide misleading data if compensatory mechanisms due to genetic alteration(s) occur within the animal (also see general comments on mice below).

Degree of scarring in mice at macroscopic and microscopic level is significantly less than in humans. Therefore relatively difficult to accurately quantitate improvements with treatments over the normal scarring response. Timepoints selected for assessment in published studies, e.g. 14 to 28 days, are typically during the granulation tissue formation phase prior to formation of a stable scar. As such these studies do not represent a suitable timepoint for evaluating the true scar reduction effects of therapies.

Initial target identification and validation. Gene modifications may elicit effects on the scarring response, inducing scarless healing or excessive scarring. Incisions most relevant for scarring, punch biopsies relevant for healing endpoints.

Outbred and inbred strains can be utilised. Gene expression data indicate molecular processes have a relevant level of comparability to humans. Modulators of the scarring response can be evaluated in the absence of any potentially confounding effects seen in transgenic animals. Incisions most relevant for scarring, punch biopsies relevant for healing endpoints.

Regeneration, improvements in scarring measured using a range of parameters (gene, protein, histological analysis, tensile strength, macroscopic appearance, etc.). Endpoints typically studied at 14 to 28 days post-wounding. Scars stable around day 70. Improvements in scarring measured using a range of parameters (gene, protein, histological analysis, tensile strength, macroscopic appearance, etc.). Endpoints typically studied at 14 to 28 days post-wounding. Scars stable around day 70 post-wounding.

Transgenic mice (knockouts and overexpressors; incisions and punch biopsy wounds)

Mouse (incisions and punch biopsy wounds)

Limitations

Utility

Endpoints studied

Model

Table 4.1 In vivo models used to evaluate scar improvement therapies

(29, 46, 47)

(22, 23, 42–45)

Refs

© Woodhead Publishing Limited, 2011 Ear wounds often used as a model for chronic healing and excessive scarring.

Degree of scarring in pigs at macroscopic and microscopic level is significantly less than in humans. Lengthy and costly studies due to timing of relevant endpoints. Red Duroc pigs do not accurately model all relevant aspects of human hypertrophic scars and require significantly long studies and therefore have an associated potentially prohibitive cost. No robust evidence of translation of findings in models to effective therapeutics in prospective, double-blind and well controlled trials in humans.

Improvements in scarring measured using a range of parameters (gene, protein, histological analysis, macroscopic appearance, etc.). Endpoints typically studied at 20 to 40 days post-wounding. Improvements in scarring measured using a range of parameters (gene, protein, histological analysis, wound width, tensile strength, macroscopic appearance, etc.). Endpoints typically studied up to 6 to 12 months post-wounding when the scars are stable. Structure of skin reported to be most similar to humans. Gene expression data indicates molecular processes have a relevant level of comparability to humans. Accepted species for wound healing studies. Large or multiple wounds possible due to size. Incisions most relevant for scarring, punch biopsies relevant for healing endpoints. Red Duroc pig reported to model aspects of hypertrophic scarring in humans.

Pig (minipigs, domestic swine and Red Duroc; incisions, excisions and punch biopsy wounds)

Rabbit (incisions and punch biopsy wounds)

Many scientific reagents are geared towards the study of mice and humans and consequently there are some limitations in terms of reagents (e.g., antibodies for immunocytochemistry) to completely compare all mechanisms to those in man. Most studies use unsuitable time points of <70 days, where scars have not matured/stabilised. Rarely used to assess normal skin wound healing on the back. Although some features of excessive scarring are modelled, the biological relevance of the ear wounds (involving cartilage) to cutaneous wounds in humans is not completely clear.

Rats demonstrate comparability to scars in humans (volunteers) at the macroscopic, microscopic and gene expression levels. Relatively easy to differentiate the effects of scar reducing agents.

Improvements in scarring measured using a range of parameters (gene, protein, histological analysis, wound width, tensile strength, macroscopic appearance, etc.). Scars stable around 80 days post-wounding.

Rat (incisions and punch biopsy wounds)

Limitations

Utility

Endpoints studied

Model

(9, 54–57)

(51–53)

(48–50)

Refs

© Woodhead Publishing Limited, 2011

Requirement of suitable infrastructure Suitable for demonstrating and expertise. safety and efficacy. Model highly relevant and translates to patient based studies. Easy to differentiate the effects of scar reducing agents on a range of clinically and scientifically relevant parameters.

Improvements in scarring measured using a range of parameters (gene, protein, histological analysis, wound width, tensile strength, macroscopic appearance, etc.).

Human volunteers (incisions, excisions and punch biopsy wounds)

Limitations

Utility

Endpoints studied

Model

Table 4.1 Continued

(13, 39, 36, 40, 58–59)

Refs

The discovery and development of new therapeutic treatments (a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

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4.3 Comparison of scarring at the macroscopic and microscopic levels between experimental incisional wound models in pre-clinical species and humans scarring response in mice 70 days following a 1 cm full thickness incisional wound on the dorsum at the macroscopic (a; arrowheads indicate ends of original wound) and microscopic (b; arrowheads indicate scar) levels. Scarring response in rats 84 days following a 1 cm full thickness incisional wound on the dorsum at the macroscopic (c; arrowheads indicate ends of original wound) and microscopic (d; arrowheads indicate scar) levels. Scarring response in pigs 168 days following a 1 cm full thickness incisional wound on the dorsum at the macroscopic (e; arrowheads indicate ends of original wound) and microscopic (f; arrowheads indicate scar) levels. Scarring response in humans 365 days following a 1 cm full thickness incisional wound on the inner aspect of the upper arm at the macroscopic (g; arrowheads indicate ends of original wound) and microscopic (h; arrowheads indicate scar) levels.

macroscopic appearance of these scars and the ability to assess a scar reduction effect within these pre-clinical models demonstrates that next to man, rats scar the worst and represent the most appropriate model (Fig. 4.3). We have also compared the gene expression profiles during the healing and scarring process in these pre-clinical species (up to 30, 000 genes per sample per time point; >300 samples; 11 time points) and compared these to profiles in man (Caucasians and Non-Caucasians; 30,000 genes per sample per time

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365 days

180 days

30 days

14 days

7 days

5 days

3 days

1 day

84 days

28 days

14 days

7 days

5 days

3 days

1 day

12 hours

6 hours

0 hours

0

Z Score normalised gene expression

Z Score normalised gene expression

Rat 2.0 1.0 0 –1.0 2.0 1.0 0 –1.0 2.0 1.0 0 –1.0

4.4 Gene expression in models of incisional wounds and scars in rat and man demonstrate molecular comparability. The 50 genes that best represent each of the k-means clusters are shown. In all three images the genes in the upper sections of each graphical representation represent those genes involved in the initial healing and immune response. Those in the middle graphs are genes involved in the granulation of the wound and the lower section graphs are genes involved in the remodelling of the scar as it matures.

point; >250 samples; 9 time points). Both heat map and cluster analysis of the expression of genes involved in all the major phases of healing and scarring have clearly demonstrated molecular comparability, particularly between rat and man, indicating that the major difference between healing and scarring in these models is time, with humans exhibiting an extended scar maturation phase (36) (Fig. 4.4). In addition, a number of genes/gene pathways have been identified from these studies in rats and man as further potential novel targets for the reduction of scarring in the skin. The relevance and translation of our studies and findings in pre-clinical models to those in man are further discussed in Chapter 17 with respect of avotermin (transforming growth factor beta 3).

4.4

Translation from pre-clinical studies to clinical efficacy

As noted above, we have demonstrated that there is significant molecular and cellular comparability between the healing and scarring process in relevant pre-clinical models and in man. Unlike other therapeutic/chronic disease indications, it is important to note that healing and scarring represent an acute biological response that is conserved across species and the progression of which is somewhat predictable. However, whilst a number of studies have reported therapeutic scar reduction in a variety of

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pre-clinical models, very few, if any, have demonstrated a translation of these findings to man in suitably designed, controlled, prospective and randomised clinical trials (9–11). Our approach for the development of therapies has focused on agents for the prophylactic reduction of scarring in man. This involves local administration of these agents to the margins of a wound at the time of surgery that leads to long-term improvements in scarring. The use of prophylactic, regenerative medicines is a novel pharmaceutical approach to scar improvement and there are a number of challenges associated with this including: designing clinical trials in what is a pioneering therapeutic area; developing and validating suitable endpoints for evaluating the effectiveness of a prophylactic drug, where there is no established baseline against which to determine improvements in scarring (since baseline would otherwise be normal skin before surgery or injury); and the fact that patients vary markedly in their propensity for scarring (37, 38). Our novel approach has been to utilise a within-subject, placebo-controlled, human volunteer model, prior to starting patient studies, not only to establish local drug safety and tolerability but also to investigate a number of other key parameters including: optimal dose(s) and dosing frequency of the drug; evaluation of a variety of relevant endpoints; effects of the drug in subjects with different demographics, e.g. sex, race, ages, etc; effects of the drug in different wound types, e.g. incisions and excisions. Studies to date have demonstrated that these prospective, double-blind, within-subject designs allow for a relevant and well-controlled approach to determining proof of concept for potential therapies. Since there are no registered pharmaceuticals for the prophylactic reduction of scarring, we have had to pioneer this area in terms of clinical trial design and so have explored a variety of potential surgical models in patient populations to define their appropriateness for demonstration of drug effects. We have successfully demonstrated a translation of scar reduction approaches from pre-clinical models to clinical studies, showing clear and robust effects with both ilodecakin (recombinant human interleukin-10; IL-10; Prevascar®) in a Phase II clinical trial and with avotermin (recombinant human transforming growth factor beta 3; TGFβ3; Juvista®) in extensive Phase II volunteer and patient based studies (39–41; and Chapter 17 on avotermin and emerging therapies).

4.5

Understanding the mechanisms of action of prophylactic scar improvement therapies

Following cutaneous injury, numerous interacting and dynamic molecular and cellular events are initiated. These include a series of cascades involved

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Advanced wound repair therapies Embryonic healing

Adult healing

Adult healing

Scar–free healing

With scar reduction therapy

Without scar reduction therapy

No haemostatic phase Low levels of TGF b1

Platelet degranulation

Platelet degranulation

Application of therapy around the time of wounding alters initial and subsequent microenvironments ‘re-routing’ the scarring response

Release of profibrotic and proinflammatory mediators, e.g. TGF b1, PDGF

Primitive/less differentiated immune cells

Rapid resolution of response Low levels of profibrotic cytokines

Absence of myofibroblasts

Release of profibrotic cytokines

Alteration of numbers/levels/ kinetics of myofibroblasts

Myofibroblast differentiation

Reduces collagen deposition

Production of excessive collagen

Cytoskeleton organised in more random fashion

Formation of cytoskeleton (actin) stress fibres

Collagen deposited in normal ‘basket-weave’ architecture

Collagen deposited in organisation more similar to normal skin

Collagen deposited in abnormal parallel organisation

Rapid wound maturation and regeneration of tissue

Increased protease activity and decreased protease inhibitors

Persistence of myofibroblasts Decreased protease activity and increased protease inhibitors

Reduction in time of wound and scar maturation

Extended wound and scar maturation including angiogenesis

More normal collagen organisation

Abnormal collagen organisation

Cytoskeleton organised in random fashion

Normal collagen organisation

Tissue regeneration

Scar improvement

Granulation tissue deposition and remodelling

Rapid cell migration

Alteration of inflammatory cell numbers/types/kinetics, and cytokines released

Inflammatory response

Neutrophils, Monocytes, Macrophages, Lymphocytes

Minimal inflammatory response

Haemostasis

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Scarring

4.5 Mechanisms and processes associated with scar-free healing, scar-forming healing and prophylactic scar reduction therapies.

in amplification, induction, repression, feed-forward and feed-back processes that result in a series of sequential and temporal microenvironments within the wound, with which resident cells and those infiltrating the wound interact (Fig. 4.5). The molecular and cellular behaviour of the wound is

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dependent on the composition of the tissue microenvironment at any one time. Therefore any alteration of the molecular or functional behaviour of cells (e.g., by appropriate therapeutic modulation) results in changes to subsequent wound microenvironments and ultimately affects the tissue response. In wound healing and scarring, like embryonic development, the system contains a number of pathways exhibiting multiple redundancy which gives robustness to the system. If minor pathways are therapeutically modulated, whilst subsequent microenvironments may be re-routed, they nevertheless result in a scarring phenotype. However, modulation of a major pathway that alters multiple microenvironments synergistically, results in significant alterations and a major ‘re-routing’ of the healing response, leading to the propagation and amplification of a phenotype of improved scar appearance (Fig. 4.5). From our studies in a range of preclinical species we have identified a number of key pathways that are central to generating a scarring response. Our use of human volunteers, in an experimental medicine context, has also rapidly allowed us to confirm which of these identified pathways are relevant in man and hence identify and progress new therapeutics into the clinical arena.

4.6

Conclusions

The reduction of scarring represents a clear medical need. Currently there are no registered pharmaceuticals for the prophylactic improvement of scarring and no single therapy is accepted universally as the standard of care. The spectrum of healing following wounding ranges from the ability to completely regenerate tissue through to the formation of hypertrophic and keloid scars. Importantly a number of studies have demonstrated that all mammalian organisms retain the ability to heal via both regenerative as well as scar-forming processes. This is key in terms of being able to therapeutically modulate the healing response in adults and reduce the severity of subsequent scarring. Our approach to the development of therapies has focused on agents for the prophylactic reduction of scarring. This has been significantly aided by our extensive studies comparing and understanding the molecular processes and scarring phenotypes in pre-clinical models, as well as our pioneering use of human volunteers both in longitudinal scarring studies and in an experimental medicine context which has rapidly allowed us to confirm which of the identified pathways are relevant in man. The translation of findings in the rat pre-clinical model to man has been shown in suitably designed and controlled prospective and randomised clinical trials. In terms of the mechanisms of action, the prophylactic administration of scar improvement therapeutics, results in significant alterations and a major

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‘re-routing’ of the healing response, resulting in the propagation and amplification of a phenotype of improved scar appearance, by virtue of a change in the architecture of the deposited collagen. The understanding of the scientific basis of scar-free and scar-forming healing and our pioneering approach to the development of therapies has allowed the identification and progression of new treatments. We have demonstrated that the development of pharmaceuticals for prophylactic scar improvement, that are additive to good surgical technique, is achievable, resulting in new therapies with a sound scientific basis and clear evidence of effectiveness in robust clinical trials. A clear example of this is further discussed in Chapter 17 in relation to the development of avotermin (recombinant human transforming growth factor beta 3).

4.7

References

1. McGrouther DA. Facial disfigurement. BMJ 1997;314:991. 2. Robert R, Meyer W, Bishop S, et al. Disfiguring burn scars and adolescent selfesteem. Burns 1999;25:581–5. 3. Newell R. Psychological difficulties amongst plastic surgery ex-patients following surgery to the face: a survey. Br J Plast Surg 2000;53:386–92. 4. Layton AM. Optimal management of acne to prevent scarring and psychological sequelae. Am J Clin Dermatol 2001;2:135–41. 5. Rumsey N, Clarke A, White P. Exploring the psychosocial concerns of outpatients with disfiguring conditions. J Wound Care 2003;12:247–52. 6. Lawrence JW, Fauerbach JA, Heinberg L, Doctor M. Visible vs hidden scars and their relation to body esteem. J Burn Care Rehabil 2004;25:25–32. 7. Valente SM. Visual disfigurement and depression. Plast Surg Nurs 2004;24: 140–6. 8. Bayat A, McGrouther DA. Clinical management of skin scarring. Skinmed 2005;4:165–73. 9. Meier K, Nanney LB. Emerging new drugs for scar reduction. Expert Opin Emerg Drugs 2006;11:39–47. 10. Reish RG, Eriksson E. Scars: a review of emerging and currently available therapies. Plast Reconstr Surg 2008;122:1068–78. 11. Reish RG, Eriksson E. Scar treatments: pre-clinical and clinical studies. J Am Coll Surg 2008;206:719–30. 12. Ferguson MW, O’Kane S. Scar-free healing: from embryonic mechanisms to adult therapeutic intervention. Philos Trans R Soc Lond B Biol Sci 2004;359:839–50. 13. McCallion RL, Ferguson MWJ. Fetal Wound Healing and the Development of Antiscarring Therapies for Adult Wound Healing. In: Clark RAF ed. The molecular and cellular biology of wound repair. New York: Plenum Press, 1996; 561–600. 14. Armstrong JR, Ferguson M.W. Ontogeny of the skin and the transition from scar-free to scarring phenotype during wound healing in the pouch young of a marsupial, Monodelphis domestica. Dev Biol 1995;169:242–60.

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15. Cowin AJ, Brosnan MP, Holmes TM, Ferguson MW. Endogenous inflammatory response to dermal wound healing in the fetal and adult mouse. Dev Dyn 1998;212(3):385–93. 16. Whitby DJ, Ferguson MW. Immunohistochemical localization of growth factors in fetal wound healing. Dev Biol 1991;147(1):207–15. 17. O’Kane S, Ferguson MW. Transforming growth factor betas and wound healing. Int J Biochem Cell Biol 1997;29(1):63–78. 18. Shah M, Rorison P, Ferguson MWJ. 2000 The role of transforming growth factors beta in cutaneous scarring. In: Garg HG, Longaker MT, eds. Scarless wound healing. New York: Marcel Dekker Inc, 2000; 213–26. 19. Cowin AJ, Holmes TM, Brosnan P, Ferguson MWJ. 2001 Expression of TGF-β and its receptors in murine fetal and adult dermal wounds. Eur J Dermatol 2001;11:424–31. 20. Ferguson MWJ, Whitby DJ, Shah M, Armstrong J, Siebert JW, Longaker MT. Scar formation: the spectral nature of fetal and adult wound repair. Plastic Reconstructive Surg 1996;97:854–60. 21. Gawronska-Kozak B. Regeneration in the ears of immunodeficient mice: identification and lineage analysis of mesenchymal stem cells. Tissue Eng 2004;10(7–8):1251–65. 22. Gawronska-Kozak B, Bogacki M, Rim JS, Monroe WT, Manuel JA. Scarless skin repair in immunodeficient mice. Wound Repair Regen 2006;14(3):265–76. 23. Beare AH, Metcalfe AD, Ferguson MW. Location of injury influences the mechanisms of both regeneration and repair within the MRL/MpJ mouse. J Anat 2006;209(4):547–59. 24. Colwell AS, Krummel TM, Longaker MT, Lorenz HP. An in vivo mouse excisional wound model of scarless healing. Plast Reconstr Surg 2006;117(7): 2292–6. 25. Schrementi ME, Ferreira AM, Zender C, Dipietro LA. Site-specific production of TGF-beta in oral mucosal and cutaneous wounds. Wound Repair Regen 2008;16:80–6. 26. Metcalfe AD, Willis H, Beare A, Ferguson MW. Characterizing regeneration in the vertebrate ear. J Anat 2006;209(4):439–46. 27. Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999;2; 341(10):738–46. 28. Su SC, Mendoza EA, Kwak HI, Bayless KJ. Molecular profile of endothelial invasion of three-dimensional collagen matrices: insights into angiogenic sprout induction in wound healing. Am J Physiol Cell Physiol 2008;295(5):C1215– 29. 29. Mori R, Shaw TJ, Martin P. (2008) Molecular mechanisms linking wound inflammation and fibrosis: knockdown of osteopontin leads to rapid repair and reduced scarring. J Exp Med 2008 Jan 21;205(1):43–51. 30. Kirkpatrick JC, Fuchs S, Hermanns IM, Peters K, Unger RE. Cell culture models of higher complexity in tissue engineering and regenerative medicine. Biomaterials 2007;28(34):5193–8. 31. Dallon JC, Ehrlich P. A review of fibroblast-populated collagen lattices. Wound Rep Regen 2008;(16):472–9. 32. Ucuzian AA, Greisler HP. In vitro Models of Angiogenesis. World J Surg 2007;(31):654–63.

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33. Harunari N, Zhu KQ, Armendariz RT et al. Histology of the thick scar on the female, Red Duroc pig: final similarities to human hypertrophic scar. Burns 2006;32(6):669–77. 34. Zhu KQ, Carrougher GJ, Gibran NS, Isik FF, Engrav LH. Review of the female Duroc/Yorkshire pig model of human fibroproliferative scarring. Wound Repair Regen 2007;15 Suppl 1:S32–9. 35. Xie Y, Zhu KQ, Deubner H et al. The microvasculature in cutaneous wound healing in the female Red Duroc pig is similar to that in human hypertrophic scars and different from that in the female Yorkshire pig. J Burn Care Res 2007;28(3):500–6. 36. Bond J, Duncan JA, Sattar A et al. The maturation of the human scar: an observational study. Plast Reconstr Surg 2008;121(5):1650–8. 2008; 121(2), 487–96. 37. Mustoe TA, Cooter RD, Gold MH et al. International clinical recommendations on scar management. Plast Reconstr Surg 2002;110:560–71. 38. Bayat A, McGrouther DA, Ferguson MW. Skin scarring. BMJ 2003;326:88–92. 39. Occleston NL, O’Kane S, Goldspink N, Ferguson MWJ. New therapeutics for the prevention and reduction of scarring. Drug Discovery Today 2008;13(21–22): 973–81. 40. Occleston NL, Laverty HG, O’Kane S, Ferguson MWJ. Prevention and reduction of scarring in the skin by transforming growth factor bea 3 (TGFβ3): from laboratory discovery to clinical pharmaceutical. J Biomater Sci Polym Ed 2008;19(8):1047–63. 41. Ferguson MWJ, Duncan J, Bond J et al. Prophylactic administration of avotermin on skin scarring: three double-blind, placebo-controlled, phase I/II studies. Lancet 2009;373(9671):1264–74. 42. Proetzel G, Pawlowski SA, Wiles MV et al. Transforming growth factor-beta 3 is required for secondary palate fusion. Nat Genet 1995;11(4):409–14. 43. Isoda M, Ueda S, Imayama S, Tsukahara K. New formulation of chemical peeling agent: histological evaluation in sun-damaged skin model in hairless mice. J Dermatol Sci 2001;27 Suppl 1:S60–7. 44. Flanders KC, Major CD, Arabshahi A et al. Interference with transforming growth factor-beta/Smad3 signalling results in accelerated healing of wounds in previously irradiated skin. Am J Pathol 2003;163(6):2247–57. 45. Cho SB, Park CO, Chung WG, Lee KH, Lee JB, Chung KY. Histometric and histochemical analysis of the effect of trichloroacetic acid concentration in the chemical reconstruction of skin scars method. Dermatol Surg 2006;32(10):1231–6. 46. Aarabi S, Bhatt KA, Shi Y et al. Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis. FASEB J 2007;21(12):3250–61. 47. Qiu C, Coutinho P, Frank S et al. Targeting connexin43 expression accelerates the rate of wound repair. Curr Biol 2003;13(19):1697–703. 48. Dang CM, Beanes SR, Lee H et al. Scarless fetal wounds are associated with an increased matrix metalloproteinase-to-tissue-derived inhibitor of metalloproteinase ratio. Plast Reconstr Surg 2003 Jun;111(7):2273–85. 49. Birch M, Tomlinson A, Ferguson MW. Animal models for adult dermal wound healing. Methods Mol Med 2005;117:223–35. 50. Colwell AS, Beanes SR, Soo C et al. Increased angiogenesis and expression of vascular endothelial growth factor during scarless repair. Plast Reconstr Surg 2005;115(1):204–12.

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51. Kloeters O, Tandara A, Mustoe TA. Hypertrophic scar model in the rabbit ear: a reproducible model for studying scar tissue behavior with new observations on silicone gel sheeting for scar reduction. Wound Repair Regen 2007;15 Suppl 1:S40–5. 52. Kryger ZB, Sisco M, Roy NK et al. Temporal expression of the transforming growth factor-Beta pathway in the rabbit ear model of wound healing and scarring. J Am Coll Surg 2007;205(1):78–88. 53. Kim I, Mogford JE, Witschi C, Nafissi M, Mustoe TA. Inhibition of prolyl 4-hydroxylase reduces scar hypertrophy in a rabbit model of cutaneous scarring. Wound Repair Regen 2003;11(5):368–72. 54. Moy LS, Peace S, Moy RL. Comparison of the effect of various chemical peeling agents in a mini-pig model. Dermatol Surg 1996;22(5):429–32. 55. Ulrich MM, Verkerk M, Reijnen L et al. Expression profile of proteins involved in scar formation in the healing process of full-thickness excisional wounds in the porcine model. Wound Repair Regen 2007;15(4):482–90. 56. Gallant CL, Olson ME, Hart DA. Molecular, histologic, and gross phenotype of skin wound healing in Red Duroc pigs reveals an abnormal healing phenotype of hypercontracted, hyperpigmented scarring. Wound Repair Regen 2004;12(3):305–19. 57. Zhu KQ, Carrougher GJ, Couture OP et al. Expression of collagen genes in the cones of skin in the Duroc/Yorkshire porcine model of fibroproliferative scarring. J Burn Care Res 2008;29(5):815–27. 58. Bond JS, Duncan JA, Mason T et al. Scar redness in humans: how long does it persist after incisional and excisional wounding? Plast Reconstr Surg 2008; 121(2):487–96. 59. Bush J, Duncan JAL, Bond JS, Durani P, So K, Mason T, O’Kane S, Ferguson MWJ. Scar-improving efficacy of avotermin administered into the wound margins of skin incisions as evaluated by a randomized, double-blind, placebocontrolled, phase II clinical trial. Plast Reconstr Surg 2010;126(5):1604–15.

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5 Monitoring chronic wounds and determining treatment P. P L A S S M A N N, University of Glamorgan, UK

Abstract: By measuring the physical dimensions of wounds, clinicians receive feedback about the success of a selected treatment approach. This chapter reviews existing wound measurement techniques and highlights underlying principles and difficulties. As an example the performance of a new hand-held, non-contact, wound measurement device is analysed. The colour of a wound provides important clues about its status to the clinician. It can be used to assess the distribution of tissue types and potentially detect the onset of infection at an early stage. The underlying fundamentals of colour measurements by digital cameras are outlined and an example classification system is presented. Key words: wound measurement, wound colour measurement, classification engines, stereo-photogrammetry.

5.1

Introduction

The management of chronic wounds such as those found in diabetic feet or leg ulcers is placing an increasing burden on health service systems (Krouskop et al., 2002). Although the underlying aetiology of a chronic wound is usually known to the wound healing specialist, there is a wide variety of treatment regimes available (Graumlich et al., 2003). The measurement of wound dimensions and colour both have the potential for assisting the correct treatment approach and thus improve clinical practice, assist in trials and reduce the risk of litigation. Even for skilled and experienced practitioners the only reliable way of establishing if a wound is responding to a chosen treatment is often to objectively and repeatedly measure the wound size. Chronic wounds, however, have an unfortunate tendency of healing very slowly. Months or years rather than days or weeks are common for the healing process. Any measurement technique used for the purpose of establishing healing progress therefore has to be very precise in order to capture small changes in a wound’s dimensions. The first part of this chapter is dedicated to current measurement practice and recent developments in this area. 130 © Woodhead Publishing Limited, 2011

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A common complication in the wound healing process is infection, which, once established, is painful and may last several weeks. During this time healing is severely compromised and the size of the wound usually increases. Any approach capable of detecting the onset of an infection at an early stage should therefore improve both patient care and healing time. There is an obvious relationship between wound colour and the inflammation caused by infection. This relationship is studied in the second part of this chapter.

5.2

Wound size measurements

5.2.1 Motivation for wound size measurements Many wounds such as leg ulcers can take several months to heal, and may recur at a later date. One way to improve both the healing of an individual wound and clinical practice, in general, is to continuously monitor wound status, e.g. its area, volume, circumference but also its colour. Because of the slow healing rate of chronic wounds, dimensional measurements on their own show only past changes in the healing rate and can therefore allow only reactive but not proactive treatment. The treatment of leg ulcers has been estimated to cost the UK’s National Health Service in excess of £300 million (approx. US $450 million) annually (Davis et al., 1992, p123). Treatment of these wounds is a matter of clinical experience and, in the past, it was often regarded as a process that could be monitored subjectively. The ever-increasing amount of interventions available to the physician today, however, make it necessary to closely monitor the lesion with objective means in order to identify the most effective treatment regime at every phase of the healing process. This need is emphasised by the trend towards evidence-based medicine and increasingly enforced by health service regulators and insurers. This in turn leads to the need of applying best practice and, failing that, running the risk of litigation in clinical malpractice suits.

5.2.2 Definitions An important factor in monitoring wound size is the question of how to define critical descriptors such as area, volume and circumference. At this point it is worthwhile to first define two parameters which are critical for all types of measurement: •

Accuracy. In the context of this chapter accuracy is defined as the offset (or bias) of a measured entity with respect to a true and known value. • Precision. This is defined as repeatability. It is usually expressed as the standard deviation of several repeated measurements of an entity. In

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order to make the precision values dimensionless (i.e. scale invariant) the standard deviation is usually expressed as a percentage of the mean of the measurements made. Circumference Intuitively, the circumference of a wound appears to be a straightforward and well-defined measure. In reality, however, the circumference of a wound is a fractal. It is an infinitely long curve with properties similar to those of a Koch curve (von Koch, 1904). Its length is scale variant, i.e. the length depends on the magnification of the wound image: the greater the magnification, the more details of the borderline can be followed and the longer the circumference becomes. This effect is identical to that created by the length of the line used for delineation. In computerised applications the outline is always made up from sections of straight lines with defined unit length. As this unit length approaches zero (which it can never fully reach) the line becomes increasingly undulated and thus longer. The measurement of circumference is therefore only meaningful if the magnification factors of images and the line drawing parameters are kept constant. As a consequence of the fractal nature of a wound’s boundary a ‘true circumference’ of a wound does not exist, i.e. it is impossible to quote figures for system accuracy. Therefore the performance of a circumference measurement device is also best described by precision (i.e. repeatability). Area In classical 2-dimensional photography the naturally curved area of a wound is projected onto a flat imaging sensor. This results in an apparent reduction of the measured area. A common approach to counteract this perspective reduction is to model the shape of the wound site, e.g. a leg as a cylinder, and to retrospectively correct the apparent size. In order to minimise the subjective aspect of this process (e.g. manually aligning a 2D wound photograph on the cylinder model) semi-3D systems have been developed that determine the shape of the wound site and automatically align the 2D photograph on the mathematically derived 3D site model. An example is the ‘Silhouette’ system manufactured by Aranz Medical Ltd in New Zealand (Jull et al., 2009). In 3-dimensional systems the problem of perspective distortion does not exist as the third dimension is integrated into the measurement process. While in these systems the fractal boundary problem reappears, the area does not approach infinity but asymptotically converges towards a true value. Accuracy figures are therefore again meaningless without keeping magnification factors of images and line drawing parameters constant. Precision is again a more suitable parameter to characterise system performance.

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Volume The volume of a wound is the space enclosed between the wound surface and the former healthy skin surface. It can in principle be determined to any degree of accuracy with the resulting figure approaching asymptotically the true value as magnification increases. In practice, however, a complication arises from the need to reconstruct or interpolate the former healthy skin surface. Since this surface has disappeared there is no way to verify a measurement result against a known ‘true’ value and no meaningful accuracy figure can be calculated, again leaving precision as the only useful parameter for describing system performance on wounds. System accuracy on well-defined models can, however, be determined if there is a known volume to compare against. In an experiment performed at the dimensional measurement facility at the UK’s National Physical Laboratory the test volume shown in Fig. 5.1 was scanned using a high performance 3D surface measurement machine under strictly controlled laboratory conditions (temperature stability, vibration proofing, calibrated against national standard, etc.). The scan resulted in a point cloud of one x, y and z position for every square millimetre of the model’s surface. A least square error fit of two shapes, sphere and paraboloid, was then applied to this data cloud to model

5.1 One of 12 calibration artefacts produced by the National Physical Laboratory (UK).

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the ‘wound bed’ and a perfect plane was used to model the ‘former healthy skin’ surface. The resulting two volumes differed by 1.5%. This figure represents the upper limit of volumetric measurement accuracy currently available under ideal laboratory conditions on static models. In reality factors such as wound flexibility, the need to reliably reconstruct the skin surface and the inferior performance of mobile scanners produce significantly worse results.

5.2.3 Development of wound measurement techniques Length and area measurements For length and depth measurements, devices such as the Kundin ruler (Kundin, 1989) are normally the preferred method, while the current ‘gold standard’ for area measurement is the practice of tracing the perimeter of a wound through a double layer of a flexible transparent sheet material (Keast et al., 2004). While the layer in contact with the wounds is discarded, the upper layer with the tracing is then measured by either placing it on metric graph paper, planimeters or by a second round of tracing using a digitising tablet (Thawer et al., 2002). This contact making method can be painful to the patient and may risk infection. In practice the use of unsuitably thick or exhausted marker pens and the high degree of dexterity required by the clinician performing the tracing significantly reduce the theoretical precision of the method (Lagan et al., 2000) and errors up to 25% have been reported (Bulstrode et al., 1986). Several attempts have therefore been made to replace the data capture phase by digital photography or video, which has the advantage of avoiding contact with the patient (Stacey et al., 1991 and Smith et al., 1992). Wound pictures are usually measured on a computer screen where they can also be enlarged so that the tracing process is freed from time restrictions and physical demands. Although area measurements produced this way tend to be more precise than those obtained by transparency tracings, the method depends on the photography skills of the clinician who has to combine a frame-filling wound picture together with a reference scale in a single image taken perpendicular to the wound site. Results are only correct if the underlying assumptions that wound and reference scale are at the same distance to the camera and that the wound site itself is flat are met (Solomon et al., 1995). Volume measurements Wounds with a significant volume have a tendency of healing first from their base before decreasing in area. At early healing stages the area might

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actually increase, although the volume is decreasing. By just making area measurement, earlier stages of the healing process may therefore be missed or misrepresented. The current gold standard for volume measurements is the practice of filling the wound with a paste like material based on alginates or silicone (Stotts et al., 1996). These pastes usually set to a flexible and non-sticking cast within a few minutes so that they can be removed from the wound and measured by either using a water displacement approach or, if the density of the material is controlled, by weighing them on precision scales. The precision of the method depends on the manual dexterity of the clinician applying the paste who has to shape the outside of the cast into a form that resembles the original healthy skin surface as closely as possible while the paste is in the process of setting. An alternative approach is the practice of covering the wound with a layer of self-adhesive transparent sheet material and to measure the amount of saline required to fill the cavity between the wound bed and the film using a calibrated syringe (Berg et al., 1990). Due to the tendency of wounds to absorb saline and the difficulty of producing the right amount of curvature in the covering sheet material this method is not only less accurate than the cast method but also significantly less hygienic, as the contaminated saline may spill out when the film is removed. In the past a number of attempts have therefore been made to design and build wound measurement instruments that are non-contact, precise and user friendly. Amongst these were laser scanners (Ibett et al., 1994), structured light-based instruments (Krouskop et al., 2002 and Plassmann et al. 1995) and stereo-photogrammetric devices (Langemo et al., 2001 and Boersma et al., 2000). These were either user-friendly but not very precise, or precise but relatively cumbersome and difficult to use instruments. Based on recent advances in digital imaging hardware and stereophotogrammetry software the author’s group have now produced a new instrument that combines the advantages of the best traditional approaches (non-contact, light-weight, mobile, visual record, easy to use, fully volumetric) with high precision and accuracy. This instrument is described in the following section.

5.2.4 Example system: the Measurement of Area and Volume Instrument System (MAVIS) measurement device Instrument description The MAVIS design (an acronym for Measurement of Area and Volume Instrument System) is based on the principle of stereo-photogrammetry. Two laterally displaced images are recorded in a process not dissimilar to

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5.2 The MAVIS-III instrument: camera, inverse cross-polarised flash, 3D lens and dual light point projector.

that of the human visual system where the left and the right eye produce 3D scene information. The amount of lateral displacement of a given point in an observed object or scene is a direct measure for the distance between that point and the camera’s focal point. Fig. 5.2 shows the instrument assembly, which consists of four main components: • • •

A good quality SLR camera with a resolution of 12 mega-pixels or more delivered by a low noise imaging array with high dynamic resolution. A dual flash with two polarising filters orientated at 90 degree angle towards each other. A stereo lens adapter that produces the two laterally displaced images via a set of mirrors. The adapter is equipped with two more polarising filters orientated at an 90 degrees angle to each other and also at 90 degrees with respect to the filters in the flash. This combination has been dubbed ‘inverse cross polarisation’ (patent pending). Rather than reducing specular reflection by using the standard cross polarisation approach used in many photographic applications (i.e., two polarisation filters attached to flash and lens rotated by 90 degrees towards each other) this

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four-filter design actually uses the specular reflections to enhance the 3D imaging results. • A targeting aid for the user is provided by a dual light point projector at the base of the unit. The projector produces two beams of light that intersect at the centre of the field of view and guarantees fully focused wound images.

Taking images The instrument is about 1kg in weight and powered only by batteries to make it safe and highly portable. All photography parameters (distance, exposure time, ISO equivalent setting, etc.) are pre-set so that the instrument is ready to take images in a few seconds as soon as the flash is fully charged. The user activates the targeting projector, aligns both its light beams in the middle of the wound to be measured to ensure that the wound is centred in the image and taken within a tolerance distance of ±10 cm. After ensuring that the projector beam spots are as circular as possible (if they are elliptical the instrument’s orientation towards the wound is not perpendicular, which may result in decreased accuracy and precision of measurements) the user releases the camera’s trigger to capture a stereo wound image. On a typical 1GB memory card the camera can store approximately 200 stereo images.

Making measurements The images are transferred from the camera to a PC or laptop by the MAVIS measurement program, which automatically recognises the presence of the camera’s USB connector and starts the downloading process. Aided by an image pan and zoom facility, users can then start to measure individual stereo images by delineating the perimeter of wounds using the computer’s mouse. The remainder of the measurement process is fully automatic and results after approximately 1 minute in a fully rendered 3D map and representation of the wound site as shown in Fig. 5.3 with the following measurement data available for storage in the software’s patient and wound data base: • •

the 3D representation of the wound the true wound surface area (in contrast to the projected area in traditional photographic wound measurement techniques) • the true circumference of the wound (again in contrast to the projected perimeter provided by planar 2D photography) • the volume of the wound.

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(c)

(b)

5.3 Stereo image of an excision wound (a) taken by the MAVIS camera, 3D mesh of the wound surface (b) and minimum surface mesh enclosing the wound volume (c).

As already mentioned above (Section 5.3.2) the volume of the wound is not ‘true’ as a plausible definition is difficult to achieve. A repeatable volume is used instead and defined as the space enclosed between the measured wound surface and a minimum surface supported by the perimeter of the wound as shown in Fig. 5.3(c). This minimum surface can be envisaged as a piece of virtual flexible rubber membrane that is pinned into place at the wound’s perimeter. Apart from being computationally more robust and producing more repeatable results than other surface reconstruction approaches, this definition has the added advantage that protruding or overgranulating tissue inside the wound can not produce ‘negative volume’. MAVIS instrument performance A set of 12 calibration bodies were produced by the UK’s National Physical Laboratory (NPL). These artefacts were produced to tight tolerance limits and dimension were validated using a high precision 3D stage measurement machine at the NPL. The 95% confidence limit (approximately 2 standard deviations) in the accuracy of all three parameters, area, volume and circumference, of the artefacts is smaller than 1% for all 12 bodies. An example is shown in Fig. 5.1, the distribution of artefact areas and volumes is shown in Fig. 5.4. Each of the 12 artefacts was photographed 10 times under deliberately poor conditions (i.e., hand-held, varying ambient lighting, random flash

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35 4B

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30

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25 7A

20

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6A

15 10

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5 0

9B

8B 12B

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6 8 Volume [cm3]

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5.4 Area and volume of the 12 calibration artefacts. Dimensions of artefacts used for calibration (triangles) are located on the perimeter of the scatter plot gamut while the control ones (circles) are on the inside.

charges times, instrument used by untrained volunteers after only two minutes of minimum instructions) in order to simulate a real life worst case scenario. The standard deviations of each set of 10 measurements provide a measure for the precision of measurements and are plotted in the graphs of Fig. 5.5. As expected from previous studies (Plassmann and Jones, 1998) the precision of all measurements decreases as the respective dimensions become smaller. In practice this is not a problem as small wounds (less than approximately 1 cm3 in volume with an area of less than 5 cm2) have already progressed significantly and can be expected to heal quickly. Fig. 5.6(a) compares the area accuracy of the instrument with that of other approaches and Fig. 5.6(b) demonstrates that its precision is equivalent to that achieved by other 3D scanners such as laser or structured light scanners.

5.3

Wound colour measurements

5.3.1 Motivation for wound colour measurements Tissue classification Segmentation approaches to wound colour measurement are usually based on manual/visual tissue classification tools such as the RYB (red, yellow, black) or the extended BRYP (black, red, yellow, pink) model (Cuzzell, 1988). A similar system is used by the PUSH tool (Pressure Ulcer Scale for Healing (Berlowitz et al., 2005) where the distribution of the four main tissue types commonly found in healing chronic wounds (epithelial, granulation, sloughy and necrotic) is visually assessed. The European Pressure Ulcer Advisory Panel in its 2004 Statement (Defloor et al., 2005)

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Std.Dev. [% of Area]

(a) 2.00% 1.50% 1.00% 0.50% 0.00%

0

5

10

15

20

25

30

35

10

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Area [cm2]

Std.Dev. [% of Volume]

(b) 10.00% 8.00% 6.00% 4.00% 2.00% 0.00% 0

2

4

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8

Volume [cm3]

Std.Dev. [% of Circumference]

(c)

5.00% 4.00% 3.00% 2.00% 1.00% 0.00% 0

5

10

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20

Circumference [cm]

5.5 Precision of area (a), volume (b) and circumference (c) measurements expressed as 1 standard deviation of the respective value. Diamonds represent the ideal case (laboratory conditions), circles are results of measurements made by untrained volunteers (worst case scenario).

suggests colour as one of the factors useful for the diagnosis and treatment of ulcers. Wound tissue segmentation based on colour is therefore directly addressing a clinical need. As already pointed out above, however, visual inspection is subjective and inaccurate with low inter-operator repeatability (Beekman et al., 2007).

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(a) 20

15

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0

rulers

tracings

photography

MAVIS

(b) min. precision (large and shallow wounds)

40 35 30 25 20 15 10 5 0

max. precision (small and deep wounds) rulers

saline

casts

3D scanners MAVIS

5.6 Typical precision values of wound area (a) and volume (b) measurement techniques quoted in literature compared with those of the MAVIS stereo-photogrammetry device. Figures expressed as 1 standard deviation of the respective value.

Detection of infection Repeated episodes of infection are a common complication in the treatment of chronic wounds; approximately half of all wounds undergo at least one episode of infection. Each episode can delay the progress of healing by several weeks. Anecdotal evidence from clinicians suggests that an infected wound looks ‘angry’, while a well-progressing lesion shows ‘nice’ pink granulation tissue. If a wound is presenting with ‘angry’ colours an infection is usually the underlying cause. Unfortunately, it is usually only at the late stage of an infection when colour changes are obvious and the patient is reporting pain, that treatment of the underlying problem commences. If the presence of

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infection could be detected at onset, wound deterioration could potentially be avoided, reducing treatment cost and patient discomfort. In this context it is important to note that although inflammation correlates with infection, it is not necessarily the sole underlying cause. In the first stages of a normal healing process, for example, inflammation is an expected reaction and part of the wound healing cascade. Other mechanisms such as irritation due to chemical or physical stimulation can also cause inflammation. Conversely, infection does not always cause inflammation if, for example, the body’s defence mechanisms are compromised. Finally, infection should not be confused with bacterial or viral colonisation of the wound. This is normal and it is only a cause for concern when the natural balance of micro-organisms is disturbed by one particular agent multiplying aggressively that the term infection is appropriate. Bearing this in mind it is likely that an image processing technique capable of identifying and classifying subtle colour changes should be able to detect the onset and presence of inflammation in a chronic wound at an early stage and should therefore be a powerful tool for facilitating proactive wound treatment.

5.3.2 Lighting Correct illumination is important for wound colour assessment. Light sources with different hues can significantly alter the appearance of a wound, while specular reflections (highlights) may render parts of it unsuitable for subsequent analysis. In principle, there are two opposing ways of dealing with the effects of lighting: the first is to control the illumination of the site as closely and as repeatably as possible, the second is not to control it at all but to include a reference object with known reflectance properties into the scene. Two examples of these two methods are outlined in the following sections.

Controlled lighting An ideal light source for the controlled lighting approach should either be significantly brighter than any other ambient light source (incandescent lamps, strip lighting, daylight through windows, etc.) or block out ambient light entirely. Blocking out ambient light by using, for example, an enclosed light box is technically the better approach, as it allows the system designer to produce extremely smooth (lambertian) and shadow-free light of constant and known colour. In practice, however, this solution is generally infeasible due to the physical size of such a lighting enclosure. The device

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also needs to be as close to the wound as possible, possibly touching the surrounding tissue, risking injury or infection. Therefore the first approach with an extremely bright light source is usually preferred. In practice this source is usually a flash gun (Lagan et al., 2000). In contrast to incandescent lamps, these guns have a colour temperature close to daylight, and in contrast to fluorescent lamps or LED lighting, they have a constant emission spectrum. This emission spectrum also tends to be constant over time with only marginal yellowing of the flash bulb after several thousand shots. Flash guns, however, have one significant disadvantage: they are point light sources. Such sources produce strong specular reflections, especially in moist areas of the wound. Specular reflections are so bright that in their direct location, information about the wound can no longer be obtained, and in their neighbourhood, information is significantly compromised by the ‘bleaching’ of all colours into white. Other data such as wound boundary definition is usually lost as well. A possible solution to this problem is to aim the light of the flash gun not at the wound but at a large reflector panel, which in turn reflects it onto the wound area. This again makes the photography setup large, bulky and difficult to transport. Another common approach to reduce specular reflection is the use of a set of two polarising filters oriented at 90 degrees towards each other (McFall, 1996). The first filter is mounted in front of the flash lamp. This will allow only, say, horizontally polarised light through to illuminate the wound. Specular reflection does not change the polarisation angle and light reflected from these areas can therefore be blocked off by using a second, now vertically polarising, filter that is mounted in front of the camera lens. Nonspecular, (i.e. lambertian) reflection, in contrast, does change the polarisation angle of the reflected light and consequently some of it will be able to pass through the filter in front of the camera lens. This double filter arrangement blocks out the majority of the light produced by the flash lamp so that a powerful gun is usually employed in this technique. While for computerised colour analysis this technique delivers excellent results, this is unfortunately not the case for the purpose of subjective inspection or visual representation. In images taken under cross-polarised lighting conditions skin appears waxen and devoid of the small micro highlights that under normal lighting conditions generate textural information about surface smoothness or roughness for the observer. This disadvantage can be overcome by taking a second photograph of the wound with at least one of the polarising filters removed. Uncontrolled lighting In this technique the photographer relies on ambient lighting alone. Coming from several sources and being reflected multiple times from walls and

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ceilings, this light tends to be more distributed than flash gun light and therefore causes less specular reflection. On the other hand, it is clear that the colours of the light sources and those of reflecting objects will change the appearance of the wound in an unknown way. The solution to this problem is to include a reference object such as the MacbethTM colour checker chart (McCanny et al., 1976) used, for example, by van der Haegen et al. (2006). Provided the assumption of homogeneous illumination over the wound area is correct (e.g. no shadows or light with different hues from different directions) the precisely known properties of the colour chart allow the restoration of the true wound colours to the limits of discernibility for a human observer with the ΔE reduced to 3.5 ± 2.9 (Wannous et al. 2008). ΔE is defined in the CIE 1976 L*a*b* colour space as any ΔE less than 1.0 being imperceptible (ISO 11664-4:2008, Colorimetry, Part 4). Apart from the assumption of homogeneous illumination this approach relies on highly precise colour reference charts which may have to be discarded after use in order to avoid cross-contamination. At a price of several tens of pounds/dollars per chart, this may be uneconomical. By using a reference chart not all of the available photograph area can be used to record the wound, resulting in a reduced definition of wound features. In the case of a 24 colour mini MacbethTM chart an area of approximately 8 cm × 6 cm is lost. On the other hand, however, the known dimensions of the chart allow the image to be scaled for dimensional analysis. An additional advantage of using an external reference chart is that display and printing devices can be retrospectively calibrated from the photographs taken to produce true colour output.

5.3.3 Digital cameras Early digital cameras could not measure colour accurately enough and in the past, wound colour has therefore been measured using colorimeters mainly developed for the printing industry (van Zuijlen et al., 2002). While being highly precise, these devices integrate the colour of an object over an area of varying size. If this area is large, these instruments miss details in the highly inhomogeneous and detailed colour patterns of wounds. If the area is small, the measurement process requires the operator to manually scan different areas of the wound, which makes the production of a spatial distribution map difficult. Fortunately, the technological progress in digital camera technology nowadays allows for fast measurement of colour at high spatial resolution and to the limits of discernibility by a human observer. In contrast to common perception the amount of pixels in a digital camera is not the defining criterion for good quality. For colour processing the camera’s dynamic range, pixel noise and optical aberrations are signifi-

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cantly more important. An introduction to CMOS sensor technology is provided by Fossum (1997). Briefly: •

•

•

Dynamic range describes the ratio between the maximum and minimum light intensities (i.e. white and black) that a camera’s sensor array can process. In a coloured object, for example, the green colour channel may already be clipped to the maximum, while the red and blue channels are not. This will result in an apparent colour shift (towards purple) that will distort all subsequent image analysis steps in this area. Pixel noise adds or subtracts random amounts of light intensities individually to each of the camera’s pixels. If a red sensitive pixel has a certain amount of intensity added, while its neighbouring green and blue sensitive pixels do not (or even have intensity removed), then the resulting colour impression in this area will shift towards green. In low quality cameras, especially under low lighting conditions, this effect produces grainy images. Optical aberrations are produced by the lens system. Low quality lens systems tend to produce chromatic aberrations. These change the colour of objects, especially at edges or boundaries. Professional ‘achromatic’ lens systems are virtually free of aberrations.

In order to minimise the above effects professional or semi-professional SLR cameras should be used for wound colour imaging. These cameras also offer the photographer the option of saving images in unprocessed and uncompressed formats (RAW format) rather than one of the popular compressed formats that inevitably introduce artefacts into the image. JPG compression artefacts, for example, are visually unobtrusive and hardly noticeable, but can produce undesired results in computerised image analysis.

5.3.4 The meaning of wound colour An underlying problem of colour metrics is not the measurement of colour itself but to correlate a particular wound colour with a particular ‘meaning’. In some instances meaning can be objectively verified (e.g. infection), although work by Basak et al. (1992) has shown that a culture from a wound swab occasionally produces different outcomes from that of a biopsy. In other instances the correlation is based on subjective human judgement (e.g. inflammation). In these cases a common approach to minimise the influence of individual subjective judgements is to use a cohort of individuals and repeatedly ask them to perform a particular classification task. The performance of a computerised colour classification system with respect to the average judgement of all human classifiers is then expressed in a Kappa (κ) value:

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5.1

where P0 stands for the agreement between judges and Pe for the expected random agreements under the Null Hypothesis that judges classify purely at random. The use of the Kappa value has been reported in previous work on wound colour segmentation (Oduncu et al., 2004). For details refer to Cohen’s (1960) original work. Kappa values can also be used to construct a ‘true’ benchmark or ‘super clinician’ against which both individual clinicians and classification engines can be measured. This ‘super clinician’ could be constructed using, for example, a weighted averaging process where the weights applied to individual clinician’s judgements are a function of their closeness to the median of all judgements. Colour classification systems can be broadly separated into systems using either segmentation or a holistic approach.

5.3.5 Approaches Segmentation approach Segmentation approaches attempt to automate the task of tissue classification in order to automate the before mentioned (see Section 5.3.1) PUSH, RYP or BRYB tools. Wannous et al. (2007) provide an overview into these approaches, and a recent paper by Veredas et al. (2010) outlines a novel hybrid approach based on neural networks and Bayesian classifiers for automatic tissue identification in wound images. The paper claims that this approach results in high performance rates with an average sensitivity of 78.7%, a specificity of 94.7% and an accuracy of 91.5% when detecting the varying tissue types. Holistic approach In the holistic approaches the entire wound area is treated as a single entity and a colour histogram of the entire wound area is produced. This histogram is then analysed with respect to a set of so-called ‘features’ that describe its shape and position. These features are then correlated with clinician’s judgement (e.g. granulating, necrotic, slough, inflamed) in order to extract meaning from the data. Correlation may be performed by computerised classification system such as artificial neural networks (ANNs), regression analysis or support vector machine (SVMs). Generally, however, the learning data base has to be large enough otherwise the generalisation capabilities of the classification algorithm may be compromised (Jain et al., 2000). A colour histogram is an approximation of the colour distribution and as shown in Fig. 5.7 may be considered as Gaussian. Therefore, standard

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inflamed

0.6 0.5 non-inflamed 0.4 0.3 0.2 0.1 0.0

yellow

pink

red

purple

5.7 Typical (idealised) hue distribution for inflamed and non-inflamed wounds in the vicinity of the red part of the visible spectrum.

descriptors can be used to define the respective departure from the normal curve in characterising wound colour distribution. These descriptors (or statistical moments) are briefly outlined below. Mean and median The mean of the histogram represents the centre point of the distribution, separating the histogram into two equally probable subsets. The median is similar, but not identical to the mean. It splits the range of the histogram into two equal sample sizes regardless of the probability. This can be an advantage over the mean in cases where the extreme probability would be artificially increased due to clipping. It is apparent from Fig. 5.7, that neither the mean nor the median of the chromaticity distribution alone are adequate to discriminate between inflamed and non-inflamed wounds. Standard deviation Standard deviation (std) represents the dynamics of the histogram, how wide around the mean the colours of the wound image are distributed. Non-inflamed wounds have a higher standard deviation than those which are inflamed (see Fig. 5.7).

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Kurtosis Kurtosis quantifies the flatness level of the distribution at the mean. Again, the expectation from Fig. 5.7 is that the kurtosis for non-inflamed wounds should be smaller than that for inflamed wounds. Kurtosis is equal to three for a normal distribution.

5.3.6 Example system: detection of wound inflammation In an attempt to develop a system for the early detection of wound infection the author’s group examined whether the colour of two wound tissue types (granulation and sloughy), would allow an automatic classification into ‘inflamed wound’ and ‘not inflamed wound’ (in: Schaefer et al. 2009, p89– 113). The underlying idea was that wound infection often goes hand in hand with inflammation and thus colour change. The above four statistical moments were used in four colour spaces with three colour channels in three colour domains: L*u*v*, L*C*h* and L*s*h*. This resulted in a total of 37 possible inputs or features for three classification systems under test. (There are not 48 = 4 × 4 × 3 features because of the duplication of L* and h* channel in the three colour domains.) Obviously each of these inputs could also be combined with each other to produce an additional input. If each input is combined with each other input this would result in a total of 666 possible combinations. The number of combinations is even higher if more than two inputs are combined. If four features of the possible 37 are combined this would result in 66045 possible unique combinations. This is the so called ‘curse of dimensionality’ common in classification systems. The amount of inputs was therefore reduced using a ‘brute force’ computational approach (i.e. testing each combination against all others). This resulted in the suggestion that for granulation tissue based classification the kurtosis of chroma C* performs most effectively, for slough tissue based classification the skewness of the u* channel and for the combination of both tissue types kurtosis of L* perform best. These four top features were used to test their respective performance on three competing classification engines based on logistic regression, a artificial neural network (ANN) and a support vector machine (SVM). Table 5.1 summarises the results together with results for the nine clinicians participating in the study. In some cases the SVM performs better than the most skilled clinician. This means that the classification of the SVM is in better agreement (i.e. having a higher kappa value in Table 5.1) with the mean opinion of the most skilled individual clinicians. In most wounds, the SVM machine is close to the top of the performance table. It is not considered a perfect decision but it is consistently better than most clinicians and could therefore be part of a future expert training system.

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Table 5.1 Comparison of logistic regression, ANN and SVM against clinician performance Individual/Tissue type

Clinician

1 2 3 4 5 6 7 8 9 Mean clinician

0.097 0.178 0.199 0.331 0.337 0.532 0.615 0.677 0.709 0.683

Logistic regression

Granulation Slough Both combined

0.509 0.531 0.127

ANN

Granulation Slough Both combined

0.282 0.196 −0.018

SVM

Granulation Slough Both combined

0.592 0.717 0.055

Super-clinician benchmark

5.4

κ

Clinician/System

1.000

Background material

5.4.1 Wound colour A general introduction to the concepts of colour measurement, calibration and processing is given in Sangwine and Horne’s ‘Colour Image Processing Handbook’ (1998). The IEEE publication ‘Colour Processing in Digital Cameras’ (Adams et al., 1998) provides information on the particular advantages and disadvantages of using colour information derived from digital cameras. This should be read in conjunction with Pointer’s practical guide on camera characterisation (Pointer et al., 2001) and Cutting and Harding’s (1994) work on the connection between colour and infection.

5.4.2 Wound size The current ‘gold standard’ for measuring wound volume is the practice of filling the wound cavity with a viscous material that hardens relatively quickly, e.g. an alginate. This was described first by Berg et al. (1990). An overview of a variety of measurement methods and their respective

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performance is given by the author (Plassmann et al., 1994) and also by Thawer et al. (2002). A number of computerised wound measurement devices have appeared in recent years. Due to the high startup costs (R&D, regulatory compliance, etc.), however, these devices are generally expensive and manufacturers find it difficult to achieve profitability. The VERG photogrammetric system produced by Vista Medical Ltd (Winnipeg, Canada), for example, has been discontinued in November 2009. Aranz Medical Ltd (Christchurch, New Zealand) manufactures the PDA based ‘Silhouette’ system which produces a 3D approximation (but not a complete 3D map) of a wound using fixed laser stripe projectors. Provided the skin surface surrounding the wound is continuous (e.g. at the lower leg) this instrument calculates reasonably precise area and circumference results. Volume figures are derived from a single cross-section of the wound and are approximations. Eykona Technologies Ltd. (Oxford, UK) has announced a new device that makes use of a ‘shade from shading’ approach where several images of the same object are merged into a single 3D representation. This technology can reproduce fine surface details in high quality. At the time of writing this device was not yet commercially available. The author’s group has founded Photometrix Imaging Ltd (Pontypridd, UK). The company produces the MAVIS system outlined in this chapter.

5.5

References

Adams J, Parulski K, Spaulding K, 1998, Color Processing in Digital Cameras, IEEE Micro, 18(6), 20–30, ISSN:0272-1732 Basak S, Dutta SK, Gupta S, Ganguly AC, 1992, Bacteriology of wound infection: evaluation by surface swab and quantitative full thickness wound biopsy culture. Journal of the Indian Medical Association, 90, 33 Beeckman D, Schoonhoven L, Fletcher J, Furtado K, Gunningberg L, Heyman H, Lindholm C, Paquay L, Verdú J, Defloor T, 2007, ‘EPUAP classification system for pressure ulcers: European reliability study’, J Adv Nurs., 60(6), 682–691 Berg W, Traneroth C, Gunnarsson A, Lossing C, 1990, ‘A method for measuring pressure sores’, Lancet, 335, 1445–1446 Berlowitz DR, Ratliff C, Cuddigan J, Rodeheaver G, 2005, ‘National Pressure Ulcer Advisory Panel. The PUSH Tool: A Survey to Determine Its Perceived Usefulness’. Advances in Skin & Wound,18(9), 480–483 Boersma SM, van den Heuvel FA, Cohen AF, Scholtens REM, 2000, ‘Photogrammetric Wound Measurement with a Three-Camera Vision System’, Proceedings of the XIXth Congress of International Archives of Photogrammetry & Remote Sensing, Amsterdam, Netherlands, 84–91 Bulstrode CJK, Goode AW, Scott PJ, 1986, ‘Stereophotogrammetry for measuring rates for cutaneous healing: a comparison with conventional techniques’, Clinical Sci, 71, 437–443

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Cohen J, 1960, ‘A coefficient of agreement for nominal scales’, Educational and Psychological Measurement, 20(1), 37–46 Cutting KF, Harding KG, 1994, ‘Criteria for Identifying Wound Infection’, Journal of Wound Care, 3(4), 198–201 Cuzzell JZ, 1988, ‘The New RYB Color Code’, American Journal of Nursing, 88 (10), 1342–1346 Davis MH, Dunkley P, Harden RM, Harding K, Laidlaw JM, Morris AM, Wood RAB, 1992, The Wound Programme, Centre for Medical Evaluation, University of Dundee Defloor T, Schoonhoven L, Fletcher J, Furtado K, Heyman H, Lubbers M, Lyder C, Witherow A, 2005, ‘Statement of the European Pressure Ulcer Advisory PanelPressure Ulcer Classification: Differentiation Between Pressure Ulcers and Moisture Lesions’, Journal of Wound, Ostomy and Continence Nursing, 32(5), 302–306 Fossum ER, 1997, ‘CMOS Image Sensors: Electronic Camera-On-A-Chip’, IEEE transactions on electronic devices, 44(10), 17–25 Graumlich JF, Blough LS, McLaughlin RG, Milbrandt JC, Calderon CL, Agha SA, Scheibel LW, 2003, ‘Healing pressure ulcers with collagen or hydrocolloid: a randomized, controlled trial’, J Am Geriatr Soc, 51, 147–154 Ibbett DA, Dugdale RE, Hart GC, Vowden KR, Vowden P, 1994, ‘Measuring leg ulcers using a laser displacement sensor’, Physiol Meas, 15, 325–332 Jain AK, Duin RPW, and Mao J, 2000, ‘Statistical Pattern Recognition: A review’, IEEE Transactions on pattern analysis and machine intelligence, 22(1), 4–37 Jull A, Parag V, Walker N, Maddison R, Kerse N, Johns T, 2009, ‘ The PREPARE Pilot RCT of Home-based Progressive Resistance Exercises for Venous Leg Ulcers’, Journal of Wound Care, 18(12), 497–503 Keast DH, Bowering CK, Evans AW, Mackean GL, Burrows C, D’Souza L, 2004, ‘MEASURE: A proposed assessment framework for developing best practice recommendations for wound assessment’, Wound Repair and Regeneration, 12, 1–17 Krouskop TA, Baker R, Wilson MS, 2002, ‘A non contact wound measurement system’, J Rehabil Res Dev, 39, 337–346 Kundin JI, 1989, ‘A new way to size up wounds’ American Journal of Nursing, 2, 206–207 Lagan KM, Dusoir AE, McDonough SM, Baxter GD, 2000, ‘Wound measurement: the comparative reliability of direct versus photographic tracings analyzed by planimetry versus digitizing techniques’, Arch Phys Med Rehabil, 81, 1110–1116 Langemo DK, Melland H, Olson B, Hanson D, Hunter S, Henly SJ, Thompson P, 2001, ‘Comparison of 2 wound volume measurement methods’, Adv Skin Wound Care, 14, 190–196 McCamy CS, Marcus H, Davidson JG, 1976, A Color-Rendition Chart, Journal of Applied Photographic Engineering, 2, 3, 95–99 McFall K, 1996, ‘Photography of dermatological conditions using polarized light’, Journal of Visual Communication in Medicine, 19(1), 5–9 Oduncu H, Hoppe A, Clark M, Williams RJ, Harding KG, 2004, ‘Analysis of skin wound images using digital color image processing: a preliminary communication’, Int J Low Extrem Wounds, 3(3), 151–156 Plassmann P, Melhuish JM and Harding KG, 1994, ‘Methods of measuring wound size: a comparative study’. Wounds: A compendium of clinical research and practice, 6(2), 54–61

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Plassmann P, Jones BF, Ring EFJ, 1995, ‘A structured light system for measuring wounds’, The Photogrammetric Record, 15, 197–204 Plassmann P, Jones TD, 1998, ‘MAVIS: a non-invasive instrument to measure area and volume of wounds’, Medical Engineering and Physics, 20(5), 332–338 Pointer MR, Attridge GG, Jacobson RE, 2001, ‘Pratical camera characterization for colour measurement’, The Imaging Science Journal, 49(2), 63–80 Sangwine SJ, Horne REN, editors. 1998, The Colour Image Processing Handbook. Optoelectronics Imaging and Sensing, London, Chapman and Hall, ISBN 0-412-80620-7 Schaefer G, Jiang J, Hassanien A, editors, 2009, Computational Intelligence in Medical Imaging: Techniques and Applications, CRC Press, ISBN 1420060597, 89–113 Smith DJ, Bhat S, Bulgrin JP, 1992, ‘Video image analysis of wound repair’, Wounds, 4, 6–15 Solomon C, Munro AR, Van Rij AM, Christie R, 1995, The use of video image analysis for the measurement of venous ulcers, Br J Dermatol, 133, 565–570 Stacey MC, Burnand KG, Layer GT, Pattison M, Browse NL, 1991, ‘Measurement of the healing of venous ulcers’, Aust N Z J Surg, 61, 844–848 Stotts NA, Salazar MJ, Wipke-Tevis D, McAdo E, 1996, ‘Accuracy of alginate moulds for measuring wound volumes when prepared and stored under varying conditions’, Wounds: A compendium of clinical research and practice, 8, 5, 159–164 Thawer HA, Houghton PE, Woodbury MG, Keast D, Campbell K, 2002, ‘A comparison of computer-assisted and manual wound size measurement’, Ostomy Wound Management, 48, 46–53 Van der Haegen Y, Naeyaert JM., 2006, ‘Consistent cutaneous imaging with commercial digital cameras’, Arch Dermatol, 142(1), 42–6 Veredas F, Mesa H, Morente L, 2010, ‘Binary Tissue Classification on Wound Images With Neural Networks and Bayesian Classifiers’, IEEE Transaction on Medical Imaging, ISSN: 0278-0062 von Koch H, 1904, (original French title: ‘Sur une courbe continue sans tangente, obtenue par une construction géométrique élémentaire’) English translation: ‘On a continuous curve without tangents, constructible from elementary geometry’ in: Classics on Fractals, Westview Press, 2004, 25–45 Wannous H, Treuillet S, Lucas Y, 2007, ‘Supervised Tissue Classification from Color Images for a Complete Wound Assessment Tool’, Engineering in Medicine and Biology Society, EMBS 2007. 29th Annual International Conference of the IEEE, 22–26 Aug. 2007, 6031–6034, ISSN: 1557-170X, ISBN: 978-1-4244-0787-3 Wannous H, Lucas Y, Treuillet S, Albouy B, 2008, ‘Fusion of Multi-view Tissue Classification Based on Wound 3D Model’, In: Advanced Concepts for Intelligent Vision Systems, Springer Berlin/Heidelberg, ISBN 978-3-540-88457-6, p924-935 DOI 10.1007/978-3-540-88458-3_84) van Zuijlen PP, Angeles AP, Kreis RW, Bos KE, Middelkoop E, 2002, ‘Scar Assessment Tools: Implications for Current Research’, Plastic and Reconstructive Surgery, 109(3), 1108–1122

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6 Functional requirements of wound repair biomaterials R. M. DAY, University College London, UK

Abstract: Wound repair biomaterials play an important role in the health and well-being of a large proportion of the population. These may be associated with an acute setting, such as surgery, or a long-term scenario, such as with chronic wounds. The diverse types of wounds that exist are matched by an equally varied selection of wound repair biomaterials, many of which are tailored to match the functional requirements of a particular wound. This chapter will describe some of the key functional requirements that exist for wound repair biomaterials related to wound pain, exudate, odour and infection. Key words: wound, odour, infection, exudate, pain.

6.1

Introduction

The functional requirements of wound repair biomaterials will depend to a large extent on the characteristics of the wound being treated. Although wound healing is a complex process, the general mechanisms required for healing are shared amongst wounds of different origins. Depending on the type of wound, the healing process can be facilitated by adapting the functional properties of the material used for the dressing accordingly. Fundamental requirements of wound dressing materials include being biocompatible, non-cytotoxic, and non-antigenic. To optimize healing, the wound should remain moist to provide an optimal environment for cell proliferation and migration, whilst avoiding excess fluid that would lead to maceration and an increased risk of infection and malodourous complications. The course of wound healing is determined by a number of factors that can influence (and be influenced by) the type of biomaterial chosen to facilitate wound closure. These include infection, mechanical force at the wound site interfering with the blood supply and delivery of nutrients, age of the patient, their nutritional status, concurrent disease, and region of the body and type of tissue affected. Chronic wounds are a major cause of morbidity, often significantly lowering an individual’s quality of life and represent a major economic burden to both the patient and healthcare system. Because of this, treatment of 155 © Woodhead Publishing Limited, 2011

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many types of chronic wound now involves the use of biomaterials that have been specifically designed to address or prevent the cause of delayed healing. Their mechanism of action can range from intervention at a molecular level through to modulation of the physical environment. Many of these materials have arisen through improved understanding of the aetiology of chronic wounds and better methods of evaluating the behaviour of individual wounds. Therefore the prospect of designing an ‘off-the-shelf’ wound repair biomaterial that is capable of simultaneously meeting all the requirements for treating any type of wound is unlikely. This chapter will cover some of the key features and problems that should be considered when developing wound repair biomaterials: pain relief, management of exudate, odour, and prevention of infection. Careful attention to each of these features will in turn promote wound healing. Each of the following sections will explain the importance of these features and describe some of the approaches being incorporated into biomaterial technology to address the problems.

6.2

Wound pain and dressing materials

Pain is common problem associated with wound management. It is an evolutionary mechanism designed to protect the wounded area from further damage. Although it is a major element of the inflammatory process, it remains a relatively poorly understood component of the tissue response to injury. The sensation, transmission and perception of pain are mediated by chemical factors released in response to injury and during the subsequent inflammatory and healing response. The density of tissue at the wound site, increased tissue turgor associated with inflammation, and the number of primary afferent nociceptors at the wound site all contribute to the extent of pain experienced. Unsurprisingly therefore, the surface area of a wound does not necessarily correlate with the extent of pain experienced, with severity varying unpredictably between patients and sites of wounds (Valenica et al., 2001). In addition to the role of endogenous factors directly associated with the healing process, pain can also result from a variety of artefactual causes secondary to the initiating insult. These include removal of dressing materials that have become adhered to the wound site, subsequent repeated application and removal of adhesive dressing materials resulting in the stripping of tissue layers, poor management of wound exudate resulting in tissue excoriation and maceration of skin surrounding the wound, and wound infection resulting in the release of additional inflammatory modulators. The importance of managing pain is sometimes overlooked. Pain plays more than a psychological role in patient response to tissue injury and can impair the wound healing response by activating a neuroendocrine stress

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response, reducing the autonomic, somatic and endocrine reflexes, resulting in catabolism, hypercoagulation and immunosuppression (Goodwin et al., 1998). Providing pain relief may enhance wound healing and reduce morbidity (Kehlet et al., 1997). Persistent wound pain is a common feature in the majority of patients with chronic wounds, producing a significant negative impact on healing rates and quality of life. Therefore the effect of wound repair biomaterials must be carefully considered to minimize and, in some circumstances, reduce the impact of pain so as to facilitate the healing process. Many studies investigating the effectiveness of wound dressing materials have included pain as one of the outcome measures, as either a subjective or objective factor; however, these studies are often inconsistent in methodological quality and the criteria used to measure and report pain have been heterogeneous, precluding many from use in meta-analytical models. Wound dressing materials designed to minimize or reduce pain have been sought for many years. One of the most extensively studied fields is pain associated with wound dressings used for burn injuries. Pain encountered with wound dressing changes for this type of injury immediately after skin grafting is reported to be particularly intense (Sharar et al., 2008). Skin-graft donor sites have traditionally been dressed with gauze-based dressings but these have the drawback of adhering to the wound surface when left for long periods, causing pain upon removal. Attempts to avoid pain caused by this procedure have included soaking the dressing before attempting removal. Thus, pain associated with this type of dressing material may be less noticeable in wounds with high exudate output, due to the necessary higher frequency of dressing changes required and the likelihood that the dressing material will not become desiccated and attached to the wound (Ubbink et al., 2008). Although a number of analgesic measures to counter pain are available, novel methods of including pain relief into wound dressing material have been sought that will provide an alternative solution to this problem, whilst reducing systemic absorption of analgesics that is associated with the risk of neurological and cardiovascular toxicity and avoiding the need for repeated injections. Local delivery of analgesic drugs might be offer one solution to this. Microspheres consisting of poly(D,L)-lactide and loaded with the local anaesthetic, bupivacaine, were shown to increase the duration of the antinociceptive effect when assessed with an acute inflammatory pain model (Fletcher et al., 1997). Although the intended primary clinical implication was a controlled-release drug delivery system for local anaesthetic solutions in the block of central and peripheral nerves, a similar application could be envisaged for the treatment of chronic wounds where the microspheres form part of the dressing material.

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Over the years, several types of wound dressings derived from biological materials have been reported to have pain-reducing benefits. Bose evaluated the use of human amniotic membrane for dressing burn wounds and described it as a simple, effective and inexpensive treatment (Bose et al., 1979). The rationale for its use was the prevention of wound desiccation due to water loss through evaporation, protecting the wound from infection, and preparing the wound for grafting. After separating the membrane from the placenta and washing in aseptic solution, the amniotic and chorionic layers were applied to burn wounds and, depending on the location and extent of the wound, covered with a wet dressing and bandage. The membrane was inspected and replaced at regular intervals, with removal helping to debride the wound in preparation for grafting. The antibacterial properties of the membrane are thought to have resulted from lysozyme and progesterone present in the amniotic fluid. Although the membranes were reported to offer relief from pain in partial-thickness burns, adherence of the membrane, even after soaking, caused pain when attempting to remove it. More recently the pain-reducing effects of Biobrane, a composite semipermeable wound dressings consisting of layers of woven non-degradable nylon mesh and an outer silastic coating to prevent vapour loss and microbial infection, has been reported (Whitaker et al., 2008). Bonding of the material to the wound bed is facilitated by type I collagen peptides bonded to the exposed nylon and silicone surfaces. When used for the treatment of burn wounds and donor sites the dressing remains attached to wound until spontaneous detachment by epithelialization of wound. When used appropriately pain levels associated with wounds dressed with Biobrane are reduced (Whitaker et al., 2008). Evidence supporting the use of dressing materials that incorporate nanocrystalline silver for wound management primarily exists due to the widely reported antimicrobial properties of silver (discussed below), but improved pain levels have also been described during dressing changes (as well as decreased frequency of dressing changes) (Fong and Wood, 2006). It is currently unclear whether the nanocrystalline silver has innate pain-reducing activity or if the effect is due to its antimicrobial and anti-inflammatory properties preventing the local release of pain-inducing chemical mediators. The pressure exerted by dressing materials on the wound can have a major influence on the healing outcome and amount of pain experienced by the patient. Although the optimal clinical management of venous ulcers is debatable, compression therapy is considered by many to be the best conservative treatment for venous leg ulcers, achieving its effect most likely through correction of the underlying venous hypertension. This approach often involves the use of either bandages or stocking-based systems. A number of randomized, controlled trails have compared the effects of

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compression bandages and stockings, including pain (during application and whilst wearing the dressing) as a subjective factor. These studies have generally shown stockings to be advantageous over bandages, especially with regards to comfort, ease of application and removal. The search for optimal compression therapy for venous leg ulcers led Amsler and colleagues to conduct a systematic and meta-analysis of randomized controlled trials comparing compression systems based on stockings with divers bandages (Amsler et al., 2009). Pain, included as a secondary endpoint in the meta-analysis, was found to be better controlled with leg compression compared with stockings (Amsler et al., 2009). However, patients treated with the Unna boot, a moist zinc-impregnated paste bandage providing compression and topical treatment, reported more pain, both during application and at home compared with the use of a hydrocolloid dressing in addition to the elastic compression (Koksal and Bozkurt, 2003). A prospective, comparative study of a combination of hydrocolloid dressing in conjunction with elastic compression stocking, versus the use of Unna’s boot, consisting of a non-compliant most paste bandage to provide compression, for the management of venous ulcers observed that the intensity of pain experienced by the patient during dressing changes and at home measured at the third week of therapy was significantly lower for patients in the hydrocolloid dressing group (Koksal and Bozkurt, 2003). From the majority of these studies it can be concluded that a direct correlation exists between the degree of complexity associated with the process of applying and removing dressing materials from the wound and the amount of pain experienced. Wound pain can be exacerbated by the dressing materials being applied either directly to the wound itself or during dressing changes. Likewise, poor flexibility and increased bulk at the wound site caused by the dressing material may result in additional pressure being applied to the wound when the patient moves. To address this problem, a prospective randomized trial comparing calcium alginate dressings with retention tape dressings on split skin donor graft sites assessed patient comfort, effectiveness and ease of use (Giele et al., 2001). An overall pain score combining a number of different indices found no significant difference at 24 hours but at 72 hours only 16% of patients with the retention tape dressing reported considerable pain versus 49% of the patients receiving alginate dressings. Similarly, during removal of the dressing, 20% of patients with the retention dressing reported moderate pain versus 50% who received calcium alginate dressings (Giele et al., 2001). The beneficial effects are believed to have arisen from the reduced intervention at the wound site required with the use of adhesive retention tape used for wound dressing. Furthermore, the tape was applied directly to the donor site wound, adhering to the normal skin at the margins but not the moist wound itself. Wound exudate escaped through the pores of the dressing material and was absorbed by overlying gauze,

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which was removed leaving the tape adhered to the skin and donor site for 14 days or until it dropped off by itself when the wound had reepithelialized. By acting as an ‘artificial scab’ the tape dressing was suggested to have reduced pain by being less bulky and stiff compared with alginate dressings (Giele et al., 2001). Studies dating back to the 1960s have demonstrated that wound healing is optimized when the local environment is maintained moist (Winter, 1962; Hinman and Maibach, 1963). This has led to a plethora of occlusive and semi-occlusive dressings consisting of hydrogels, alginates, hydrocolloids, foams and films that have features designed to retain moisture and accelerate healing. A variety of mechanisms associated with wound hydration have been proposed to account for the accelerated healing observed with such materials. These include moisture retention at the wound site increasing the rate of re-epithelialization of wounds by facilitating epidermal migration; development of good granulation tissue in the wound base; increased oxygen tension in the wound environment; prevention of wound desiccation to maintain an electrical gradient between the wound and adjacent nonwound environment; and retention of wound fluid that contains growth factors capable of stimulating cell proliferation (Helfman et al., 1994). As well as facilitating tissue healing by retaining the growth factor rich fluid at the wound site, some of the dressings are reported to reduce the amount of pain experienced by the user. A systematic review of the use of moist wound healing dressings used for split-thickness skin graft donor sites found moist dressings to be significantly favourable over non-moist dressings for both increasing healing rates and lowering pain scores without increasing infection rates (Beam, 2007). Regulation of tissue temperature at the wound site may also provide pain relief, especially as raised tissue temperatures is a typical feature of the inflammatory response. The ability to cool burn wounds has long been recognized as being beneficial to the relief of pain. In addition to relieving pain, cooling of fresh burn wounds has been shown clinically and histologically to limit tissue damage and allow more rapid healing in deep dermal wounds (Venter et al., 2007). As well as providing a moist environment, hydrogels are able to cool the skin surface for several hours, which can provide pain relief at the wound site and reduce the inflammatory response (Jandera et al., 2000; Helfman et al., 1994). Changes in temperature reported are typically between 1.5 and 4°C depending on the water content (and thus evaporative cooling capacity) in the different types of hydrogel used (Jandera et al., 2000; Coats et al., 2002). The optimal temperature of the coolant applied to burn wounds is unclear. Normal skin temperature is 32°C and the analgesic threshold is 28°C (Coats et al., 2002). The application of hydrogel dressings alone with exposure to air movement can reduce the skin temperature by as much as 10°C (Coats et al., 2002). Venter and

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colleagues investigated measuring intradermal temperatures relative to tissue histology after applying ice water (1–8°C) or tap water (12–18°C) in a burn model (Venter et al., 2007). The cooling produced by tap water led to less necrosis and faster wound healing compared with the wounds cooled with ice water and continued to be beneficial even when cooled 30 minutes after wounding (Venter et al., 2007). The use of dressings on large burns to induce cooling remains a controversial practice, since even small reductions in skin temperature (1°C) can exacerbate burn-induced hypothermia (Martineau and Shek, 2006). The risk of such hypothermia is raised when dressings cover a large surface area/mass ratio, such as with children. Looking into the future, hybrid biomaterial-tissue-engineered dressings may offer additional methods of treatment associated with reduced pain. Tissue-engineered skin equivalents have been found to accelerate the speed of healing and reduce the amount of pain associated with healing donor sites. The living skin equivalent was made from type I bovine collagen and cultured allogeneic cells (keratinocytes and fibroblasts) isolated from human neonatal foreskin and considered to mimic the activity of autografts (Muhart et al., 1999). At present, this type of treatment is not routinely used due to expense and lack of availability, but it may feature more prominently as the field of tissue engineering expands. Despite the development of many new types of wound dressing materials, a systematic review of literature on the efficacy of modern wound dressings in healing of chronic and acute wounds by secondary intention performed by Chaby and colleagues found insufficient evidence to indicate that modern dressings provide improved pain relief in the dressing of acute or chronic wounds over saline or paraffin gauze dressings (Chaby et al., 2007). Relatively simple remedies, such as transparent materials to enable wound inspection or dressings designed to allow removal without causing trauma, would fulfill some the functional requirements to reduce pain associated with wound repair biomaterials.

6.3

Exudate management

Wound exudate consists of fluid leaked from capillaries and contains a complex mixture of water, electrolytes, nutrients, inflammatory mediators, white cells, proteases, growth factors and waste products (wound exudate itself and possibly dressing degradation products). At the cellular level, the amount of fluid leaking from the blood vessels into a wound is determined by the increased permeability of the capillaries and the hydrostatic and osmotic pressures across the vessel wall. Larger wounds and increased capillary leakage or tissue oedema may amplify the amount of exudate produced (Harding, 2008). In healthy individuals, the amount of exudate typically decreases as the wound progresses towards healing (Thomas,

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1997). But increased fluid leakage through skin can arise from a number of causes, including increased capillary permeability during the early inflammatory response following tissue injury, chronic inflammation, malignancy, wound bed infection, transudate resulting from raised capillary pressure in the lower limb, lymphorrhoea, and oedema due to low blood protein levels or intravenous fluid overload (Vuolo et al., 2004). Other types of wounds that are associated with high volumes of fluid output include fistulae as well as wounds with a large surface area. Although wound exudate can prove problematical for management by the practitioner, it plays an essential role in the normal healing process of all wounds, especially during the inflammatory and proliferative phases of acute wound healing where it provides nutrients and growth factors for the proliferating and migrating cells required for healing. Following research conducted in the 1960s, a moist healing environment is now generally considered to be most conducive to accelerating healing by preventing dehydration of the cells (Winter, 1962). However, excessive exudate production associated with wounds that do not heal as expected may further impair the healing process. Excessive production of (or failure to clear) wound exudate can result in maceration and excoriation of healthy tissue in the periwound region, impairing migration of cells across the wound surface and delaying healing. Fluid trapped on the skin surface results in swelling of the keratinocytes and weakening of the stratum corneum making it more susceptible to trauma, such as that caused by the removal of adhesive dressing material (Fletcher, 2002). The presence of multiple factors can increase the risk of maceration and include the volume of exudate, bacterial infection, and the levels of histamine, proteolytic enzymes and inflammatory cytokines (Gray and Weir, 2007). One of the most extensively studied groups mediators that are associated with impaired wound healing are the matrix metalloproteinases (MMPs). These are endopeptidases that require divalent cations, such as Ca2+ or Zn2+, to function as proteases that remodel the extracellular matrix. Dysregulation or overproduction of MMPs is thought to play a significant role in impaired wound healing (Wysocki et al., 1993; Palolahti et al., 1993; Trengove et al., 1999). Their presence in chronic wound exudate is thought to contribute to the damage of healthy tissue by disrupting the balance of tissue synthesis towards tissue degradation, resulting in a delayed healing process (Rayment and Upton, 2009; Baker and Leaper, 2000). Traditional dressing materials, such as gauze, are designed to keep the wound dry by absorption of wound exudate whilst preventing entry of bacteria. Fluid and exudate are absorbed by the fibres of the dressing but regular changes are required to prevent maceration of healthy tissue and infection by bacteria. Furthermore, if the dressing is allowed to dry in situ, the gauze will adhere to the wound, causing patient discomfort when

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removed. This type of dressing allows no control of the absorption of wound exudate or balance of the moisture level at the wound surface. Since the water vapour transmission rate (WVTR) from skin varies dramatically depending on the type of wound and stage of healing, consideration of this should be a critical feature of all biomaterials used to dress wounds. Fluid balance is especially important in burn wounds and venous leg ulcers, where the fluid conserving function of normal skin is destroyed and large volumes of fluid are lost through evaporation and exudation. This can result in a decrease in body temperature and increase in metabolic rate (Quinn et al., 1985). The WVTRs measured on exposed venous leg ulcer site and full thickness burns are similar, approximately 80 g m−2 h−1 compared with values in normal skin of 8–9 g m−2 h−1 (Wu et al., 1996). The values recorded depend on the depth of wound. In order to maintain a desired moist environment, whilst avoiding fluid accumulation, the water vapour transmission rate must be matched to the type of wound to which the dressing is being applied (Wu et al., 1996). The amount of moisture evaporating from the surface of a dressing can be quantified as the moisture vapour transmission rate (MVTR). In wounds with a high amount of exudate the dressing material will need a high absorptive capacity to remove the excessive fluid. Wounds with low amounts of exudate can be managed with dressings that have a high moisture vapour transpiration rate (MVTR), which allows the fluid to evaporate from the wound site. Users of this type of biomaterial need to ensure moisture vapour transmission is not blocked by the use of tapes and additional dressing materials over the primary dressing (Jones and Milton, 2000). Conversely, wounds with a low exudate, which run the risk of drying out, may require dressings that are less absorbent and capable of conserving moisture. A variety of different biomaterials currently exist that are used as dressings designed to absorb and retain fluid, whilst maintaining an appropriate fluid balance in wound environment. These include hydrocolloids, alginate and carboxymethylcellulose (CMC) fibres (Boateng et al., 2008). When hydrated the materials turn into a gel, which ideally stays intact to prevent leakage and peri-wound maceration. Alginates and CMC fibres have a high absorptive capacity making them ideal for heavily exudating wounds by reducing the number of dressing changes required (Boateng et al., 2008). Since wound exudate potentially contains a number of factors that promote tissue healing, selective absorption of fluid from wound exudate has been proposed as a method of increasing the concentration of these factors to promote tissue healing (Achterberg et al., 1996). However, this approach may also increase the concentration of proteolytic enzymes, such as MMP, which could lead to further tissue destruction (Trengove et al., 1999; Yager and Nwomeh, 1999). Nevertheless, attempts have been made to address the imbalance between tissue synthesis and degradation in chronic wounds. Because

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of their critical role in chronic wounds, a number of studies have investigated the use of wound dressing biomaterials to modulate MPP activity in the wound exudate to facilitate healing of chronic, non-healing wounds. Absorption and sequestration of MMP onto the dressing material is one approach that has been explored. This has included the application of peptide recognition sequences of elastase to the cotton-based cellulose fibres of wound dressing gauze, which function to remove elastase by sequestering the MMP to the dressing fibres (Edwards et al., 1999). Similarly, cotton gauze has been modified by oxidation, phosphorylation, and sulfonation in an attempt to enhance elastase affinity by ionic or active site uptake and reduce its activity in wound fluid (Edwards et al., 2001). As well as absorbing factors that may impede wound healing, attempts to modify the chronic wound environment by inactivating proteases is another area of active research. Schönfelder and colleagues tested a range of dressings from different biomaterial groups to investigate their influence on the proteolytic enzyme elastase released from neutrophils. The materials tested were Suprasorbs C (a collagen foam product consisting of collagen type I from bovine origin) (Suprasorbs C), Tabotamps (a dressing from oxidized regenerated cellulose [ORC]), and a mixture of collagen and ORC (Promograns). All of the tested materials were able to reduce the concentration and activity of elastase, as well as showing antioxidant activity (Schönfelder et al., 2005). The authors suggest the latter is significant because reactive oxygen species (ROS) induce increased concentration of MMPs at an mRNA level, so dressing materials capable of scavenging ROS is likely to prove beneficial for wound healing. Alternative approaches to tackling increased MMP in the wound exudate include inactivating protease activity using dressing materials that contain biomaterials that act as an alternative or competitive substrate for the MMPs (such as collagen), or that bind the divalent cations essential for MMP activity (such as negatively charged oxidized regenerated cellulose) so decreasing excess metal ions in chronic wound fluid (Cullen et al., 2002a; 2002b; Eming et al., 2008; Rayment et al., 2008). Consideration should also be given to patient-focussed factors when selecting the dressing material to manage wound exudate. These include the reduced quality of life patients with exudating wounds endure, caused by leakage resulting in soiling of clothes, feelings of uncleanliness, discomfort and malodour (Fletcher, 2002). Management of excess exudate also places extra burden on healthcare resources in terms of time required for frequent dressing changes and dressing materials.

6.4

Prevention and control of infection

The skin surface provides an important innate barrier function. This is often lost as a result of wounds allowing an entry route for infectious agents that

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colonize contaminated external surfaces that would normally be prevented from entering underlying sterile tissue environments. This effect may be compounded if concurrent disease is present that alters the immune response, rendering the individual more susceptible to infection (Zedler et al., 1999). Once the innate skin barrier has been breached, leukocytes from the innate and acquired immune system infiltrate the damaged tissues to eliminate the microbial infection resulting in an inflammatory response. Early identification of infection can be difficult to determine in wounds where visibility is obscured by the dressing material. As a consequence, key clinical signs, such as increased pain, presence of pus and malodour, are commonly used as indicators of infection (Cutting and Harding, 1994). Enzymes secreted by leukocytes as part of the immune response may contribute to further damage in the surrounding tissue; therefore the inflammatory process needs to recede before the regenerative phase of healing can progress. This process is interrupted when the wound becomes infected, resulting in the inflammatory phase becoming chronic and delayed healing. Prevention (rather than treatment) of wound infection is therefore a primary goal to allow complete healing to progress in a normal manner. Colonization of wounds by bacteria (also called bioburden or bacterial burden) is a common feature, especially with chronic wounds containing devitalized tissue. Loss of the epidermal barrier function exposing subcutaneous tissue, along with poor blood flow and tissue hypoxia contribute to this process (Falanga, 2004). Wounds are prone to colonization by a diverse microbial flora arising from three main sources: exogenous microorganisms in the environment, normal skin microflora, and endogenous sources primarily from the gastrointestinal, oropharyngeal and genitourinary mucous membranes (Bowler et al., 2001). Patients with acute disease processes such as surgery, trauma and burns have an increased risk of developing nosocomial infection and it is estimated 30% of patients in intensive care units are affected by this type of infection (Vincent, 2003). Chronic wounds provide an optimal environment for infection by bacteria. Certain strains of bacteria that infect wounds have the capacity of producing biofilms that protect the microorganisms from host immune effector cells and antibiotics. The biofilm appears as a thick layer of slime and consists of an exopolysaccharide matrix secreted by the bacteria that acts as a protective barrier and facilitates bacterial colonization. The precise role of biofilms play in chronic wounds is uncertain. Evidence from in vitro studies suggest that biofilms can reduce the viability of keratinocytes, increase apoptosis and impede wound closure (Kirker et al., 2009). This effect would delay closure of the primary barrier between internal tissues and the external environment providing an entry route for further infection. The nutrient-poor environment of chronic wounds stimulates bacteria to form biofilms. Fibrin deposited in the wound also facilitates physical

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attachment of the biofilm to the wound bed. Once a wound is infected, biofilms can develop rapidly. In vivo studies involving inoculation of a porcine partial-thickness wound model with Pseudomonas aeruginosa demonstrated the formation of a biofilm within 72 hours (Serralta et al., 2001). In vitro studies have observed biofilms of Pseudomonas aeruginosa developing at 5 hours after inoculation and mature biofilm existing by 10 hours (Harrison-Balestra et al., 2003). However, in vivo most wounds do not become chronically infected and this is likely to be the result of factors such as the availability of bacterial nutrients, the time taken for fibrin formation to facilitate biofilm attachment, the effects of the host immune system and administration of antiseptics, which will impair the formation of biofilms (Harrison-Balestra et al., 2003). To counter the problem of bacterial colonization of wounds, many dressing biomaterials have been designed to elute antimicrobial compounds. These include iodine, chlorohexidine, and silver ions. The materials are designed to deliver a therapeutic dose of the active compound in a controlled and sustained manner so as to avoid toxicity. Silver has been used as an antimicrobial compound for many years and a wide range of dressing materials incorporating silver now exist (Klasen, 2001; Thomas and McCubbin, 2003a; Thomas and McCubbin, 2003b). Incorporation of the antimicrobial activity of silver ions into dressing materials can be achieved via several different mechanisms. These include the exchange of silver ions between the dressing and the wound, the release of silver sulfadiazine into the wound, absorption of exudate in the dressing, and local antimicrobial action in the dressing (Lansdown et al., 2005). Despite the effectiveness of silvercontaining antimicrobial dressing materials, there are some considerations regarding the use of compounds delivering silver ions. The use of topical agents containing silver ions has been associated with localized and generalized argyria – the development of irreversible pigmentation of the skin (Drake and Hazlewood, 2005). Questions have also arisen from in vitro studies regarding the cytotoxicity of silver dressings, which have indicated marked cytotoxicity towards human skin keratinocytes and dermal fibroblasts (Du Toit and Page, 2009; McCauley et al., 1989, 1992; Poon and Burd, 2004). These findings have been substantiated using tissue explant culture and mouse excisional wound models, where it has been suggested this effect might contribute to delayed wound healing or inhibition of epithelialization (Burd et al., 2007). However, the models used do not necessarily provide a close approximation of the events occurring with humans in vivo and should be interpreted with caution. Other approaches to controlling the bacterial burden of a wound include the creation of an adverse environment. This approach is thought to provide the antibiotic properties observed when dressing wounds with honey. Suggestions for the mechanism underlying this effect include the establishment

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of a hyperosmotic environment (Lusby et al., 2005) or the production of hydrogen peroxide when the honey is mixed with wound exudate (Henriques et al., 2006). Modulation of the pH in the wound environment may also be used to control colonization of bacteria. Hydrocolloid dressings produce an acidic environment (pH 5), which has been shown to inhibit growth of P. aeruginosa and S. aureus (Varghese et al., 1986). New approaches to preventing wound infection are continuing to evolve. One approach that also avoids dressing changes that may cause discomfort to the patient and damage the delicate healing tissue, is the use of an occlusive bioresorbable dressings that provides topical antibiotic treatment has been proposed (Elsner and Zilberman, 2010). This dressing materials consist of a composite of polyglyconate mesh coated with porous poly(DL-lacticco-glyolic acid) matrix. With the use of resorbable materials intended to reduce the need for frequent dressing changes.

6.5

Odour management

Chronic wounds associated with offensive malodour is a cause of significant physical and emotional distress to the patient (Price, 1996). Malodour is frequently considered as a problem associated with exudate facilitating bacterial infection and is often encountered with malignant cutaneous wounds. The loss of vascularity and occlusion of blood vessels associated with increased tumour mass, which leads to reduced oxygen diffusion and hypoxic tissue. The wound then becomes colonized by anaerobic bacteria whose metabolic processes produce volatile fatty acids that give rise to the odour (Alvarez et al., 2007). Anaerobic and aerobic bacteria rapidly colonize necrotic tissue. Lipid metabolized by anaerobes (Bacteroides fragilis, Bacteroides prevotella, fusobacterium nucleatum, Clostridium perfringens and anaerobic cocci) results in the production of volatile fatty acids (proprionic, isobutyric, butyric, isovaleric and valeric) (Alvarez et al., 2007). Therefore key considerations for the management of odour include the removal of necrotic tissue, prevention or reduction and management of underlying infection, control of wound exudate, thorough wound cleaning and increased frequency of dressing changes (Harding, 2008; Alvarez et al., 2007). The traditional approach of adding fragrances (e.g. oil of peppermint) to the dressing material only serves to mask the odour and like dressings incorporating carbon or charcoal designed to absorb the odour, do not offer a long-term solution as they do not deal with the underlying cause. Effective treatment for malodorous wounds includes eradication of anaerobic infection using topical administration of metronidazole (Sparrow et al., 1980). For fungating tumours combination of metronidazole with wound dressings such as petroleum gauze, calcium alginate, hydrofiber or foam dressings has also been shown to be effective (Kalinski et al., 2005). Some dressings

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themselves, such as hydrocolloids and gelatin, produce a characteristic odour as they decompose in situ after gelling over the wound but this is eliminated once the dressing has been irrigated from the wound.

6.6

Future trends

The field of wound care biomaterials is continuing to evolve as new materials are developed and our understanding of wound biology improves. The availability of more types of biomaterial will not necessarily correspond with improved wound healing rates. The use of new biomaterials for wound healing requires validation in multi-centred trials. Improvement in wound dressing technology is frequently hampered because of the poor methodological quality of many clinical studies, limiting the availability of multicentre based evidence indicating the most effective wound repair materials. Where available, validated and standardized tools should be used in studies that will provide better data for metanalysis regarding the recommended use of biomaterial dressings. A standardized approach is already available for the laboratory testing and evaluation of wound care materials, for example via the Surgical Materials Testing Laboratory (SMTL). The service provides independent product examination or evaluation to ensure compliance of submitted materials with nationally or internationally agreed standards. An example of this relates to the guidelines on the fluid handling properties of dressings provided by manufacturers, which tend to lack of consistency in describing the level of absorbency between different dressing materials (standard methodology, weight by product or weight by area). To overcome this, standard in vitro tests conducted by the STML have been devised to determine parameters such as fluid absorbency, moisture vapour transmission rates, fluid handling capacity and gelling properties. Better exudate management, not only in terms of the volume of fluid, but also its constituents is likely to benefit from a variety of exciting studies in wound repair biomaterials research. Improved understanding of the specific mediators that impede wound healing enables selective targeting of these compounds thus allowing the beneficial factors to remain in situ (Eming et al., 2008; Schönfelder et al., 2005; Rayment et al., 2008). Better methods for monitoring wound healing are sought. The materials used for many wound repair biomaterials obscure visual inspection of the wound. Indirect assessment of the wound is one way this could be overcome, for example the use of smart materials that indicate the release of volatile gases from infected wounds could rely on technology similar to that already available for sampling gases (Armani et al., 1996). The use of biomaterials for tissue engineering of wounds is a rapidly growing field covering an increasing variety of materials. Despite the

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continual development of synthetic materials, biological materials remain at the forefront of this area of research (Sabolinski et al., 1996). Biological materials offer advantages of improved biocompatibility, despite drawbacks such as mechanical integrity and the potential risks of zoonotic infection with animal-derived material that can be avoided with synthetic materials. With an increasingly improved understanding of the immunological mechanisms underlying biocompatibility, it is likely that synthetic materials will eventually supersede these materials. This in turn will open up opportunities for developing materials that provide better solutions to the functional requirements outlined in this chapter.

6.7

Sources of further information and advice

There are numerous websites that provide further information and advice on the functional properties of wound care biomaterials. The following are websites relevant to wound biomaterials that have been selected as Top Sites by Biomat.net (http://www.biomat.net). The Wound Healing Society (http://www.woundheal.org/) is a non-profit organization composed of clinical and basic scientists that seeks to advance the science and practice of wound healing. It provides a forum for interaction among scientists, physicians, licensed practitioners, industrial representatives and government agencies. The Society publishes the leading journal in this area, Wound Repair and Regeneration. The Wound Care Information Network (http://www.medicaledu.com/) provides free unbiased wound care information for patients, clinicians and hospital administrators. WoundSource: The Kestrel Wound Product Sourcebook (http://www. woundsource.com/) is provided by Kestrel Health Information, Inc., a Vermont-based company. It provides wound care professionals with comparative information on over 1000 products manufactured by over 150 wound care companies. The Woundbiotech web site (http://www.bu.edu/woundbiotech/index.html) was established by Professor Vincent Falanga at Boston University School of Medicine. The site provides information about the wound healing process in different types of wounds, disseminates information about established wound care products and information on the development and use of novel therapeutic products for wound healing.

6.8

References

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Alvarez O M, Kalinski C, Nusbaum J, Hernandez L, Pappous E, Kyriannis C, Parker R, Chrzanowski G, and Comfort C P (2007) ‘Incorporating Wound Healing Strategies to Improve Palliation (Symptom Management) in Patients with Chronic Wounds’, Journal of Palliative Medicine, 10, 1161–1189. Amsler F, Willenberg T, and Blättler W (2009) ‘In search of optimal compression therapy for venous leg ulcers: A meta-analysis of studies comparing divers bandages with specifically designed stockings’, Journal of Vascular Surgery, 50, 668–674. Armani M E H, Payne P, and Persaud K C (1996) ‘Multifrequency measurements of organic conducting polymers for sensing of gases and vapours’, Sensors and Activators B, 1–3, 137–141. Baker E A and Leaper D J (2000) ‘Proteinases, their inhibitors and cytokine profiles in acute wound fluid’, Wound Repair and Regeneration, 8, 392–398. Beam J W (2007) ‘Management of Superficial to Partial-Thickness Wounds’, Journal of Athletic Training, 42, 422–424. Boateng J S, Matthews K H, Stevens H N E, and Eccleston G M (2008) ‘Wound Healing Dressings and Drug Delivery Systems: A Review’, Journal of Pharmaceutical Sciences, 97, 8, 2892–2923. Bose B (1979) ‘Burn wound dressing with human amniotic membrane’, Ann R Coll Surg Engl, 61, 444–447. Bowler P G, Duerden B I, and Armstrong D G (2001) ‘Wound microbiology and associated approaches to wound management’, Clin Microbiol Rev, 14, 244–269. Burd A, Kwok C H, Hung S C, Chan H S, Gu H, Lam W K, and Huang L (2007) ‘A comparative study of the cytotoxicity of silver-based dressings in monolayer cell, tissue explant, and animal models’, Wound Repair Regen, 15, 94–104. Chaby G, Senet P, Vaneau M, Martel P, Guillaume J C, Meaume S, Teot L, Debure C, Dompmartin A, Bachelet H, Carsin H, Matz V, Richard J L, Rochet J M, SalesAussias N, Zagnoli A, Denis C, Guillot B, and Chosidow O (2007) ‘Dressings for acute and chronic wounds: a systematic review’, Arch Dermatol, 143, 1297– 1304. Coats T J, Edwards C, Newton R, and Staun E (2002) ‘The effect of gel burns dressings on skin temperature’, Emerg Med J, 19, 224–225. Cullen B, Smith R, McCulloch E, Silcock D, and Morrison L (2002a) ‘Mechanism of action of PROMOGRAN, a protease modulating matrix, for the treatment of diabetic foot ulcers’, Wound Repair Regen, 10, 16–25. Cullen B, Watt P W, Lundqvist C, Silcock D, Schmidt R J, Bogan D, and Light N D (2002b) ‘The role of oxidised regenerated cellulose/collagen in chronic wound repair and its potential mechanism of action’, Int J Biochem Cell Biol, 34, 1544–1556. Cutting K and Harding K (1994) ‘Criteria for identifying wound infection’, Journal of Wound Care, 3, 198–201. Drake P L and Hazelwood K J (2005) ‘Exposure-related health effects of silver and silver compounds: a review’, Ann Occup Hyg, 49, 575–585. Du Toit D F and Page B J (2009) ‘An in vitro evaluation of the cell toxicity of honey and silver dressings’, J Wound Care, 18, 383–389. Edwards J V, Batiste S L, Gibbins E M, and Goheen S C (1999) ‘Synthesis and activity of NH2- and COOH-terminal elastase recognition sequences on cotton’, J Pept Res, 54, 536–543.

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Edwards J V, Yager D R, Cohen I K, Diegelmann R F, Montante S, Bertoniere N, and Bopp A F (2001) ‘Modified cotton gauze dressings that selectively absorb neutrophils elastase activity in solution’, Wound Repair Regen, 9, 50–58. Elsner J J and Zilberman M (2010) ‘Novel antibiotic-eluting wound dressings: An in vitro study and engineering aspects in the dressing’s design’, J Tissue Viability, 19, 54–56. Eming S, Smola H, Hartmann B, Malchau G, Wegner R, Krieg T, and Smola-Hess S (2008) ‘The inhibition of matrix metalloproteinase activity in chronic wounds by a polyacrylate superabsorber’, Biomaterials, 29, 2932–2940. Falanga V (2004) ‘The chronic wound: impaired healing and solutions in the context of wound bed preparation’, Blood Cells Mol Dis, 32, 88–94. Fletcher D, Le C P, Guilbaud G, and Le V R (1997) ‘Antinociceptive effect of bupivacaine encapsulated in poly(D,L)-lactide-co-glycolide microspheres in the acute inflammatory pain model of carrageenin-injected rats’, Anesth Analg, 84, 90–94. Fletcher J (2002) Exudate theory and the clinical management of exuding wounds. Nursing times.net. Available from: http://www.nursingtimes.net/nursing-practiceclinical-research/exudate-theory-and-the-clinical-management-of-exudingwounds/200107.article [Accessed 16 March 2010]. Fong J and Wood F (2006) ‘Nanocrystalline silver dressings in wound management: a review’, Int J Nanomedicine, 1, 441–449. Giele H, Tong A, and Huddleston S (2001) ‘Adhesive retention dressings are more comfortable than alginate dressings on split skin graft donor sites – a randomised controlled trial’, Ann R Coll Surg Engl, 83, 431–434. Goodwin S A (1998) ‘A review of preemptive analgesia’, J Perianesth Nurs, 13, 109–114. Gray M and Weir D (2007) ‘Prevention and treatment of moisture-associated skin damage (maceration) in the periwound skin’, J Wound Ostomy Continence Nurs, 34, 153–157. Harding K (2008) ‘Would Exudate and the Role of Dressings: A Consensus Document’, International Wound Journal, 5, iii–12. Harrison-Balestra C, Cazzaniga A L, Davis S C, and Mertz P M (2003) ‘A woundisolated Pseudomonas aeruginosa grows a biofilm in vitro within 10 hours and is visualized by light microscopy’, Dermatol Surg, 29, 631–635. Helfman T, Ovington L, and Falanga V (1994) ‘Occlusive dressings and wound healing’, Clin Dermatol, 12, 121–127. Henriques A, Jackson S, Cooper R, and Burton N (2006) ‘Free radical production and quenching in honeys with wound healing potential’, J Antimicrob Chemother, 58, 773–777. Hinman C D and Maibach H (1963) ‘Effect of air exposure and occlusion on experimental human skin wounds’, Nature, 200, 377–378. Jandera V, Hudson D A, de Wet P M, Innes P M, and Rode H (2000) ‘Cooling the burn wound: evaluation of different modalites’, Burns, 26, 265–270. Jones V and Milton T (2000) ‘When and how to use hydrocolloid dressings’, Nurs Times, 96, 5–7. Kalinski C, Schnepf, M, Laboy, D, Hernandez L, Nusbaum J, McGrinder B, Comfort C, and Alvarez O M (2005) ‘Effectiveness of a topical formulation containing metronidazole for wound odor and exudates control’, Wounds, 17, 84–90.

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Kehlet H (1997) ‘Multimodal approach to control postoperative pathophysiology and rehabilitation’, Br J Anaesth, 78, 606–617. Kirker K R, Secor P R, James G A, Fleckman P, Olerud J E, and Stewart P S (2009) ‘Loss of viability and induction of apoptosis in human keratinocytes exposed to Staphylococcus aureus biofilms in vitro’, Wound Repair Regen, 17, 690–699. Klasen H J (2001) ‘Historical review of the use of silver in the treatment of burns. I. Early uses’, Burns, 26, 117–130. Koksal C and Bozkurt A K (2003) ‘Combination of hydrocolloid dressing and medical compression stockings versus Unna’s boot for the treatment of venous leg ulcers’, Swiss Med Wkly, 133, 364–368. Lansdown A B, Williams A, Chandler S, and Benfield S (2005) ‘Silver absorption and antibacterial efficacy of silver dressings’, J Wound Care, 14, 155–160. Lusby P E, Coombes A L, and Wilkinson J M (2005) ‘Bactericidal activity of different honeys against pathogenic bacteria’, Arch Med Res, 36, 464–467. Martineau L and Shek P N (2006) ‘Evaluation of a bi-layer wound dressing for burn care I. Cooling and wound healing properties’, Burns, 32, 70–76. McCauley R L, Linares H A, Pelligrini V, Herndon D N, Robson M C, and Heggers J P (1989) ‘In vitro toxicity of topical antimicrobial agents to human fibroblasts’, J Surg Res, 46, 267–274. McCauley R L, Li Y Y, Poole B, Evans M J, Robson M C, Heggers J P, and Herndon D N (1992) ‘Differential inhibition of human basal keratinocyte growth to silver sulfadiazine and mafenide acetate’, J Surg Res, 52, 276–285. Muhart M, McFalls S, Kirsner R, Elgart G W, Kerdel F, Sabolinski M L, HardinYoung J, and Eaglstein W H (1999) ‘Behavior of tissue-engineered skin: a comparison of a living skin equivalent, autograft, and occlusive dressing in human donor sites’, Arch Dermatol, 135, 913–918. Palolahti M, Lauharanta J, Stephens R W, Kuusela P, and Vaheri A (1993) ‘Proteolytic activity in leg ulcer exudate’, Exp Dermatol, 2, 29–37. Poon V K and Burd A (2004) ‘In vitro cytotoxicity of silver: implication for clinical wound care’, Burns, 30, 140–147. Price E (1996) ‘Wound care: The stigma of smell’, Nurs Times, 92, 71–72. Quinn K J, Courtney J M, Evans J H, Gaylor J D, and Reid W H (1985) ‘Principles of burn dressings’, Biomaterials, 6, 369–377. Rayment E A, Dargaville T R, Shooter G K, George G A, and Upton Z (2008) ‘Attenuation of protease activity in chronic wound fluid with bisphosphonatefunctionalised hydrogels’, Biomaterials, 29, 1785–1795. Rayment E A and Upton Z (2009) ‘Finding the culprit: a review of the influences of proteases on the chronic wound environment’, Int J Low Extrem Wounds, 8, 19–27. Schönfelder U, Abel M, Wiegand C, Klemm D, Elsner P, and Hipler U (2005) ‘Influence of selected wound dressings on PMN elastase in chronic wound fluid and their antioxidative potential in vitro’, Biomaterials, 26, 6664–6673. Serralta V W, Harrison-Balestra C, Cazzaniga A L, Davis S C, and Mertz P M (2001) ‘Lifestyles of bacteria in wounds: presence of biofilms?’, Wounds, 13, 29–34. Sharar S R, Miller W, Teeley A, Soltani M, Hoffman H G, Jensen M P, and Patterson D R (2008) ‘Applications of virtual reality for pain management in burn-injured patients’, Expert Rev Neurother, 8, 1667–1674.

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Sabolinski M L, Alvarez O, Auletta M, Mulder G, and Parenteau N L (1996) ‘Cultured skin as a “smart material” for healing wounds: experience in venous ulcers’, Biomaterials, 17, 311–320. Sparrow G, Minton M, Rubens R D, Simmons N A, and Aubrey C (1980) ‘Metronidazole in smelly tumours’, Lancet, 1, 1185. Thomas S (1997) ‘Assessment and management of wound exudate’, Journal of Wound Care, 6, 327–330. Thomas S and McCubbin P (2003a) ‘A comparison of the antimicrobial effects of four silver-containing dressings on three organisms’, J Wound Care, 12, 101–107. Thomas S and McCubbin P (2003b) ‘An in vitro analysis of the antimicrobial properties of 10 silver-containing dressings’, J Wound Care, 12, 305–308. Trengove N J, Stacey M C, MacAuley S, Bennett N, Gibson J, Burslem F, Murphy G, and Schultz G (1999) ‘Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors’, Wound Repair Regen, 7, 442–452. Ubbink D T, Vermeulen H, Goossens A, Kelner R B, Schreuder S M, and Lubbers M J (2008) ‘Occlusive vs gauze dressings for local wound care in surgical patients: a randomized clinical trial’, Arch Surg, 143, 950–955. Valencia I C, Falabella A, Kirsner R S, and Eaglstein W H (2001) ‘Chronic venous insufficiency and venous leg ulceration’, J Am Acad Dermatol, 44, 401–421. Varghese M C, Balin A K, Carter M, and Caldwell D (1986) ‘Local environment of chronic wounds under synthetic dressings’, Arch Dermatol, 122, 52–57. Venter T H, Karpelowsky J S, and Rode H (2007) ‘Cooling of the burn wound: the ideal temperature of the coolant’, Burns, 33, 917–922. Vincent J L (2003) ‘Nosocomial infections in adult intensive-care units’, Lancet, 361, 2068–2077. Vuolo J (2004) ‘Current options for managing the problem of excess wound exudate’, Prof Nurse, 19, 487–491. Whitaker I S, Prowse S, and Potokar T S (2008) ‘A critical evaluation of the use of Biobrane as a biologic skin substitute: a versatile tool for the plastic and reconstructive surgeon’, Ann Plast Surg, 60, 333–337. Winter G D (1962) ‘Formation of the scab and the rate of epithelialisation of superficial wounds in the skin of the young domestic pig’, Nature, 193, 293–294. Wu P, Nelson E A, Reid W H, Ruckley C V, and Gaylor J D (1996) ‘Water vapour transmission rates in burns and chronic leg ulcers: influence of wound dressings and comparison with in vitro evaluation’, Biomaterials, 17, 1373–1377. Wysocki A B, Staiano-Coico L, and Grinnell F (1993) ‘Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9’, J Invest Dermatol, 101, 64–68. Yager D R and Nwomeh B C (1999) ‘The proteolytic environment of chronic wounds’, Wound Repair Regen, 7, 433–441. Zedler S, Bone R C, Baue A E, von Donnersmarck G H, and Faist E (1999) ‘T-cell reactivity and its predictive role in immunosuppression after burns’, Crit Care Med, 27, 66–72.

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7 Tissue-biomaterial interactions S. D O W N E S and A. A. M I S H R A, University of Manchester, UK

Abstract: Tissue engineering skin remains a major scientific and clinical challenge. Wound healing can be categorised into distinct phases and the stages of healing. The processes are controlled using a series of cellular and biochemical responses. In this chapter, we describe the clinical problems and the biological interactions that occur in wound healing. The processes by which novel materials can be modified, designed and developed are addressed. The underlying fundamental clinical problems are linked to the future development of new tissue engineering strategies using biomaterials. Commercialisation issues such as the regulatory route and the development of new medical devices are also considered. Key words: skin, tissue engineering, biomaterials, surface treatments.

7.1

Introduction: definitions

In order to characterise tissue-biomaterial interactions, it is imperative to lay down precise definitions for each of the terms present. Biomaterial: A biomaterial is a synthetic or natural material that is used to repair, replace or augment diseased or damaged tissue within the human body. In the specific case of advanced wound care, biomaterials would be any materials that are utilised in the treatment of wounds, which stimulates the skin’s self regeneration capabilities or, perhaps in the future, may replace the function of skin (non-remodelling skin graft replacement). Tissue: A contiguous organisational level of cells. Tissues interact to form organs, organs interact to form systems. In the case of wounds, the tissue in question would be the epidermis, dermis and subcutaneous tissue. The organ would be the skin as a whole, and the physiological system would be the integumentary system. On a macro-scale, the analysis in this chapter will focus on the interaction between the biomaterial and the epidermis or dermis tissues. However, as with most systems in biology, there is a hierarchy of processes that must be analysed in further detail. 174 © Woodhead Publishing Limited, 2011

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Overview of tissue-biomaterial interactions

An accepted overview of events at the tissue-biomaterial interface can be utilised (Williams, 1987) in order to fully characterise the process. The interactions at the interface may be broken down into four major groups, that occur concurrently but whose effects are visible over a range of time scales. For advanced wound repair therapy biomaterials, therefore, the prominence of each interaction is related to their effective time scale. The four interactions may be classed as: i. Interactions between the wound and the biomaterial surface. Exudate from the wound, proteins and biochemical agents present at the wound site will interact with the biomaterial surface. Proteins from the wound site are significant in this stage. The effective time scale is in the order of seconds to minutes. ii. The biomaterial response to the wound environment. The properties of the biomaterial determine the manner in which it reacts to the wound. A porous biomaterial may wick exudate from the wound and become saturated, whilst another biomaterial may be biodegraded by the biochemicals present at the wound site. The effective time scale is in the order of minutes to hours. iii. The tissue response to the biomaterial. The biocompatibility and physical properties of the biomaterial determine the extent to which the wound site reacts to it. A porous biomaterial may wick exudate and cause a dry wound site. If a biomaterial is not biocompatible with the wound, the tissue response can range from abnormal function to cytotoxic apoptosis. The effective time scale is seconds to days. iv. The fourth stage is the host systemic response. This is the response of the body as a whole to a biomaterial. Such reactions are rare in the case of wound repair; however, there may be certain instances where individuals are allergic to materials used. The allergic reaction would be classed as a stage four interaction between the biomaterial and the tissue (even though the reaction is systemic rather than local). The effective time scale can be seconds to years. The four stages of interaction between the biomaterial and tissue occur concurrently. However, there is an amount of interaction and interdependence between the different stages, as outlined above with the porous biomaterial wicking exudate example. A certain event during one stage may trigger or affect the outcome of another stage, and self-reinforcing cycles may be formed. Due to the time scales, and the rather unique nature of wound care implants/treatments, it is necessary to examine each stage in differing levels of detail. The most pertinent stages to wound care are the first and third,

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with surfacial interactions and local tissue responses being both the most prominent and important to consider.

7.3

Interactions at the biomaterial surface

A scheme for interactions at the biomaterial surface was proposed by Jiao and Cui (2007), wherein a layer of moisture or water molecules adheres to the biomaterial surface. This layer of moisture forms as soon as the biomaterial is exposed to the air (if humid), or when placed onto the wound site. The formation of this layer is nearly instantaneous, and precedes the rapid attachment of proteins onto the biomaterial surface. The attachment of proteins is a major step in the biological interactions at the tissue-biomaterial interface. After the attachment of proteins (a process known as adsorption), cells attach onto the surface as the adsorbed layer of proteins act in a biointegrative manner. Late stage interactions are focused on tissue-biomaterial interaction in bulk layers. The interaction of biochemical molecules and proteins present at the wound site and the biomaterial occurs over a very short time scale. Studies (Vroman, 1977) have shown that proteins attach to the biomaterial surface in a matter of seconds post-implantation. The attachment of proteins in particular depends on different driving mechanisms. A change in the Gibbs free energy of the system (increase in entropy or enthalpy) will result in a negative Gibbs free energy value for the attraction force between the protein and the biomaterial surface. Electrostatic attraction is another factor. If the biomaterial has a charge, either negative or positive, it will attract certain proteins, which will attach to its surface. This is made possible due to the bipolar nature of many proteins, and their subsequent attraction to any charged surface. Surface topography is a major consideration in protein adsorption. Work by Rechendorff et al. (2006) shows that fibrinogen, a major biochemical at wound sites, is particularly affected by the surface topology of polymeric biomaterials that it comes in contact with. Benchmarked by bovine serum albumin, Rechendorff et al. showed fibrinogen was more susceptible to changes in surface topography. The results additionally showed that this increased protein adsorption was not merely due to a corresponding increase in surface area with roughness, but that the increased adsorption was in response to increased roughness as a separate factor. This suggests that protein adsorption is indeed affected by the roughness of the biomaterial surface, and that smoother surfaces will result in lower degrees of adsorption, over and above the anticipated decrease from a lower surface area. A major determinant in the amount of protein adsorbed onto the biomaterial surface is the concentration of proteins at the wound site. A bloody

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wound would have a higher concentration of serum proteins, fibrinogen and glycoproteins that may adsorb onto the biomaterial surface. Thus the biomaterial used in advanced wound therapies must be adapted for the intended stage of usage. The protein concentrations present in chronic wounds are, for example, much lower than those at bloody wounds. Another driver is the hydrophilicity of the biomaterial. A highly hydrophilic material will not experience protein adsorption as rapidly as a hydrophobic material. A corollary to a lower rate of adsorption is also a lower amount of total protein adsorption onto the material. Hydrophilicity cannot be measured directly as a material property, hence contact angles are used. A biomaterial with a water droplet contact angle of above 90 degrees is considered hydrophobic, and will experience rapid protein attachment. This is due to the relative difference in hydrophilicity between the proteins and the biomaterial. Yet another driver is the solubility of proteins at the wound site. At different stages in the wound healing cascade, different proteins are present. The solubility of these proteins varies, and must be taken into consideration as less soluble proteins are liable to adsorb and accumulate onto the biomaterial surface. The significance of protein adsorption onto biomaterial surfaces is an oft-debated issue. Whilst empirical evidence as to the detrimental effect of the phenomenon is available, no theory has thus far been put forward as to the mechanism. Magnani (Magnani et al., 2002) showed that the adsorption of proteins onto biomaterial surfaces disrupts the normal functioning and properties of the material. Bioactive coatings are especially rendered ineffective by protein adsorption onto the surface of the biomaterial. The composition of the protein layer attached to the biomaterial surface changes over time. Vroman et al. describe the change in composition, as the lighter proteins from plasma/exudate, in particular fibrinogen (abundant in wound tissue) can be displaced at a later stage by kininogen. When designing a biomaterial for an advanced wound repair application, especially if bioactive in nature, it is imperative to know the degree of protein adsorption onto the surface and thus indirectly the level of disruption of the biomaterial’s function. There is presently no unified theory which allows the prediction of the rate of protein adsorption onto a biomaterial surface. Therefore there is a need to empirically test each biomaterial before it is used in an advanced wound repair application. The most common method to achieve this is to use radioactive labelling to attach heavy elements such as I-125 to proteins (Slack and Horbett, 1988). The test is an accurate quantitative measure of how much protein adsorbs onto the biomaterial surface. By conducting the test over time, it is possible to determine the profile of protein adsorption onto the biomaterial surface. Alternative techniques such as isomorphous replacement followed by Fourier transform infrared spectroscopy allow the observation of protein adsorption onto the

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biomaterial surface in real time. Quartz crystal microbalance is another approach that may be utilised, where the resonance of a crystal is affected by the adsorption of proteins to a surface. This results in a change in resonance, giving a measure for adsorption. The degree of polymer adsorption is determined in part by the intrinsic properties of biomaterials (namely, hydrophilicity). Quantitative testing by Brash and Uniyal (Brash and Uniyal, 1976) showed that fibrinogen attachment levels vary between 0.034 μgcm−2 and 1.09 μgcm−2 for a range of polymers going from most hydrophilic to most hydrophobic. Interestingly, the work of Castillo et al. (Castillo et al., 1984) showed that the structure of proteins adsorbed in a layer changes, and in particular the macromolecular conformation is affected by the change in microstructure. Over time, the nature of the protein layer changes as the conformation of the proteins change and desorption may occur. Amiji et al. (Amiji and Park, 1993) investigated the ability of PEO (polyethylene oxide) to act as a steric stabiliser on polymeric biomaterial surfaces in order to increase the steric repulsion force in the interfacial system. The result was that protein adsorption decreased on the surface, due to the movement of the shear layer (a bulk layer of immobilised ions) outward, and thus presenting a physical barrier to the attachment of proteins to the biomaterial surface. The PEO molecules attached to the surface of the biomaterial extend outwards in chains, due to being confined in a small area. A layer of PEO chains thus extends outwards from the surface, shifting the shear layer outward and stabilising the surface by preventing adsorption through steric forces between the shear layer and the protein. Such steric stabilisation is more reliable than electrostatic repulsion, as the amphiphilic nature of proteins discounts the universal efficacy of electrostatic forces to dissuade adsorption. Steric stabilisation also has the ability to prevent the adsorption of bacteria onto the biomaterial surface. This will be investigated in further detail later on in this chapter, as infection at the tissue-biomaterial interface is examined. In conclusion, protein adsorption onto the biomaterial surface occurs rapidly once a biomaterial is introduced into a wound site. The adsorption of proteins modifies the normal functioning of the biomaterial, and may particularly disrupt the workings of bioactive or biointegrative coatings. In order to prevent protein adsorption, a design involving steric stabilisation may be utilised, which would yield a non-fouling biomaterial surface. A non-fouling surface would also prevent the adsorption of bacteria onto the biomaterial prior to, and after, placement on the wound.

7.3.1 Biological interactions at the tissue-biomaterial interface If proteins adsorb to the biomaterial surface, cells will begin to attach to the biomaterial, encouraged by the presence of proteins. A key point is that

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the composition of the adsorbed protein layer will affect both the composition and the degree of cell attachment to the biomaterial. Wilson et al. (2005) suggest that the composition of the protein layer directly affects the composition of cells attached. They posit that key extracellular proteins, in particular fibronectin, can affect cell migration, attachment and even postadherence morphology. Whilst cellular responses are limited to the composition of the adsorbed protein layer and thus a direct correlation exists between them, it is evident that the composition of the protein layer is in turn dependent upon the intrinsic material properties of the biomaterial. Thus through control of the biomaterial properties of advanced wound therapy products, cellular responses may be controlled indirectly. In the case of advanced wound therapies, it is vital to note the specific protein groups and cells involved in the wound healing cascade. Biological interactions at the tissue-biomaterial surface are characterised by two subdivisions: specific and non-specific. Specific interactions are those caused by precise antigen-antigen reactions between the adsorbed proteins on the surface and the cells migrating onto the surface. Precise ligand reactions take place between the extracellular proteins and the cells, leading to the preferential attachment of certain types of cells onto the surface. Non-specific interactions are those caused by generic reactions that do not involve antigenic or ligand reactions. An unmodified, unstabilised hydrophobic surface would result in the non-specific adsorption of proteins from the wound site onto the biomaterial surface. This in turn would lead to the non-specific attachment of cells to proteins (on the macro-scale). The cell-protein interactions are called non-specific because when considered overall, there is no preferential attachment. It is important to note that on the chemical level, however, the cells still attach to proteins via ligand reactions. Specificity of reactions can thus be utilised, or discouraged, based on the requirements of biomaterials in advanced wound therapy applications. A modified biomaterial surface, composed of steric hindrance groups (such as PEO), however, including specific ligand attachment points for specific proteins, could lead to the preferential attachment of certain cell types. Alternatively the method could be reversed in order to promote attachment of non-specific proteins, whilst inhibiting the attachment of specific proteins and thus inhibiting the integration of certain cells. This is a very important principle, as it may be utilised to prevent new skin growth integrating into the biomaterial via surface interactions. If the attachment of epidermal cells, keratinocytes and protein precursors to the biomaterial surface can be avoided, the integration of newly regenerated tissue into the biomaterial may be avoided. If such precautions are not taken, removal of the biomaterial from the interface may result in relapsed tissue trauma as the wound is re-opened.

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7.3.2 Biointegration Whilst some tissue-biomaterial interactions are considered to be detrimental to the long-term healing of the wound, in other cases it is desirable to increase the amount of biointegration between the biomaterial and tissue. The considerations of biointegration can range in extent from a few seconds to the lifetime of a patient. Stynes et al. (2008) consider the full range of time scales. Clinical attempts at biointegrative devices for advanced wound repair have failed due to localised inflammation and fibrous encapsulation. This is the process of wound healing enveloping the implant with new tissue, isolating it from the body’s natural tissue. Work conducted thusfar on the issue has focused on both sides of the effect, with biointegration being considered in terms of both problems and benefits. Successful biointegration begins with biocompatibility. Biocompatible materials are those that do not have a detrimental effect upon either the local tissue or the body as a whole. For a biomaterial to have a high degree of biointegration with skin, it must fulfil the criteria of being noninflammatory, non-immunological and non-carcinogenic. Biointegration attempts may lead to failure of the implant if adequate precautions are not taken. Von Recum et al. (Von Recum, 1984) suggest a scheme of biointegrative failure at the tissue-biomaterial interface. Their work suggests that epidermal layer healing prevents the formation of a seal around the implant, due to the body creating new tissue surrounding the implant and preventing its interaction with other tissue (marsupialisation). This prevents long-term integration. Mechanical forces may inhibit dermal regeneration seals from forming at the tissue-biomaterial interface. Eventually, infection causes the implant to fail. Present attempts to create a biointegrative skin implant have not been successful for the reasons listed above. Von Recum studied percutaneous devices designed for long-term trans-epidermal and trans-dermal applications (such as intravenous needles). As such, parallels exist between percutaneous devices and skin implants, as both are in contact with epidermal tissue for extended periods of time. Von Recum’s theorised answer to the question of successful biointegration was the use of pores in the biomaterial surface, which would allow the ingrowth of cells. The theory shadows the approach used by tissue engineers, whose scaffolds include open macroporosity in order to encourage cell in-migration and proliferation. Thus an advanced wound therapy product which is intended to biointegrate with epidermal tissue must include an element of porosity. The biggest challenge posed by biointegration is that the implant must preferentially integrate with the epithelium at the edges of the wound, whilst preventing the epithelium from regrowing to the extent where the implant is marsupialised.

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The benefits of biointegration are numerous. The main advantage is that chronic wounds may be healed entirely in a controlled manner, as the biomaterial remodels into the patient’s own skin over time. The integration of the biomaterial into the skin, forming a seal around the biomaterial, means that the wound site moves from being non-functional to functional. For all intents and purposes, the patient would not be troubled by the wound site. Another benefit of biointegration is that the issues surrounding the need to change biomaterials over time are avoided. By designing a therapeutic product that is integrated into the very tissue it is designed to heal, the tissue sustains the product by providing it with the necessary functions (draining of exudate, etc.). Biointegration of epithelial cells is improved by the use of modified biomaterial surfaces, notably the inclusion of basement membranes. These membranes, such as laminin-5, and collagen-4, must be permanently attached to the biomaterial surface via the use of plasma surface modification techniques (Stynes et al., 2008). With a stable attachment of basement membranes on the biomaterial surface (as opposed to the transitory instantaneous protein biofilm normally formed), epithelial cells can be preferentially attached onto the biomaterial surface. The biomaterial surface and the tissue can thus be integrated to a higher extent. A major challenge in biointegration is the mechanical forces present on the epidermis layer overlying the interface. The proposed theoretical solution to this is to anchor the dermal layer to the biomaterial; however, researchers have so far been unable to fixate or adhere the tissue and biomaterial in such a manner. Pendegrass et al. (2006) used biomimicry via structured, controlled porous transcutaneous implants which utilised the same structure as the interface between deer antler and deer skin. By using controlled porosity at a specific geometry in terms of the placement of the implant into dermis, it was possible to create a better epidermal seal between the dermal tissue and the biomaterial.

7.3.3 Biomaterial response to tissue environment Biomaterials for skin repair are wide ranging, from hyaluronan, alginate, polymers and collagenic materials. The most commonly used are polymeric or collagen in nature, as these two groups of materials have suitable chemical and mechanical properties for skin repair applications. The conditions present at the wound site do not ordinarily cause the accelerated degradation of either biopolymers or collagen. Wound exudate doesn’t contain any chemical agents that can degrade polymers chemically; however, the enzyme collagenase is present (as it is used to remodel wounds during ordinary wound healing). Thus the use of a collagen matrix based

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biomaterial must take into account the presence of collagen degrading enzymes at a wound site. There are conditions under which biopolymers may begin to degrade at the wound site, due to the generation of free radicals within the polymer matrix and the subsequent free-radical de-polymerisation. Hypothetical causes of such an incidence could be any event that causes the entropic or enthalpic contribution to the energy of the system to increase. This includes mechanical shock, elevated temperatures or from sources such as x-rays or ultrasound. However, it is worth nothing that such scenarios are primarily academic in nature, and in practice would be unlikely to occur to an extent high enough to cause bulk degradation of the biomaterial surface. Williams (1987) makes special note of polymers that include hydrolysable crosslinks, particularly those polymers which are hydrophilic. Polyamides and poly (ester-urethanes) come under this category, but degrade slowly over a period of time. Williams also hypothesises that certain cellular interactions with the biomaterial may trigger degradation of the polymer, as cells release enzymes which can break down polymers, and the fractions or remnants of the degraded polymer act as catalysts for the reaction. The rate of degradation will be much higher if a porous structure is used due to the increased surface area.

7.4

Tissue response to biomaterial

Localised tissue response to the implanted biomaterial is one of the most important factors in the healing of hard to heal wounds. The major considerations are inflammation, infection, immunogenicity, cytotoxicity (necrosis) and interactive healing mechanisms.

7.4.1 Inflammation Inflammation is an abnormal tissue state where blood and plasma migrates into tissues at a higher than normal concentration. Inflammation results in (and is diagnosed by) the four classic symptoms of rubor, calor, dolor and tumour. These correspond to redness, heat, pain and swelling respectively. Inflammation occurs when leucocytes migrate into the tissue-biomaterial (wound site) interface. Macrophages soon follow, and prolong the inflammation. In addition, cytokines may propagate a higher degree of inflammation, and result in chronic inflammation. In such conditions, implant failure may occur. The degree of inflammation is related to the intrinsic material properties of the biomaterial utilised. An inflammatory reaction to a biomaterial could result in abnormal wound healing, or in the case of chronic wounds, may delay or disrupt healing.

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7.4.2 Immunogenic reaction Immunogenicity or immunoreactivity results from the biomaterial being detected by the body’s immune system as a foreign object. Immunoreactive biomaterials, especially wear particles, are detected by antigenic reactions on cells. A biochemical cascade then occurs, whereby T-helper cells migrate towards the biomaterial. This immune response can result in rejection of the biomaterial, and non-union between the biomaterial and the wound site. For bio-integrative products, this would be a significant hindrance to successful functioning. Immunogenicity may also be brought about by biomaterials that have become damaged or oxidised and therefore no longer non-immunogenic.

7.4.3 Infection Infection may occur from a high density of bacteria being present on the biomaterial surface. Staphylococcus Epidermidis, Methycilin resistant staphylococcus aureus (MRSA) and other bacteria are a major concern for polymeric products in wound repair applications. Bacteria are beneficial at low levels, and especially so in chronic wounds (Edwards and Harding, 2004), as they may in fact speed up or improve chronic wound healing. In cases where clinical symptoms of infection were not found in chronic wounds, healing was substantially harder. However, at very high levels, bacteria may cause infection and ultimately localised necrosis. This necrotic tissue inhibits wound healing, and is a major cause of chronic wounds. A primary safety criteria for a wound healing biomaterial is that it must be totally sterile when introduced to the wound, to avoid infection from harmful bacteria. However, bacteria will be naturally present at the wound site, and will adhere to the material surface.

7.4.4 Toxicity To some extent, all materials have a degree of toxicity (Stynes et al., 2008); however, to be biointegrative, a biomaterial must have a low level of cytotoxicity. The biomaterial must additionally have good wear characteristics, as small particles of any material are immunogenic within a certain size range due to macrophage phagocytosis. However, in instances where the biomaterial is toxic to the surrounding cells, there can be apoptotic cell death followed by bulk tissue necrosis. Such an event would hinder healing in a normal wound, and would cause regression in a chronic wound.

7.4.5 Carcinoma Biomaterials used must additionally be non-carcinogenic. Biomaterials that are pre-approved by the FDA including Polylactic Acid (PLLA),

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Polylactic-co-galactic acid (PLGA), Polycaprolactone (PCL), Polymethyl Methacrylate (PMMA), etc., have a long track record of use in the human body and are proven to be non-carcinogenic according to current medical knowledge and regulatory standards.

7.5

Conclusion

Tissue-biomaterial interactions are an important consideration for advanced wound repair therapies. The interactions may be split into four major categories, the most pertinent of which are the initial interactions at the tissuebiomaterial interface and the reaction of the local tissue to the introduction of a biomaterial. Surface interactions are interrelated, and occur over a range of timescales. The first stage is the near instantaneous adsorption of moisture and proteins to the biomaterial surface. These proteins then allow and affect the attachment of a layer of cells from the epithelial layer. The final stage may be the partial or total integration of the biomaterial and the tissue; however, in practice this is rarely achieved due to fibrous encapsulation of the implant, adverse cellular responses or bacterial infection at the interface. The ability to control the physico-chemical properties of biomaterials, particularly with techniques such as plasma surface modification, allows scientists to control the nature of tissue-biomaterial interactions. By controlling protein adsorption at the biomaterial surface by modifications, such as altering surface charge, or by selecting the geometry of the biomaterial (surface roughness, topography, porosity), scientists may control the extent of tissue-biomaterial interactions. Products may be designed to be either non-integrative or biointegrative depending on the aims of the treatment. As advanced wound care therapies increase in sophistication and refinement, controlling the interaction between cells and biomaterials is becoming increasingly important. The goal of creating a successful, long-term biointegrative wound dressing is only possible, if the specific tissuebiomaterial interactions are fully understood, characterised and controlled. Scientific advances, in the study of biointegration, raises the potential to create wound dressings that function as a temporary wound closure system and then slowly remodel over time into the patient’s own skin. Then the technology to help solve the problem of chronic and hard-to-heal wounds could be developed.

7.6

References

Amiji M., Park K. (1993). “Surface modification of polymeric biomaterials with poly(ethylene oxide), albumin, and heparin for reduced thrombogenicity” J. Biomater. Sci., Polymer Edition, 4, (3): 217–234(18).

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Brash J. L., Uniyal S. (1976). “Adsorption of albumin and fibrinogen to polyethylene in presence of red cells” Trans. Am. Soc. Artif. Intern. Organs. 22: 253–259. Castillo E. J., Koenig J. L., Anderson J. M. and Lo J. (1984). “Characterization of protein adsorption on soft contact lenses”, Biomaterials 5: 319. Edwards R., Harding K. (2004). “Bacteria and wound healing”, Curr. Opin. Infect. Dis. 17: 2, 91–96. Jiao Y. P., Cui F.Z. (2007). “Surface modification of polyester biomaterials for tissue engineering” Biomed. Mater. 2: R24–R37. Magnani A., Peluso G., Margarucci S. and Chittur K. K. (2002). “Protein adsorption and cellular/tissue interactions” Integrated Biomaterials Science, edited by Barbucci R. Kluwer Academic/Plenum Publishers. Pendegrass C. J., Goodship A. E. and Blunn G. W. (2006). Development of a soft tissue seal around bone-anchored transcutaneous amputation prostheses. Biomaterials 27: 4183–4191. Rechendorff K., Hovgaard M. B., Foss M., Zhdanov V. P. and Besenbacher F. (2006). “Enhancement of protein adsorption induced by surface roughness” Langmuir 22 (26): 10885–10888. Slack S. M., Horbett T. A. (1988). “The Vroman effect” proteins at interfaces II, Chapter 8, pp. 112–128. Stynes G. et al. (2008). “Tissue compatibility of biomaterials: benefits and problems of skin biointegration” Anz J. Surg. 78: 654–659. Von Recum A. F. (1984). Applications and failure modes of percutaneous devices: a review. J. Biomed. Mater. Res. 18: 323–336. Vroman L., Adams A. L., Klings A. L., Fisher G. C., Munoz P. C. and Solensky R. P. (1977). “Proteins at interfaces”, Ann. N.Y. Acad. Sci. 283: 65. Williams D. F. (1987). “Review of tissue-biomaterial interactions” J. Mater. Sci. 22: 3421–3445. Wilson Cameron J., Clegg Richard E., Leavesley David I. and Pearcy Mark J. (2005). “Adsorption of proteins and calcium phosphate materials bioactivity”, Tissue Engineering 11 (1–2): 1–18.

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8 Polymeric materials for chronic wound and burn dressings A. AG A RWA L, J. F. M c A N U LT Y, M. J. S C H U R R, C. J. M U R P H Y, and N. L. A B B O T T, University of Wisconsin-Madison, USA

Abstract: The correct balance of moisture in the wound healing environment has widely been accepted to increase the rate of wound healing and has resulted in the design of advanced moisture-retentive polymeric wound dressings. This chapter reviews various types of wound dressings currently used in clinics, and identifies design elements that promote optimal control of moisture in burns and chronic wounds. Polymer dressings for different wound types and different stages of wound healing are described with a focus on polymeric materials that give them their specific attributes. Key words: wound dressings, polymers, moisture, burns, chronic wounds, infection.

8.1

Introduction

The varied nature of wounds has resulted in the development of a wide range of wound dressings that target different aspects of the wound healing process (White and McIntosh, 2009, Joshua et al., 2008, Ovington, 2007, Nelson and Bradley, 2007, Morin and Tomaselli, 2007, Okan et al., 2007, Hanna and Giacopelli, 1997, Stashak et al., 2004). Acute wounds occur as a result of accidental trauma or burn injury, or as a result of invasive medical procedures. Chronic wounds are also exceedingly common and have many different etiologies and are associated with many disease processes including diabetes mellitus, arterial insufficiency, venous stasis disease, radiation therapy, pyoderma gangrenosum and other etiologies. Wounds may be partial thickness, i.e. lacking an epidermis but retaining viable dermis. Alternatively they may be full thickness with exposure of subcutaneous fat. Any vital structure can be exposed at the base of an acute or chronic wound. These complex wounds are particularly concerning because of the exposed tendon, bone, blood vessels or other vital structures. Chronic wounds and burns are often painful, may have associated infection, and have varied amount of associated drainage. There is no single dressing that creates an optimal environment for healing all types of wounds nor indeed for all of 186 © Woodhead Publishing Limited, 2011

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the various stages of healing of wounds (Harding et al., 2000, Chaby et al., 2007). Prior to the 1960s, woven gauze dressings were used for all wound types. Gauze dressings absorb wound exudate and maintain a dry wound environment, leading to eschar formation. It was assumed that a dry wound environment would reduce microbial burden (Jones, 2006). In 1962, Winter (1962) demonstrated in a porcine wound model that keeping the wound surface moist by covering it with an occlusive film increased the rate of epithelialization by 50%. The findings were validated in human wounds in 1963 (Hinman and Maibach, 1963). Subsequently, a paradigm shift towards moist wound healing has taken place in advanced burns and chronic wound care. It is now widely accepted that inadequate moisture in the wound bed results in poor wound healing (Okan et al., 2007). Proteolytic and fibrinolytic enzymes involved in autolytic debridement of the wound, and other soluble cytoactive factors required for cellular growth and migration become inactive in a dry environment. The formation of eschar further slows the ability of regenerative cells to migrate from the wound periphery into the wound center, hindering re-epithelialization. The eschar needs to be broken down and removed by the inflammatory process before regenerative cells such as keratinocytes are able to move in and regenerate the epithelium. The correct balance of moisture in the wound healing environment has been shown to increase the rate of epithelialization (Winter, 1962, Okan et al., 2007, Hinman and Maibach, 1963). An optimal moisture balance, usually typified by isoosmotic and iso-oncotic fluid media, in the wound has also been shown to soothe nerve endings, and thus minimize or eliminate pain (Alvarez, 1988, Okan et al., 2007). This chapter reviews the variety of polymeric materials that have been used in the design of advanced wound dressings. A specific focus is directed to identification of the design elements of each dressing that promote optimal control of moisture in burns and chronic wounds. Polymer dressings for different wound types and different stages of wound healing are described. The organization of this chapter is similar to that of several previously published reviews on wound dressings and the interested reader is directed to those prior publications for additional perspectives (Morin and Tomaselli, 2007, Hanna and Giacopelli, 1997, Stashak et al., 2004, Okan et al., 2007, Harding et al., 2000).

8.2

Advanced moisture-retentive wound dressings

Traditional dressings such as gauze, lint, or cotton wool, are non occlusive and dry out after absorbing wound exudate (Jones, 2006). Once this happens, they adhere to the wound bed. Even if they do not dry out, new blood

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vessels and granulation tissue can grow into the dressing structure (Rogers et al., 1999), thereby resulting in dressing adherence. Such adherence can lead to wound trauma, often accompanied by bleeding during painful dressing removal (Jones, 2006) as well as result in poor control over time of moisture and ionic strength at the wound surface. As this chapter demonstrates, the correct wound dressing must be used for the appropriate indication. For example, gauze may be the ideal dressing in an infected wound with a large amount of drainage. The gauze can be changed as often as needed to control the drainage until such time as the drainage decreases and it may be more appropriate to switch to an alternative dressing. To choose the optimal dressings for healing a wound, the wound must be evaluated regularly for changes in size, depth, amount of exudates, infection, necrotic tissue, condition of the surrounding tissue, and comfort level of the patient (Hanna and Giacopelli, 1997). With respect to moisture, advanced wound dressings can be organized into three categories: moisture absorbing, moisture maintaining, and moisture donating. Chronic or non-healing wounds are often stalled in the inflammatory process and therefore produce exudates for long periods. As an example, this is particularly true in the case of venous leg ulcers. In such cases, highly absorbent dressings are ideal and an outer compressive layer is essential for re-epithelialization. If wound tissues are adequately moist with minimal exudate production, such as in the debridement phases of healing or with partial thickness burns, dressings capable of maintaining the natural level of tissue hydration are needed. Alternatively, if tissue moisture levels are depleted as in diabetic foot ulcers, a dressing that can restore tissue hydration by providing moisture is required, but compression may be contraindicated. Today, clinicians have access to a comprehensive range of moist interactive polymeric wound dressings. Table 8.1 lists the performance parameters of an ‘ideal wound dressing’, as outlined by Tuner et al. (Turner, 1979). Unfortunately no single dressing can accomplish these goals. Fortunately dressings are continuously evolving and improving. Technology has progressed beyond a single layer of gauze. Modern wound dressings frequently comprise more than one polymeric layer. The layer of the dressing in direct contact with the wound is referred to as the contact layer of the dressing. The contact layer can radically change the characteristics of the dressing. Ideally in most wounds, the contact layer allows communication of the wound bed with deeper layers of the dressing but is nonadherent and does not cause epithelial debridement when dressings are changed. The contact layer should control pain, protect vital structures such as tendons, and allow drainage to pass through to the secondary layer. The remaining layers of the dressing are referred to as secondary dressings, and generally consist of fluffs or gauze padding that may function to provide support to the primary

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Table 8.1 Characteristics of an ideal wound dressing • • • • • • • • • • •

Creates a moist environment Removes excess exudate Prevents desiccation Promotes autolytic debridement Allows gaseous exchange Impermeable to microorganisms Prevents particulate contamination Nontoxic to beneficial host cells Provides mechanical protection Non-traumatic application and removal Cost-effective

dressing, provide barrier to bacteria and water, absorb wound fluid, or seal the wound in occlusive or semi-occlusive manner. There may also be an outer, compressive layer (Hanna and Giacopelli, 1997).

8.3

Polymeric materials in moist wound healing dressings

A central goal underlying the design of new dressing materials is to keep wound moisture at the optimal level (Ovington, 2007, Okan et al., 2007). If wound exudate levels exceed the absorptive capacity of a dressing, maceration of the surrounding skin can occur, leading to skin breakdown and wound enlargement. If the wound is too dry, it becomes desiccated and healing is delayed. As noted above, the large variation in wound moisture levels necessitates the choice of a dressing that interacts with the given wound characteristics to result in optimal wound healing. More than 400 different advanced wound dressings were listed in a recent issue of a wound care products buyer’s guide lists, including 25 alginates, 55 foams, 50 hydrocolloids, 51 hydrogels, and 24 transparent films (Motta, 2005). Presented below is an overview of the various types of wound dressings currently used in clinics along with the polymeric materials that give them their specific attributes. Table 8.2 (modified from Okan et al. (2007)) provides a summary of each polymer dressing type, the advantages and disadvantages, and the types of wounds for which they are indicated or contraindicated.

8.3.1 Polyurethane transparent films Based on Winter’s (1962) findings of increased epithelialization rate in wounds covered with occlusive films, the first occlusive polymeric dressings

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Advantages Elastic; conforms to wound shape; pain relief; prevent scab formation; allow continuous inspection; autolytic debridement High absorbency; long wearing times; semipermeable; thermal insulation; padding; easily shaped to wound; absorb bacteria High absorbency; hold exudate under high compression; conforms to irregular wound surface; painless removal Moisture donor effect; resist drying out; cooling analgesic effect; autolytic debridement; allow continuous inspection; painless removal

Description

Thin polyurethane semi-permeable sheet bonded to acrylamide or with acrylic adhesive layer; transparent

Absorbent hydrocellular centre with semiocclusive outer layer

Multiple layers; hydropolymer foam + woven acrylate + polyurethane backing; absorb, gel, wick, and transpire fluid

Cross-linked hydrophilic polymers with 90–95% water content; semipermeable; nonadherent; semi-transparent

Polymer dressing

Films

Foams

Hydropolymer foams

Hydrogels

Frequent dressing changes; selective gram negative bacterial proliferation; low absorbing capacity

Opaque

Nonadherent; require secondary dressing; opaque; stick to wounds; leak exudate under high compression

Minimal capacity to balance moisture; fluid accumulation; leakage; infection

Disadvantages

I: Dry, sloughy wounds with mild exudate; partial thickness wounds C: Exuding and ischemic ulcers

I: Highly exuding ulcers C: Dry wounds

I: Exuding ulcers, deep cavity wounds C: Dry wounds

I: Partial thickness wounds; IV catheter sites C: Exuding or infected wounds

Indications (I)/ Contraindication(C)

Table 8.2 Characteristics of different classes of polymeric wound dressings (adapted from Okan et al. (2007))

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Absorb exudate; gelation remove bacteria; painless removal; hemostatic; autolytic debridement

Absorb exudate; fiber strength allow packing wounds; non-adhesive; painless removal; fibers trap bacteria; autolytic debridement Atraumatic removal from the wound and surrounding skin; fluid impermeable; prevent maceration; long wearing times; transparent

Carboxymethylcellulose fibers available in sheets and ropes

Soft silicone sheets or gels; some bound to polyamide net; designed as wound contact layer with secondary dressings

Hydrofibers

Silicone

No secondary dressing required; painless removal; gas impermeable when dry promoting epithelialization; gelation with exudate aid debridement and remove bacteria

Non-adhseive polysaccharide consisting of mannuronic and galuronic acid residues; available in fiber and non-woven form; gel with exudate

Polyurethane film bonded to pectin/ gelatin/ carboxymethylcellulose gellable mass

Calcium alginates

Hydrocolloids

Require secondary absorbing dressing

Require secondary dressing/tape

Require secondary dressing/tape; foul odor and appearance of gel confused with infection

Leakage with excess fluid; may cause overhydration; foul odor and appearance of gel confused with infection

I: Second degree burns, donor sites, chronic leg ulcers, pediatric patients C: Moderate to heavy exuding wounds (usable with secondary dressing); deep wounds

I: Moderate to heavy exudate; partial and full thickness cavernous wounds with bacterial burden C: Dry and lightly exuding wounds

I: moderately exudating wounds, hemostasis postdebridement C: Diabetic foot ulcers

I: lightly exuding ulcers; burns; partial thickness wounds C: Ischemia, infection, vasculitis

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developed to maintain a moist wound healing environment were 0.2 mmthick polyurethane films (Thomas, 1996). They remain available today in both adhesive and non-adhesive formats. Adhesive films are coated with a layer of acrylic adhesive on one side. Non-adhesive films require a secondary dressing to adhere to the wound. Polyurethane films are designed to simply adhere to the skin surrounding the wound and maintain the moisture that is present within the wound environment. Film dressings are semipermeable, in that they are permeable to water vapor and oxygen, but impermeable to liquid water and microorganisms (Thomas, 1996). These so-called ‘retention films’ maintain the moisture balance in the wounds and provide pain relief by preventing dehydration of the wound surface and bathing the exposed nerve endings in physiological wound secretions. In shallow wounds, eschar formation is prevented and epidermal regeneration takes place at an enhanced rate, compared with traditional dry dressings (Hormbrey et al., 2003). Polyurethane film dressings are most commonly used for treating superficial wounds, including intravenous catheter sites (Hoffmann et al., 1992) and split skin graft donor sites (Hormbrey et al., 2003). They are also used to cover newly healed wounds to provide a layer of added protection from mechanical trauma (Jones and Milton, 2000). The transparency of the polyurethane films allows monitoring the wound healing without removing the dressings. These dressings have strong adhesive properties that allow them to be placed over moving surfaces such as joints, but they also can be associated with tearing of the normal surrounding skin upon removal, unless the adhesive bond is weakened by stretching of the dressing prior to removal (Campbell et al., 2000). Recently developed removal techniques help to minimize this issue (Hormbrey et al., 2003) but do not eliminate it entirely. Prevention of absorption of wound exudate is another issue with these polymeric film dressings. Fluid accumulation below the films can lead to maceration of surrounding skin. The build-up of fluid can also break the seal to the external environment, facilitating bacterial proliferation (Hoffmann et al., 1992). They are therefore used for wounds with negligible or low-levels of wound exudate (Jones and Milton, 2000).

8.3.2 Foam dressings Foam dressings are designed to deal with moderate to high levels of wound exudate. They vary in composition and levels of absorbency. Simple foam dressings are made with a porous hydrocellular polyurethane center that is laminated with a semi-occlusive film backing (Fletcher, 2005). Fluid is taken into the open cells of the foam structure by physical absorption. The foams do not liquefy or break down after absorbing the exudate. They therefore leave no particulate matter in the wound. They adhere to the surrounding

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skin, but do not adhere to the wound bed and thus allow non-traumatic removal. The semi-permeable film backing on the foam structure allows water vapor and gases to pass through the dressing while providing a barrier to liquid water and bacteria. The fluid-handling characteristics of foam dressings rely on two physical processes – absorbency and moisture vapor transmission (MVT) (Carter, 2003). Absorbency is determined largely by the porosity of the foam (i.e. volume fraction of the pores); the MVT rate is determined by the permeability of the backing film. After initial fluid uptake into the foam structure, the evaporation of the fluid through the backing film largely controls the fluid management in the dressing. The MVT keeps the dressing from becoming saturated. As evaporation takes place through the film backing, additional moisture can be removed from the wound through absorption into the foam (Carter, 2003). Through MVT, the total amount of moisture removed from a wound can exceed the absorption capacity of the foam. More permeable backing films permit higher evaporation rates thus providing the potential for longer wear times. Second generation foams have variable pore sizes (Groves and Lawrence, 1985). Smaller pore sizes impart greater moisture retention properties. Larger pore sizes allow for increased moisture exchange between wounds and the dressing, which can be particularly important for healing of ulcers. Some foam dressings have a secondary coating at the dressing/ wound surface junction such as an adhesive or soft silicone layer. The hydrophobic silicone layer prevents adhesion to the wound bed and reduces trauma and pain during dressing removal (Malone, 1987). Foam dressings are suggested for use in ulcers with high levels of exudate (Payne et al., 2009, Zimny et al., 2001, Bergan et al., 2006). They can be easily shaped to fit wounds of any size and depth. They are contraindicated in dry wounds where they may adhere to a wound bed if the exudation rate is low or has decreased during use (Malone, 1987). Under high compression, such as body weight or a secondary dressing, foam dressings may get crushed and thus lose their ability to hold the exudate within the foam cores. This may result in maceration of the surrounding skin area. The opacity of the dressing also requires that the dressing be removed for wound inspection.

8.3.3 Hydropolymer foam dressings Hydropolymer foam dressings are a new class of foam dressings that absorb and retain wound exudate even under high compression. These dressings are complex constructs composed of three separate layers. The wound contact layer is a hydropolymer – a polyurethane polymer with a strong affinity for water; the second layer is a superabsorbent acrylate ‘wicking

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layer’, and the third outer layer is an adhesive polyurethane backing (Ovington, 2000). In the wound contact layer, polyurethane is foamed to create closed cells. The foam swells and expands when fluid is absorbed, allowing it to fill the irregular contours of the wound. The polyurethane foam can take up to 15 times its weight in fluid (Ovington, 2000). The middle layer, the so-called wicking layer, consists of woven acrylate viscose/rayon material. Acrylate is a highly absorbent material, capable of absorbing up to 300 times its own weight. It gels upon contact with the exudate fluid, thus retaining the absorbed exudate. The viscose/rayon material also wicks exudate to the semipermeable polyurethane backing (the outer layer) which permits transfer of moisture vapor and gases into the atmosphere (Ovington, 2000). Through the combined mechanisms of absorption, gellation, wicking and evaporation, hydropolymer dressings remain moist and do not allow fluid back into the wound even when used under high compression (Carter, 2003). This combination of materials helps prevent maceration of surrounding skin, minimize dressing leakage, and increase dressing wear time. Hydropolymer dressings have been demonstrated to provide better fluid handling performance under high compression in exuding ulcers when compared to alginate and hydrocolloids dressings (see below) (Thomas, 1997, Schulze et al., 2001, Curt and Holger, 2005).

8.3.4 Hydrogels Hydrogels are composed of three-dimensional networks of hydrophilic polymers with a water content that is typically 90 to 95% by mass. Polymers frequently used in synthetic hydrogel dressings include polyvinyl pyrrolidone, polyacryalmide, or polyethylene oxide (Eisenbud et al., 2003). The hydrogels are formed by covalently crosslinking polymers using free radical reactions, chemically or by photoactivation using UV light or electron beams (Eisenbud et al., 2003). Depending on the extent of crosslinking and the degree of hydration, hydrogels can be created in physical forms that range from amorphous gels to semi-stiff sheets (Eisenbud et al., 2003, LayFlurrie, 2004). Hydrogel sheet dressings have sufficient structural integrity to function without a secondary dressing. They are used as primary dressings for shallow, flat wounds. Depending on the amount of drainage in the wound, hydrogel sheets have a significant advantage in that they may remain in place up to four days. In contrast to hydrogel sheets, amorphous hydrogel dressings can conform to the irregular contours of a wound bed, but must be held in place by secondary dressings. Amorphous hydrogels are available in tubes, spray bottles and foil packets. Hydrogels pre-applied to ordinary cotton gauze

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pads are also available, and are particularly useful in tunneled and undermined wounds because the dressing is able to be easily placed into and can fill in the potential space. The most useful attribute of hydrogel dressings has been their ability to hydrate the wound surface and resist drying of the wound (Thomas and Hay, 1995). They may be useful in those cases with exposed structures that are susceptible to dehydration such as tendon. Hydrogels also exhibit a ‘moisture donor’ effect for necrotic wounds that require debriding. By increasing the moisture content of the necrotic tissue and increasing collagenase production, hydrogels facilitate autolytic debridement (Flanagan, 1995). Hydrogel dressings interact with aqueous solutions by swelling to an equilibrium value. As they swell, they also trap wound debris and bacteria in the gel matrix, thus potentially reducing wound bioburden. They have water vapor permeability comparable to a semipermeable membrane (Thomas and Hay, 1995), thus providing some dynamic moisture balance in the wound. Hydrogel dressings are easily and painlessly removed from the wound bed because the moist interface between the dressing and the wound prevents dressing adherence. Hydrogel dressings are not useful in wounds with excessive exudate, and hydrogel sheets should not be used in wounds that are infected. By virtue of their water content, hydrogel dressings have a cooling influence on the wound that provides an analgesic effect and is hypothesized to reduce the inflammatory process (Jandera et al., 2000). Application of hydrogel dressings on wounds therefore results in almost immediate reduction in pain. The cooling effect may last for some six hours, and is shown to be particularly beneficial in burns and partial-thickness wounds (Coats et al., 2002). Because hydrogels are crosslinked, it is possible to use them to encapsulate drugs or biomolecules and utilize them for the delivery of topical wound medications (e.g. metronidazole and silver sulfadiazine) (Joshua et al., 2008). The loading and release of the drugs from the hydrogels can be controlled by the degree of chemical crosslinking in the gel. These qualities are discussed in more detail in Chapter 14 of this book on ‘Drug delivery dressings’. Several clinical studies (Lay-Flurrie, 2004) report on the benefits of hydrogel wound dressings with respect to providing a moist healing environment, reducing pain, and promoting autolytic debridement that leads to quicker healing and cost savings when compared to saline gauze dressings (Agren, 1998, Flanagan, 1995). The most frequent uses of hydrogel dressings have been in the management of pressure ulcers (Bale et al., 1998), diabetic foot ulcers (White and McIntosh, 2009), and burns (Coats et al., 2002, Jandera et al., 2000). Hydrogel dressings are thought to be best indicated

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for use on clean wounds during the inflammatory and debridement phases of wound healing (Lay-Flurrie, 2004, Stashak et al., 2004).

8.3.5 Hydrocolloids Hydrocolloid dressings consist of a thick, self-adhesive layer of a gellable mass that is applied to a flexible polyurethane film. The gellable mass is composed of a hydrocolloid dispersed with the aid of a ‘tackifier’ such as mineral oil and terpene resin (Stashak et al., 2004). The hydrocolloids typically combine gelatin, pectin and carboxymethyl cellulose (CMC) particles either suspended in polyisobutylene or embedded in an elastic mesh. Hydrocolloids are able to absorb fairly large amounts of wound fluid, and they are often referred to as hydroactive dressings (Eaglstein, 1985, Ferrari et al., 1995). The principal advantage offered by hydrocolloid dressings is that in their intact state, they are virtually impermeable to water vapor and therefore promote formation of a moist healing wound environment. The impermeable nature provides a protective covering and helps prevent the spread of pathogenic microorganisms (Eaglstein, 1985). These dressings are oxygen impermeable, which is claimed to increase the rate of epithelialization and collagen synthesis and decrease the pH of the wound exudates, thus reducing bacterial counts (Stashak et al., 2004). In presence of exudates, hydrocolloids absorb liquid and form a gel (Ferrari et al., 1995). As they do so, they become more permeable to moisture vapor which further increases their ability to cope with wound exudate (Stephen, 2008). The first hydrocolloid dressings (1982–83) did not perform well when exudate levels in wounds were high. Revised formulations containing new compositions of hydrocolloids have been developed for wounds with high levels of exudates. Hydrocolloid dressings are most commonly associated with the treatment of ulcerative conditions such as pressure sores and lower extremity ulcers, where they have been shown to be more effective than gauze dressings with respect to ulcer healing, pain involved in dressing changes, absorption capacity, side-effects, and cost (Heyneman et al., 2008). They also offer benefits for the management of several acute wounds, including superficial and partial thickness burns, donor sites, surgical wounds, superficial trauma, and post-operative pediatric wounds (Stephen, 2008). The principal benefit of hydrocolloid dressings in application to burn and donor sites is the reduction of wound pain. For use in heavily exuding wounds hydrocolloids dressings have been largely replaced by other products such as foams and alginates but they remain a mainstay for use on lightly exuding wounds.

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8.3.6 Calcium alginate Calcium alginate dressings are made from salts of alginic acid obtained from algae (Phaeophyceae sp.) found in seaweed. They are known for absorbing excess wound exudate and forming a non-adherent gel, which accelerates wound healing by promoting a moist wound healing environment, facilitating debridement, and helping to prevent trauma to the wound bed and the surrounding skin (Fanucci and Seese, 1991). Alginic acid is a polysaccharide consisting of mannuronic and galuronic acid residues. The sodium and calcium salts of alginic acid are prepared by the alkaline extraction of seaweed cell walls. The resultant colloidal solution of sodium alginate is precipitated by addition of calcium chloride, and the precipitate is re-dissolved using sodium carbonate to generate alginate with a mixture of sodium and calcium counter ions (Thomas, 2000). Various alginate dressings are available and differ in chemical and physical properties, depending on the proportion of mannuronic and galuronic acid residues (which depends on its botanical source) and the content of calcium and sodium ions (Seymour, 1997). A high content of mannuronic acid promotes gelling, and high galuronic acid content promotes fiber integrity for packing. Originally, alginate dressings were available as a loose fleece formed from calcium alginate fibers. More recently, they have also been fabricated with fibers woven to form a more cohesive structure, which increases the fabric’s strength when soaked with wound fluid. Other dressings have been produced from freeze-dried alginate (Thomas, 2000). Alginate fibers absorb wound exudate to form a gel matrix. Ion exchange occurs between the calcium ions of the alginate fibers and sodium ions in the exudate. When a significant proportion of the calcium ions in the fibers have been replaced by sodium, the fibers swell to form a gel on the wound surface. The gellation of the fibers also permits easy removal of the dressing (Thomas, 2000). Some dressing formulations also contain a significant proportion of sodium alginate to improve the gelling properties of the dressing. Alginate fibers have been shown to absorb and retain bacteria during formation of the gel matrix, which can be removed during dressing changes (Fanucci and Seese, 1991). The gel absorbs moisture and maintains an appropriately moist environment for optimal healing. Alginate dressings can absorb up to 20 to 30 times their weight in wound fluid (Seymour, 1997). Calcium alginate dressings are used on moderate to heavily exudative wounds during the transition from debridement to repair phase of wound healing (Seymour, 1997, Joël et al., 2002). Dry wounds should not be treated with these dressings because they have no hydrating properties. Calcium alginate dressings have also been demonstrated to have haemostatic property. The dressings improve clotting in wounds and are also used

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to promote hemostasis during various surgical procedures (Segal et al., 1998). The calcium ions released from the dressings are known to promote the activation of prothrombin in the clotting cascade (Segal et al., 1998). The entangled fibrous structure of the dressing further contributes to the effectiveness in blood coagulation. Zinc has been added to some alginate dressings to increase their hemostatic qualities. In addition to their moisture handling capabilities, alginate dressings have also been suggested to promote healing via a direct modulatory effect on wound macrophages. Some dressing have the potential to activate human macrophages to secrete pro-inflammatory cytokines within the chronic wound bed that is hypothesized to generate a pro-inflammatory signal which may initiate a stalled inflammatory phase of wound healing in chronic wounds (Thomas et al., 2000). Reportedly, dressings increase epithelialization and granulation tissue formation (Sayag et al., 1996). Because of these attributes, calcium alginate dressings are considered bioactive.

8.3.7 Hydrofiber dressings These dressings are characterized by absorptive fibers that minimize adhesion to the wound, increase absorption, retain exudate, and permit painless removal. Hydrofiber dressings are composed of highly absorbent sodium carboxymethylcellulose (CMC) fibers, and are available as sheets or ropes. CMC hydrofibers become fully hydrated with wound exudate and form a continuous, strong, cohesive gel. Compared to alginate fiber dressings, hydrofiber dressings provide better retention of wound exudate and absorbed bacteria (Waring and Parsons, 2001, Masahiro et al., 2004). CMC is not bioresorbable, thus leaving no particulate matter in the wound bed. Carboxymethylcellulose fibers have been shown to adhere to fibrin and fibronectin in the wound bed (Richters et al., 2004). Upon gelation with wound exudate, fibres uptake neutrophils into the gel matrix. This is proposed to promote autolysis and outgrowth of the keratinocytes (Richters et al., 2004). Hydrofibers have good fiber strength and can be packed loosely into sinuses (Kastl et al., 2009). Like foams, they are also non-adhesive and require a secondary dressing to stay in place. In a clinical trial, performance of the hydrofiber dressings in partial thickness burn wounds resembled (was similar to) that of cadaver skin (a commonly used dressing for burn patients) with respect to adherence to the wound, wound infection, healing time, and need for wound excision and healing time (Vloemans et al., 2001). During the healing process, the fibers dry out due to evaporation, and the dressing could be easily removed as a dry ‘crust.’ The removal can be performed without disturbance to the new epidermis. In venous ulcers, hydrofiber dressings demonstrated a 130% increased chance of healing compared to

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gauze dressings, with significant reduction in health care costs (Guest et al., 2005, Guest and Ruiz, 2005).

8.3.8 Silicone Soft silicone gel and sheet dressings are specifically designed for atraumatic removal from the wound and surrounding skin to improve the performance of the dressing with regard to pain management (Williams, 1995, Morris et al., 2009). The hydrophobic silicone layer prevents the dressing from adhering to the wound surface. Silicone maintains contact with the wound without causing friction and shear, thereby reducing tear force on removal. Non-adhesive soft silicone dressings are particularly useful for painful wounds, such as second degree burns, donor sites and chronic leg ulcers, and are used in pediatric patients (Morris et al., 2009, White and Morris, 2009, Zillmer et al., 2006, Cunningham, 2005). Soft silicone dressings act as an interface layer between the wound bed and the secondary absorbent dressing, and are used to provide an optimal environment for wound healing. Importantly, these primary wound dressings address the issue of adherence, trauma, and pain (Morris et al., 2009, Zillmer et al., 2006). Some foam dressings (as described above) have a secondary coating of a soft silicone layer at the wound interface to reduce trauma to the wound bed and pain during dressing removal (Malone, 1987). Dressings made of medical grade silicone gel bound to a soft and pliable polyamide net are also available (White and Morris, 2009). They do not adhere to the moist wound surface but do adhere to the adjacent healthy skin, sealing the wound margins and ensuring lateral fluid impermeability to the surrounding skin and minimizing maceration. These soft silicone sheets mold to the uneven surface of skin and tend to spread peeling forces on removal. The open net structure allows exudate to pass directly into a secondary absorbent dressing, maintaining a well-balanced moisture environment. The primary dressing can be left in place for extended periods of time while allowing secondary dressing changes, as required, thus avoiding disturbance to the wound-bed (White and Morris, 2009).

8.4

Infection control by polymeric wound dressings

Microbial infections are encountered in almost all cases of chronic wounds, resulting in prolonged hospitalization and frequent dressing changes (Bowler, 2002). Prior to 1962, it was thought that maintenance of a dry wound would kill bacteria and thus reduce infection. Subsequently, it was realized that dry wounds slow healing processes by inactivating key enzymes. However, an initial concern with moisture retentive dressings was that a

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sealed, moist environment would promote bacterial proliferation and infection. Several reports have documented that the incidence of wound infections associated with moisture retentive dressings is actually lower than that observed with ointments and gauze dressings (Field and Kerstein, 1994, Hutchinson and McGuckin, 1990, Hutchinson and Lawrence, 1991, Rosenfeldt et al., 2003, Madden et al., 1989). Several attributes of modern dressings are thought to be responsible for reduction bacterial colonization and wound infection. First, semi-occlusive dressings provide barriers to the entry of bacteria into the wound-bed, thus reducing incidence of secondary infections. Second, the sealing of the wound maintains a fluid circulation that prevents colonization of bacteria. Third, promotion of autolytic debridement of the wound bed and activation of macrophages suppress tissue necrosis and prevents the formation of biofilms in the wound. Lastly, some occlusive dressings lead to changes in the pH of the wound fluid (to mildly acidic) which kills the bacteria and reduces microbial burden. Exudate absorbing dressings such as foams, hydrocolloids, and hydrofibers further remove bacteria from the wound surface as exudate is absorbed into the dressings (Bowler et al., 1999). Moreover, dressings with high-levels of fluid retention can immobilize the bacteria in the polymer matrix, reducing the incidence of cross-infection. They have therefore been cited as useful for wound bed preparation in cases where there is severe microbial colonization (Schultz et al., 2003). Moisture retentive hydrocolloid dressings were shown to reduce the dispersal of bacteria by 20% when compared to gauze dressings when the dressings were removed from simulated wounds and clinical wounds (Lawrence, 1994). Fibrous absorbent dressings, such as hydrofiber and calcium alginate dressings, have been shown to be particularly efficient in trapping bacteria as their fibers gel in wound exudate. Gelation can also block fluid flow, thereby further immobilizing bacteria within the gel matrix. Several recent studies have compared the bacterial retention properties of carboxymethylcellulose (CMC) fibers and alginate fiber dressings (Bowler et al., 1999, Masahiro et al., 2004). A study in an infected animal wound model found that although CMC and alginate dressings did not show any difference in the reduction of bacterial counts in tissue biopsies, the CMC dressings retained up to ~85% of bacteria trapped within the dressings when soaked in saline, compared to ~30% by one alginate dressing and ~60% by another alginate dressing (Masahiro et al., 2004). Investigations using electron microcopy have demonstrated that CMC hydrofibers become fully hydrated with wound exudate and form a continuous, strong, cohesive gel, providing high retention of wound exudate (Waring and Parsons, 2001). In contrast, alginate fibers do not fully hydrate and form a weak gel with patches of gel and fibers (Walker et al., 2003). Thus, hydrofiber

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dressings may theoretically have an advantage in reducing cross-infection by wound-pathogens in clinical settings. Although semi-occlusive dressings that provide moisture balance reduce the rates of infection in chronic wounds as compared to gauze dressings, bacterial proliferation has frequently been found under these dressings (Wynne et al., 2004). The use of antibacterial agents, such as silver, iodine, polyhexamethylene biguanide (PHMB) and chlorhexidine based compounds is therefore common during treatments with these dressings (Landis, 2008). The latest trends are to incorporate these antibacterial agents into wound dressings that provide sustained release of these antimicrobial agents so as to reduce the frequency of dressing changes, reducing patient pain and improve overall healing outcomes (Mueller and Krebsbach, 2008, Blome-Eberwein et al., 2010).

8.5

Conclusion

Since the adoption of the moist-wound healing concept almost three decades ago, polymeric materials used in wound dressings have advanced substantially (Douglas et al., 2004). Following the early use of polyurethane films for sealing wounds, dressing agents with more desirable physical properties and more complex composite dressings composed of materials such as hydrocolloids, hydrofoams, and alginates have been developed to maintain a dynamic moisture balance in the wound microenvironment. Their development has also focused on non-adherence to wounds and painless dressing changes. Hydrogels were developed to hydrate dry wounds and restore wound healing. Continued use of these polymeric dressings has provided insight into their advantages as well as their limitations such as skin stripping, adhesion to wounds, maceration and drying. This has led manufacturers to develop second and third generation dressings. Some variants include dressings with improved wound contact surfaces to reduce adhesion. Absorptive fibers and next-generation hydrocolloids were developed to minimize adhesion to wound bed (permiting painless removal and reduced epithelial losses), and increase absorption. Specialized wound contact materials such as soft silicone sheets have been purposely developed for non-traumatic dressing changes. As knowledge in this area evolves based on clinical data, more sophisticated dressings with advanced polymeric materials are expected to be developed. A selected menu of commercially available advanced wound care dressings is provided in Table 8.3. Dressings must be chosen with care keeping in mind the major objective of the dressing being applied. For example, skin graft donor sites usually heal without any further intervention but have a moderate amount of drainage and are associated with significant pain in those with burn injury that require skin grafts. The donor site dressing should be selected to control

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Table 8.3 List of some commercially available advanced wound care dressings, classified according to their polymer functions Polymer type

Commercial dressing name

Company

Semi-permeable films

Opsite Flexifix Tegaderm Polyskin II; Blisterfilm Mepore Film Bioclusive

Smith & Nephew 3M Covidien Molnlycke Healthcare AB Systagenix

Foams

Biatain; Contreet Foam Tegasorb Lyofoam CarraSmart COPA Mepilex; Mesorb

Coloplast 3M ConvaTec/CVL Carrington Covidien Molnlycke Healthcare AB

Hydropolymer foams

Allevyn Alione Sof-Foam; Tielle

Smith & Nephew Coloplast Systagenix

Hydrocolloids

Replicare Comfeel Tegaderm Hydrocolloid DuoDerm Nu Derm Ultec

Smith & Nephew Coloplast 3M ConvaTec/CVL Systagenix Covidien

Hydrogels

Intrasite; Flexigel; Cutinova Hydro Tegaderm Hydrogel Tegagel Nu Gel Carrasyn Curasol Aquaflo

Smith & Nephew 3M ConvaTec/CVL Systagenix Carrington Health point Covidien

Alginate

Algisite M Comfeel Plus; SeaSorb Soft Tegaderm Alginate KALTOSTAT Fortex Silvercell; Fibracol Carraginate CURASORB Sorbsan Melgisorb

Smith & Nephew Coloplast 3M ConvaTec/CVL Systagenix Carrington Covidien Dow Hickam Molnlycke Healthcare AB

Hydrofiber

Aquacel

ConvaTec/CVL

Silicone sheets

CICA-CARE Mepitel; Mepitac; Mepilex

Smith & Nephew Molnlycke Healthcare AB

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drainage, maintain the moist healing environment, and minimize pain. Likewise, gauze may be appropriate for the initial therapy of infected wounds until the drainage decreases and a switch is made to an alternative. Additionally the cost of the dressing must be balanced with the cost of the dressing change. For example, a more expensive dressing that can last more than one day may be a cheaper alternative if the dressing change requires a home visit from a nurse or other health care provider. Finally, no dressing is optimal for all the different types of wound or for all stages of the wound healing process. As summarized by Okan et al., ‘clinical creativity and trial and error are needed to find a treatment plan that works best for the wound and, ultimately, for the holistic care of the patient’ (Okan et al., 2007).

8.6

Future trends

The latest trends in advancement of polymeric wound dressings are to include bioactive agents, such as ionic or nanocrystalline silver (Lu et al., 2008, Blome-Eberwein et al., 2010) or analgesics (Finn et al., 2008) that offer additional anti-bacterial and anti-inflammatory actions in chronic wounds. In addition, protease-modulating matrix dressings have been proposed to significantly accelerate healing times (Shingel et al., 2006, Romanelli et al., 2007). Other trends include the development of composite dressings of synthetic polymers and biologic materials, including collagen, hyaluronic acid and chondritin sulfate (Veves et al., 2002, Meaume et al., 2008). The addition of these substances reportedly increases epithelialization and granulation in burns and chronic wounds. These various trends in the development of biomaterials and active wound dressings have been discussed in detail in recent publications (Shu-Fen et al., 2008, Gottrup et al., 2009, Shores et al., 2007) and are reviewed in other chapters in this book.

8.7

Acknowledgements

Authors acknowledge NIH grant# 1RC2AR058971-01 from NIAMS. A.A. acknowledges support via a fellowship from the Ewing Marion Kauffman Foundation.

8.8

References

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Fletcher J (2005), ‘Understanding wound dressings: Foam dressings’, Nursing Times, 101, 50–51. Gottrup F, Jorgensen B & Karlsmark T (2009), ‘News in wound healing and management’, Current Opinion in Supportive & Palliative Care, 3, 300–304. Groves A & Lawrence J (1985), ‘Silastic foam dressing: An appraisal’, Annals of the Royal College of Surgeons of England, 67, 116–118. Guest J F & Ruiz F J (2005), ‘Modelling the cost implications of using carboxymethylcellulose dressing compared with gauze in the management of surgical wounds healing by secondary intention in the US and UK’, Current Medical Research and Opinion, 21, 281–290. Guest J F, Ruiz F J, Mihai A & Lehman A (2005), ‘Cost effectiveness of using carboxymethylcellulose dressing compared with gauze in the management of exuding venous leg ulcers in Germany and the USA’, Current Medical Research and Opinion, 21, 81–92. Hanna J R & Giacopelli J A (1997), ‘A review of wound healing and wound dressing products’, The Journal of Foot and Ankle Surgery, 36, 2–14. Harding K G, Jones V & Price P (2000), ‘Topical treatment: which dressing to choose’, Diabetes/Metabolism Research and Reviews, 16, S47-S50. Heyneman A, Beele H, Vanderwee K & Defloor T (2008), ‘A systematic review of the use of hydrocolloids in the treatment of pressure ulcers’, Journal of Clinical Nursing, 17, 1164–1173. Hinman C D & Maibach H (1963), ‘Effect of air exposure and occlusion on experimental human skin wounds’, Nature, 200, 377–378. Hoffmann K K, Weber D J, Samsa G P & Rutala W A (1992), ‘Transparent polyurethane film as an intravenous catheter dressing: A meta-analysis of the infection risks’, The Journal of the American Medical Association, 267, 2072–2076. Hormbrey E, Pandya A & Giele H (2003), ‘Adhesive retention dressings are more comfortable than alginate dressings on split-skin-graft donor sites’, British Journal of Plastic Surgery, 56, 498–503. Hutchinson J J & Lawrence J C (1991), ‘Wound infection under occlusive dressings’, Journal of Hospital Infection, 17, 83–94. Hutchinson J J & McGuckin M (1990), ‘Occlusive dressings: A microbiologic and clinical review’, American Journal of Infection Control, 18, 257–268. Jandera V, Hudson D A, de Wet P M, Innes P M & Rode H (2000), ‘Cooling the burn wound: evaluation of different modalites’, Burns, 26, 265–270. Joël B, Sylvie M, Marie-Thérèse R & Serge B (2002), ‘Sequential treatment with calcium alginate dressings and hydrocolloid dressings accelerates pressure ulcer healing in older subjects: A multicenter randomized trial of sequential versus nonsequential treatment with hydrocolloid dressings alone’, Journal of the American Geriatrics Society, 50, 269–274. Jones V & Milton T (2000), ‘When and how to use adhesive film dressings’, Nursing Times, 96, 3–4. Jones V J (2006), ‘The use of gauze: will it ever change?’ International Wound Journal, 3, 79–86. Joshua S B, Kerr H M, Howard N E S & Gillian M E (2008), ‘Wound healing dressings and drug delivery systems: A review’, Journal of Pharmaceutical Sciences, 97, 2892–2923.

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Kastl K G, Betz C S, Siedek V & Leunig A (2009), ‘Effect of carboxymethylcellulose nasal packing on wound healing after functional endoscopic sinus surgery’, American Journal of Rhinology & Allergy, 23, 80–84. Landis S J (2008), ‘Chronic wound infection and antimicrobial use’, Advances in Skin & Wound Care, 21, 531–540. Lawrence J C (1994), ‘Dressings and wound infection’, American Journal of Surgery, 167, 21S–24S. Lay-Flurrie K (2004), ‘The properties of hydrogel dressings and their impact on wound healing’, Professional Nurse, 19, 269–273. Lu S, Gao W & Gu H Y (2008), ‘Construction, application and biosafety of silver nanocrystalline chitosan wound dressing’, Burns, 34, 623–628. Madden M R, Nolan E, Finkelstein J L, Yurt R W, Smeland J, Goodwin C W, Hefton J & Staiano-Coico L (1989), ‘Comparison of an occlusive and a semi-occlusive dressing and the effect of the wound exudate upon keratinocyte proliferation’, The Journal of Trauma, 29, 924–931. Malone W (1987), ‘Wound dressing adherence: a clinical comparative study’, Archives of Emergency Medicine, 4, 101–105. Masahiro T, Shinichi H, Yoshiyuki Y, Yasutoshi S & Philip B (2004), ‘Comparison of bacteria-retaining ability of absorbent wound dressings’, International Wound Journal, 1, 177–181. Meaume S, Ourabah Z, Romanelli M, Manopulo R, De Vathaire F, Salomon D & Saurat J-H (2008), ‘Efficacy and tolerance of a hydrocolloid dressing containing hyaluronic acid for the treatment of leg ulcers of venous or mixed origin’, Current Medical Research and Opinion, 24, 2729–2739. Morin R J & Tomaselli N L (2007), ‘Interactive dressings and topical agents’, Clinics in Plastic Surgery, 34, 643–658. Morris C, Emsley P, Marland E, Meuleneire F & White R (2009), ‘Use of wound dressings with soft silicone adhesive technology’, Paediatric Nursing, 21, 38– 43. Motta G (Ed.) (2005) WOUNDSOURCE: The Kestrel wound product sourcebook, Bristol, Vermont, Kestrel Health Information Inc. Mueller S W & Krebsbach L E (2008), ‘Impact of an antimicrobial-impregnated gauze dressing on surgical site infections including methicillin-resistant Staphylococcus aureus infections’, American Journal of Infection Control, 36, 651– 655. Nelson E A & Bradley M D (2007), ‘Dressings and topical agents for arterial leg ulcers’, Cochrane Database of Systematic Reviews. Okan D, Woo K, Ayello E A & Sibbald G (2007), ‘The role of moisture balance in wound healing’, Advances in Skin & Wound Care, 20, 39–53. Ovington L G (2000), ‘Evolution in the Rainforest: The case for hydropolymer dressings’, Advances in Skin & Wound Care, 13, 4–8. Ovington L G (2007), ‘Advances in wound dressings’, Clinics in Dermatology, 25, 33–38. Payne W, Posnett J, Alvarez O, Brown-Etris M, Jameson G, Wolcott R, Dharma H, Hartwell S & Ochs D (2009), ‘A prospective, randomized clinical trial to assess the cost-effectiveness of a modern foam dressing versus a traditional saline gauze dressing in the treatment of stage II pressure ulcers’, Ostomy Wound Management, 55, 50–55.

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Richters C, du Pont J, Mayen I, Kamperdijk E W A, Dutrieux R P, Kreis R W & Hoekstra M J (2004), ‘Effects of a hydrofiber dressing on inflammatory cells in rat partial-thickness wounds’, Wounds, 16, 63–70. Rogers A, Walmsley R, Rippon M & Bowler P (1999), ‘Adsorption of serum-derived proteins by primary dressings: implications for dressing adhesion to wounds’, Journal of Wound Care, 8, 403–406. Romanelli M, Dini V & Romanelli P (2007), ‘Hydroxyurea-induced leg ulcers treated with a protease-modulating matrix’, Archives of Dermatology, 143, 1310–1313. Rosenfeldt F L, Negri J, Holdaway D, Davis B B, Mack J, Grigg M J, Miles C & Esmore D S (2003), ‘Occlusive wrap dressing reduces infection rate in saphenous vein harvest site’, The Annals of Thoracic Surgery, 75, 101–105. Sayag J, Meaume S & Bohbot S (1996), ‘Healing properties of calcium alginate dressings’, Journal of Wound Care, 5, 357–362. Schultz G S, Sibbald R G, Falanga V, Ayello E A, Dowsett C, Harding K, Romanelli M, Stacey M C, Teot L & Vanscheidt W (2003), ‘Wound bed preparation: a systematic approach to wound management’, Wound Repair & Regeneration, 11, S1–S28. Schulze H, Lane C, Charles H, Ballard K, Hampton S & Moll I (2001), ‘Evaluating a superabsorbent hydropolymer dressing for exuding venous leg ulcers’, Journal of Wound Care, 10, 511–518. Segal H C, Hunt B J & Gilding K (1998), ‘The effects of alginate and non-alginate wound dressings on blood coagulation and platelet activation’, Journal of Biomaterials Applications, 12, 249–257. Seymour J (1997), ‘Alginate dressings in wound care management’, Nursing Times, 93, 49–52. Shingel K I, Di Stabile L, Marty J & Faure M (2006), ‘Inflammatory inert poly(ethylene glycol)-protein wound dressing improves healing responses in partial- and full-thickness wounds’, International Wound Journal, 3, 332– 342. Shores J T, Gabriel A & Gupta S (2007), ‘Skin substitutes and alternatives: A review’, Advances in Skin & Wound Care, 20, 493–508. Shu-Fen L, Mark H, Chee-Jen C, Wen-Yu H & Ling-Ling L (2008), ‘A systematic review of silver-releasing dressings in the management of infected chronic wounds’, Journal of Clinical Nursing, 17, 1973–1985. Stashak T S, Farstvedt E & Othic A (2004), ‘Update on wound dressings: Indications and best use’, Clinical Techniques in Equine Practice, 3, 148–163. Stephen T (2008), ‘Hydrocolloid dressings in the management of acute wounds: a review of the literature’, International Wound Journal, 5, 602–613. Thomas A, Harding K G & Moore K (2000), ‘Alginates from wound dressings activate human macrophages to secrete tumour necrosis factor-[alpha]’, Biomaterials, 21, 1797–1802. Thomas S (1996), ‘Vapour-permeable film dressings’, Journal of Wound Care, 5, 271–274. Thomas S (1997), ‘Assessment and management of wound exudate’, Journal of Wound Care, 6, 327–330. Thomas S (2000), ‘Alginate dressings in surgery and wound management – part 1’, Journal of Wound Care, 9, 56–60.

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9 Dry wound healing concept using spray-on dressings for chronic wounds S. J O L LY and S. J O L LY, Clinogen Ltd, UK

Abstract: Dry wound healing using spray-on dressings offers a viable method of advanced wound healing for both chronic and acute wounds. The chapter discusses the concept of dry wound healing and seven key considerations when assessing wound dressings and how dry wound healing addresses each of these issues, including pH and maintenance of humidity. The chapter includes case studies demonstrating the cost benefits of a protein-based spray-on dressing and protein-rich skin repair cream compared to the use of traditional wound management protocol, and the versatility of the system and the positive environmental benefits with respect to low disposal costs of this biodegradable system. Key words: dry wound healing, spray-on dressing, protein, pH, maintenance of humidity, gaseous exchange, non-adherence, chronic wounds.

9.1

Introduction

Current wound healing follows the theory proposed by George D Winter (1–3). This methodology has been widely adopted as the norm for advanced wound healing. Winter’s pioneering work was based on porcine acute wounds. He studied the rate of epithelialisation in experimental wounds cut into the skin of healthy pigs; these wounds were covered with polythene film and compared to wounds with a natural scab exposed to the air. Winter found that epithelialisation occurred more quickly in the former. In exposed wounds epidermal cells migrated from hair follicles and the wound edges, whereas in covered wounds epidermal cells migrated through serous exudate forming a new epidermal layer above the dermis. The principle of moist wound healing led to the development of the first ‘scientific’ wound dressings to support optimal healing processes. These include products such as hydrogels to retain or bring moisture to the wound, hydrocolloids to absorb small amounts of excess moisture without drying the wound bed, absorbent foams, alginates, adhesive dressings, non-adhesive dressings and silicone-based low adherent dressings. Winter’s research was limited to acute, superficial wounds but the results have been used as a basis of moist wound healing for all types of wound of 209 © Woodhead Publishing Limited, 2011

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varying aetiologies. This has led to moist wound healing becoming the gold standard of current clinical care and product development. However, moist wound healing theory does not provide a basis for satisfactory management of every type of wound as will be demonstrated in the case studies below. As an example, whilst it has been demonstrated that a moist environment at the wound site has been shown to aid the rate of epithelialisation in superficial wounds (4–6), it was found that excess moisture at the wound site also causes maceration of the periwound skin (7). Recent research has highlighted a more complex set of requirements for effective wound healing (8–9). Based on such research an ideal wound dressing needs to: • • • •

maintain the right level of humidity enable the gaseous exchange necessary to wound healing (supply of oxygen and release of carbon dioxide) maintain the correct temperature and pH to promote the healing process provide appropriate protection from bacterial infection without causing additional trauma (e.g. by adhering to wound tissue, depositing particulates (e.g. fibres) into the wound or requiring frequent changes of dressing which disturb the wound and increase the risk of infection).

As a result, there is now a huge range of wound dressing materials and methods of delivery of dressings (10). One recent development is the use of hydrogel dressings, particularly for dry wounds that have low to medium exudate (11–13). Due to their high water content and soft elastic consistency, hydrogels are ideal candidates for depositing as a thin film onto a wound either by syringe or spray. This kind of dressing has a number of advantages: • • •

• • •

It is impervious to the transit of microorganisms to the wound. It provides excellent physical protection, having a high conformance to the contours of the wound and surrounding tissue. It is permeable, allowing the movement of air and moisture to maintain an appropriate environment for healing; it also allows exudate to escape. It is easily applied and flexible with the option to strengthen the dressing, for example by repeated spraying. It is adherent but can be easily peeled away from the wound without causing further injury (unlike some fibre-based dressings). Hydrogels are also suitable as slow-release carriers of therapeutic agents to promote healing.

As a result, an increasing number of dressings are now being sprayed onto wounds as a gel or film rather than being incorporated into a conventional fibre bandage.

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Proteins such as collagen can be deposited on to a wound as gels or films as well as conventional fibre dressings. Protein-based dressings are one of the most promising wound dressing materials, particularly given the ability of materials like collagen to promote fibroblast growth and affect macrophage activity, thus promoting wound healing (14–20). The authors’ research has focussed on developing a protein-based spray-on dressing which promotes more effective wound healing. The following sections look in more detail at the ideal properties of wound dressings and how these can be met by a protein-based spray-on dressing, as well as reviewing a number of case studies illustrating the use of these dressings.

9.2

The key properties of an ideal wound dressing

Outlined below are the key properties of an ideal wound dressing and how a protein based spray-on dressing addresses each of these key elements.

9.2.1 Maintenance of humidity at wound surface It is important to maintain the natural humidity of the tissue and not to introduce a stimulus that may cause an increase in humidity. Traditional theory recommends that the wound bed is kept in high humidity to encourage epithelialisation of cells and to avoid the formation of a scab on the wound site. Our observations have found that a higher than normal humidity is often as a result of the exudate being trapped in the wound bed; this can lead to further complication in the healing process – see Section 9.2.7 Maintenance of pH of the wound. A protein based spray-on dressing supports the body’s natural humidity and also allows excess exudate to flow away from the wound site freely. By using a protein-based spray-on dressing the wound is not only protected without the need for a secondary dressing, but also enables any exudates to flow away without impeding re-epithialisation.

9.2.2 Gaseous exchange Supply of oxygen and release of carbon dioxide should be uninhibited for healing to take place. Traditional occlusive dressings do not allow free gaseous exchange, this can lead to slower healing times as it is essential for oxygen to reach the wound site. A protein-based spray-on dressing provides a permeable membrane that allows the wound to breathe freely. Furthermore, the presence of oxygen inhibits anaerobic bacteria which thrive in oxygen deficient surroundings. By enabling the wound to have free gaseous exchange, phagocytosis occurs.

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9.2.3 Protection from trauma Trauma to the wound site and surrounding skin is minimised by leaving the wound site undisturbed. Physical protection from trauma of a wound site is essential in wound healing. However, the manner in which this is interpreted can lead to an excessive amount of trauma to the wound site and surrounding skin. One of the primary causes of trauma to a wound site is repeated changes of dressings. Once this is taken out of the equation, minimized or managed, this enables the wound to heal unhindered (21). The spray-on dressing forms its own dressing over the wound and builds up after repeated applications leaving the wound undisturbed. The use of a protein based spray-on dressing in most cases negates the need for a secondary dressing. By its very nature the dressing provides a protective layer over the wound site in most cases where the patient is within their own home.

9.2.4 Non-adherence Repeated application and removal of many dressings cause trauma to the wound site and surrounding skin causing an inflammatory response. Nonadherence is an essential consideration not only to the wound site but also to the surrounding skin. The advent of low adherence dressings has, to some extent, reduced this issue when referring to the wound site. However, the problem of a secondary dressing often causes an inflammatory response to the surrounding skin. A protein-based spray-on dressing enables the skin’s natural healing mechanism to work unimpeded. This type of dressing does not require removal, therefore any issues surrounding the problem of adherence to the wound site are immaterial. The spray-on dressing promotes the healing process by providing a framework for the wound to heal.

9.2.5 Provision of thermal insulation With the use of a dry wound healing environment it is important to avoid an artificially warm environment which may be caused by most occlusive dressings. Our findings suggest that wounds heal best at body temperature. The simplest way to achieve this is not to over-dress the wound. Traditional dressings often cause the wound site to overheat, which may reduce the proliferative response of lymphocytes. Studies have suggested that wounds heal most effectively at normal core body temperature; in the case of pressure ulcers a faster reduction in the mean surface area occurs when the wound bed is 36–38°C (22) and wound healing is slowed when temperatures fall below normal core body temperature or rise above 42°C (22). Ideally the wound should be allowed to heal at the body’s own temperature. By maintaining the temperature of the wound bed to as close to core body

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temperature as possible, the wound will be able to heal at a more uniform rate.

9.2.6 Freedom from particulate contamination The healing process is often impeded by the use of fabric dressings, which may give rise to an inflammatory response. Traditional fabric dressings are known to leave particulates in the wound. In many cases this can lead to an inflammatory response, which in turn will delay the healing process. Fibres from certain dressings may become incorporated into wounds and have an adverse effect on wound healing. The presence of debris may delay wound healing and act as a focus for infection. Contamination of wounds with foreign materials, such as dressing components, may elicit an inflammatory response whilst present within the wound (23–30). Protein based spray-on dressings are specially formulated to be compatible with the tissue fluid and not increase any inflammatory response. The introduction of a dressing that does not leave particulates in the wound ensures that the wound site can heal unimpeded. By covering the wound with a protein spray dressing the wound site is protected from any secondary dressings, should these be required.

9.2.7 Maintenance of pH of the wound Exudate, which is highly alkaline, should be allowed to flow away freely from the wound, as this creates a detrimental environment at the wound site and surrounding skin. Where the wound site of a chronic wound is kept in an alkaline environment, then healing is impeded. Chronic wound exudate has a different composition from that of an acute wound. Research has shown that the pH of chronic wound exudate is more alkaline than would be ideal for wound healing to take place and cells such as fibroblasts prefer an acidic environment (31). By removing the exudates from the wound site rather than remaining in situ, the pH of the wound bed can be restored to close to its natural pH, this enables the wound to heal.

9.3

Using protein-based spray-on dressings in practice

The concept of dry wound healing using a protein-based spray-on dressing and protein-rich skin repair cream addresses each of the seven key properties of an ideal wound dressing. As moist wound healing is not always suitable for the treatment of chronic wounds, we have demonstrated the use of the principle of dry wound healing for this type of wound in the case studies that follow in this chapter.

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In most cases, the use of a protein-based spray-on dressing is patient or carer managed as it requires frequent application. In the first instance the chronic wound site is cleaned with a sterilising solution to remove any ointments or debris from previous treatments. This principle does not advocate creating an acute wound, therefore the debridement should be kept to a minimum unless excessive necrotic tissue is present. Following the wound being cleaned and dried, the spray is applied and allowed to dry naturally. The spray is reapplied eight times per day at equal intervals during waking hours so that the dressing builds up a film over the wound site. By using a spray to deliver the protein-based dressing to the wound site it is possible to ensure that the entire wound receives an application of the dressing. Due to the nature of the spray and its composition, in the case of chronic wounds there is little or no discomfort felt upon application. By allowing any exudate to flow away from the wound it is possible to move the chronic wound from an alkaline pH to a mildly acidic pH. It is essential that the exudate is removed from the periwound skin as this is often a contributory factor in inflammation and maceration to that area. The periwound skin should be treated with a protein-rich skin repair cream four times a day, which helps to reduce trauma and inflammation. A protein-based spray-on dressing forms its own protection for the wound site and after repeated applications builds into a thick layer leaving the wound undisturbed. As wound healing progresses, this film is automatically rejected as new tissue grows at the wound site. As the film becomes thicker the patient is then advised to use the protein-rich skin repair cream on the free edge of the film. The process is repeated until the wound has healed inwards from its extremities and upwards from the base of the wound. Finally, the patient is advised to continue use of the protein-rich skin repair cream until the wound site and periwound skin heal to minimise scarring.

9.4

Case studies

9.4.1 Case study 1 – leg ulcer caused by industrial injury Mr Y, a diabetic, suffered an injury to the calf in a workplace accident causing an acute wound. He initially attended his local surgery for an assessment and was advised that he would need to attend a wound clinic on a weekly basis. The dressing protocol followed Winter’s theory of moist wound healing. Initially the wound was treated with an occlusive dressing, which was renewed at the wound clinic on a weekly basis. In this case the type of dressing used caused trauma and inflammation to the wound site as a result of so called ‘rip and pull’ trauma from repeated dressing changes; the condition of the wound gradually worsened leading to it being classified as a chronic ulcer after 1 month. The dressing protocol was revised to

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9.1 27 Jul 2001 Inflamed periwound area and moderately exudating wound following 7 months treatment using moist wound healing.

three-layer compression bandages which continued for a further 7 months. There was continuous exudation and the patient felt a great deal of discomfort and loss of concentration at work. See Fig. 9.1. Seeing no improvement over an 8-month period, the patient opted to follow a dry wound healing protocol comprising a protein-based spray-on dressing and protein-rich skin repair cream. The patient used both products as directed – application of the spray-on dressing to the wound site and inflamed skin surrounding wound site 8 times a day and allowed to dry for 10 minutes, and then the protein-rich skin repair cream applied to inflamed but non-broken skin surrounding the wound site applied 4 times a day. With the formation of a thick film, the patient was advised to ensure that the free edge of both the wound and film was kept hydrated. Within 1 week of following the above protocol a significant improvement was visible; the trauma to the skin surrounding the wound site caused by repeated dressing changes was significantly improved and the skin appeared much less inflamed. After 26 days the film had built up, see Fig. 9.2. After the first thick film layer had formed, the patient was advised that the frequency of application of the spray-on dressing be reduced to 6 times a day with continued use of the protein-rich skin repair cream. We advised the patient that the thick film should be allowed to remain in place and should

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9.2 22 Aug 2001 Build-up of film on wound after 26 days and less inflamed periwound.

not be physically removed, when it lifted away naturally then the patient was to increase use of the spray to 8 times a day; in this particular case the film remained intact. The wound was fully healed within 67 days from the change in wound management with minimal evidence of the initial injury. The protocol meant that the patient was in control of his own wound management with periodic visits to us to review the healing process. Initial application was spraying the wound site 8 times a day until a thick film formed over the wound site, and the use of protein-rich skin repair cream 4 times a day on the fragile and traumatised skin surrounding the ulcer (see Figs 9.2 to 9.4). This process continued until full healing was achieved. Table 9.1 shows the comparative costs of the treatment received prior to following the dry wound healing protocol using a protein-based spray-on dressing and the cost once the patient switched from moist wound healing.

9.4.2 Case study 2 – leg ulcer caused by accident in the home Mrs R scratched both her legs on a wicker basket whilst at home. The patient attended her local surgery and received treatment based on the

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9.3 31 Aug 2001 Periwound area and wound site are reduced in size.

9.4 2 Oct 2001 Complete healing achieved after 67 days using proteinbased spray-on dressing.

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Table 9.1 Estimated cost to NHS using moist wound healing vs. cost of dry wound healing protocol Moist wound healing costs over 243 days

Dry wound healing over 67 days

1 hour nursing time @ £41 per dressing change 3-layer compression bandage @ £6 per dressing Cost of secondary dressing @ £3 per dressing Miscellaneous costs – gloves, sterilisation, infection control, disposal @ £1 Patient cost to NHS*

Weekly 10 minute review @ £7

£68

£210

Spray @ £1.28 per day

£86

£105

Cream @ 0.82 per day

£55

Miscellaneous costs – gloves, sterilisation, infection control, disposal @ £1 per week

£10

£1785

Dressing cost per day

£7.35

£1435

£35

Equivalent dry wound healing cost Dressing cost per day

£219 £3.27

*consultant cost not included

traditional wound management theory of moist wound healing; this continued for a period of four years with no visible improvement. The patient attended a wound clinic on a weekly basis and had a variety of dressings applied to her original wounds, the final dressings used were multi-layer compression bandages. The original wound sites were scratches; however, as time progressed the variety of dressings used on the patient resulted in chronic leg ulcers on both legs. The main problems with treating this patient with multi-layer compression bandages renewed on a weekly basis resulted in high levels of exudate remaining at the wound site; as chronic wound exudate is alkaline this caused trauma, maceration and inflammation to the periwound skin and led to the breakdown of the area. After this time she began a patient-controlled regimen using both a protein-based spray-on dressing for her chronic wounds and protein-rich skin repair cream for fragile, inflamed periwound skin. The results below show the progress of healing over a period of 10 weeks on her right leg and ankle. The regimen consisted of the spray-on dressing being applied to the wound site 8 times a day for the first 30 days and then reduced to 6 times

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a day evenly throughout the day. The periwound skin was treated using a protein-rich skin repair cream which helped to rehydrate, nourish and strengthen the skin.The main principle was for the patient to keep the wound uncovered as much as possible and to follow the seven key principles outlined above. When the patient needed to dress the wound, a sterile gauze dressing was used to act as a barrier to the elements. As the wounds began to progress through the phases of healing in a dry environment, the wound did not resemble what is traditionally expected when following Winter’s (1–3) principles of moist wound healing. When the wound began to heal, the wound bed started to dry and the amount of exudate produced began to reduce. Any exudate that leaked into the periwound skin was wiped away from the skin so that it would not cause damage to the periwound skin. The next indication that was the formation of thick layer of protein film over the wound bed. Our observations showed that beneath the thick film the wound was beginning to granulate and epithelialisation had started. As we were following a process of dry wound healing, it was inevitable that the spray film would become loose and come away from the skin. The wound bed exhibited positive signs of healing, which was evident in the size of the wound being visibly reduced. The patient was advised that application of the protein-based spray-on dressing should be continued as the formation of the film and its shedding would occur a number of times until the wound site had healed both upwards and inwards. As the wound healed, the patient continued to use the protein-rich skin repair cream on the surrounding skin to ensure that this strengthened and supported remodelling of the edges of the wound site. Once the wound was fully healed it was recommended that the patient continue use of the protein-rich skin repair cream to minimise the risk of scarring at the wound site and periwound skin. The figures below show the progression of healing over a three-month period. Figure 9.5 show the state of the wound after the patient had received 4 years of multi-layer compression bandaging following moist wound healing theory. The patient began a regimen following the dry wound healing protocol using a protein based spray-on dressing and protein-rich skin repair cream; the patient initially used the spray-on dressing 8 times a day on the wound site and surrounding skin whilst also applying the protein-rich skin repair cream to any fragile skin surrounding the wound site 4 times a day. As the wound dried and a thick film formed the patient was advised to reduce the number of applications of the spray to 6 times a day – see Fig. 9.6. The patient was advised to increase frequency of application of the spray to 8 times a day when the thick film naturally came away from the skin. As the size of the wound reduced the patient continued to apply

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9.5 Jun 2002 Inflamed skin and heavily exudating wound following treatment using moist wound healing.

the protein-rich skin repair cream 4 times a day until the area was fully healed. After 70 days the wound had healed completely – see Fig. 9.7 and the patient has not suffered any further ulceration to the area. Table 9.2 shows the overall comparative costs of the treatment received prior to following the dry wound healing protocol and the cost once the patient switched from moist wound healing – the figures apply to the treatment of both legs. The dressing cost per leg is summarised in Table 9.2.

9.4.3 Case study 3 – pressure ulcer in hospitalised patient Mrs P is a 79 year old suffering from dementia and was bedridden due to being immobile and knee amputation surgery. She developed a severe pressure ulcer to the sacrum, which was managed by being cleaned on a daily basis and packed with a debriding agent, and then covered with a sterile gauze pad and bandage. The pressure ulcer was merely being managed in this manner over the 3 years that the patient had been bedridden and showed no signs of healing. Due to the severity of the pressure ulcer Mrs

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9.6 Jul 2002 Wound and periwound appear less inflamed and reduced in size.

P’s physician decided to follow a protocol of dry wound healing using a protein-based spray-on dressing and protein-rich skin repair cream, see Fig. 9.8. The patient’s wound was cleaned to remove all traces of the debriding agent and to ensure that there was no particulate contamination from any other dressing material used and this was left to dry. The spray-on dressing was applied and allowed to dry for 10 minutes then the protein-rich skin repair cream was applied to the surrounding inflamed. The patient’s carer was advised to use the following protocol: application of spray-on dressing 8 times a day, allow to dry and then apply protein-rich skin repair cream to the surrounding inflamed skin. Due to the nature of the chronic wound and its position, the wound was shielded using a sterile non-adherent pad which was not allowed to come into contact with the wound site; this ensured that the wound was not disturbed further and followed many of the seven key principles of an ideal wound dressing, i.e. non-adherence, gaseous exchange, freedom from particulate contamination, thermal insulation and protection from trauma.

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9.7 Aug 2002 Complete healing achieved after 10 weeks using dry wound healing concept. Table 9.2 Estimated cost to NHS using moist wound healing vs. cost of dry wound healing protocol for both legs Moist wound healing costs over 1460 days

Dry wound healing over 70 days

Nursing time @ £41 per dressing change 3-layer compression bandage @ £6 per dressing Cost of secondary dressing @ £3 per dressing Miscellaneous costs – gloves, sterilisation, infection control, disposal @ £1 Patient cost to NHS*

£3,744

Weekly 10 minute review @ £7 Spray @ £1.28 per day

£179

£1,248

Cream @ 0.82 per day

£115

Dressing cost per day per leg

£17,056

£416

£22,464 £7.69

Miscellaneous costs – gloves, sterilisation, infection control, disposal @ £1 per week Equivalent dry wound healing cost Dressing cost per day per leg

*consultant cost not included

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£20

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9.8 8 Oct 2009 Pressure ulcer at the sacrum of patient bedridden for 3 years.

9.9 24 Oct 2009 Wound has entered proliferation phase and exhibits signs of granulation and remodelling.

Over the following 16 days the pressure ulcer began to enter the proliferation phase of wound healing and exhibit signs of granulation and remodelling, see Fig. 9.9. Application on the protein-based spray continued as directed. During the next 15 days the wound continued to granulate and exhibited signs of healthy tissue appearing a pink/red colour, see Fig. 9.10. Given the severity of the pressure ulcer and the frail condition of the patient, complete healing was achieved after 217 days after moving to a dry wound healing protocol, see Fig. 9.11. © Woodhead Publishing Limited, 2011

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9.10 8 Nov 2009 Granulation continues and healthy tissue is visible.

9.11 13 May 2010 Compete healing achieved after 217 days.

9.5

Conclusions

As these case studies suggest, a deeper understanding of the wound healing process and the range of criteria a dressing should meet allows development of novel materials and delivery systems for wound dressings. Innovations such as protein-based, spray-on dressings allow more rapid and effective healing of dry wounds. Such dressings may also have additional benefits. The dry wound healing concept using a protein-based spray-on dressing in most cases enables the patient to be put in control of their own wound

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management. As a result, valuable resources including nursing time can be employed elsewhere. The concept has been successfully used in many first aid situations. For example, following sports injuries such as grazes the immediate application of a protein-based spray-on dressing shortens the inflammatory phase and hastens epithelialisation. Healing time is significantly reduced as the wound is left open and allowed to dry; the protocol follows the key seven principles outlined above. Finally, there are potential environmental benefits. Traditional wound management uses a series of ointments and dressings that are renewed periodically, this may be as often as three or four times a week. Due to the nature of the waste, which is classified as a ‘bio-hazard’, this waste requires incineration, which not only has a high cost attached but also a significant adverse environmental impact. A protein-based spray-on dressing, in most cases does not require the use of a secondary dressing, is biodegradable and therefore has minimal effect on the environment.

9.6

References

1. Winter G D (1962) Formation of the scab and the rate of epithelialisation of superficial wounds in the skin of the young domestic pig. Nature 193(4812): 293–4. 2. Winter G D (1963) Effect of air exposure and occlusion on experimental human skin wounds. Nature 200: 378–9. 3. Winter G D and Scales J T (1963) Effect of air drying and dressings on the surface of a wound. Nature 197: 91–2. 4. Eaglstein W H, Davis S C, Mehle A L and Mertz P M (1988) Optimal use of an occlusive dressing to enhance healing. Effect of delayed application and early removal on wound healing. Archives of Dermatology 124(3): 392–5. 5. Agren M S, Karlsmark T, Hansen J B and Rygaard J (2001) Occlusion versus air exposure on full-thickness biopsy wounds. Journal of Wound Care 10(8): 301–4. 6. Parnham A (2002) Moist wound healing: does the theory apply to chronic wounds? Journal of Wound Care 11(4): 143–6. 7. Cutting K F and White R J (2002) Maceration of the skin and wound bed. 1: Its nature and causes. Journal of Wound Care 11(7): 275–8. 8. Leaper D and Harding K (eds) (1998). Wounds: biology and management. Oxford University Press, Oxford. 9. Janis J and Attinger C (2006). Current concepts in wound healing. Plast. Reconstr Surg 117(7 Supplement): 4S–5S. 10. Gupta B and Edwards J (2009). Textile materials and structures for wound care products. In Rajendran S (ed.), Advanced textiles for wound care. Woodhead Publishing Limited, Cambridge. 11. Balakrishnan B, Mohanty M, Umashanker P and Jayakrishnan A (2005). Evaluation of an in-situ forming hydrogel wound dressing. Biomaterials 26(2): 6335–42.

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12. Peng H, Martineau L and Hung A (2008). Hydrogel-elastomer composite biomaterials for use as a wound dressing. J Mater Sci Mater Med 19(4): 1803–13. 13. Schoukens G (2009). Bioactive dressings to promote wound healing. In Rajendran S (ed.), Advanced textiles for wound care. Woodhead Publishing Limited, Cambridge. 14. Eisenbud D, Huang N, Luke S and Silberklang M (2004). Skin substitutes and wound healing: current status and challenges. Wounds 16(4): 2–17. 15. Wollina U, Schmidt W, Kronert C, Nelskamp C, Scheibe A and Fassler D (2005). Some effects of a collagen-based matrix on the microcirculation and would healing in patients with chronic venous leg ulcers. Int J Low Extreme Wounds 4(4): 214–24. 16. Ehrenreich M and Ruszczak Z (2006). Uptake on tissue-engineered biological dressings. Tissue Eng. 12(6): 2407–24. 17. Shingel K, Di Stabile L, Marty J and Faure M (2006). Inflammatory inert poly(ethylene glycol)-protein wound dressing improves healing response in partial and full-thickness wounds. Int Wound J 3(4): 332–42. 18. Zeugolis D, Paul G and Attenburrow G (2006). Reformed collagen fibres. In Anand S et al. (eds), Medical textiles and biomaterials for healthcare, Woodhead Publishing Limited, Cambridge. 19. Ji J, Borisov O, Ingham E, Ling V and Wang Y (2009). Compatibility of a protein topical gel with wound dressings. J Pharm Sci 98(2): 595–605. 20. Brett D (2008). A review of collagen and collagen-based wound dressings. Wounds 20(12); 35–43. 21. Sharp C A, McLaws M (2002). Wound dressings for surgical sites [protocol]. The Cochrane Library, Issue 1. 22. Kloth L C, Berman J E, Dumit-Minkel S (2000). Effects of a normothermic dressing on pressure ulcer healing. Advances in Skin Wound Care; 13: 2, 69–74. 23. Chakravarthy D, Rodway N, Schmidt, S et al. (1994). Evaluation of three new hydrocolloid dressings: retention of dressing integrity and biodegradability of absorbent components attenuate inflammation. J Biomed Mater Res; 28: 10, 1165–73. 24. Halbert A R, Stacey M C, Rohr J B (1992). The effect of bacterial colonization on venous ulcer healing. Australas J Dermatol; 33: 2, 75–80. 25. Danielsen L, Balslev E, Doring G et al. (1998). Ulcer bed infection. Report of a case of enlarging venous leg ulcer colonized by Pseudomonas aeruginosa. APMIS; 106: 7, 721–6. 26. Madsen S M,Westh H, Danielsen L (1996). Bacterial colonization and healing of venous leg ulcers. APMIS; 104: 12, 895–9. 27. Robson M C, Mannari R J, Smith P D, Payne W G (1999). Maintenance of wound bacterial balance. Am J Surg; 178: 5, 399–402. 28. Lookingbill D P, Miller S H, Knowles R C (1978). Bacteriology of chronic leg ulcers. Arch Dermatol; 114: 1765–8. 29. Skog E, Arnesjo B, Troeng T et al. (1983). A randomized trial comparing cadexomer iodine and standard treatment in the out-patient management of chronic venous ulcers. Br J Dermatol; 109: 77–83. 30. Whiston R J, Hallett M B, Davies E V et al. (1994). Inappropriate neutrophil activation in venous disease. Br J Surg; 81: 5, 695–8. 31. Greener B, Hughes A A, Bannister N P and Douglass J (2005) Proteases and pH in chronic wounds, Journal of Wound Care 14: 2.

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10 Assessing the effectiveness of antimicrobial wound dressings in vitro J. VAU G H A N, R. B E N S O N and K. VAU G H A N, Smith & Nephew Research Centre, UK

Abstract: During the development of an antimicrobial wound dressing a number of properties must be displayed by the product in order to support regulatory submission or marketing material. To provide the evidence for these purposes a battery of tests must be performed which demonstrate efficacy of the antimicrobial dressing. Along with the choice of test method there are many other factors that need to be taken into account depending on the physical properties of the dressing and its intended application. In this chapter, microbiological considerations will be covered in order to provide a brief overview of this complex and perhaps less understood area of product development. Key words: microbiology, antimicrobial, bacteria, micro-organism, log reduction, zone of inhibition, bacterial barrier, time-kill kinetic assay, biofilm.

10.1

Introduction

Due to an increased level of awareness and concern surrounding wound infection, particularly with regard to the rise of antibiotic-resistant organisms, it has become increasingly commonplace for wound dressings to be augmented with the addition of an antimicrobial. Examples of a selection of such dressings can be found in Table 10.1. Their efficacy is most often demonstrated through in vitro testing. There are many different tests which can be carried out to do this and traditionally many are based upon variations of just a couple of tests. This chapter will explore some of the most common tests which are used to demonstrate antimicrobial activity in vitro. It is not intended to explore the many published variations of these tests and we will only touch on testing based upon molecular approaches and those which assess biofilms, the latter being relatively underutilised compared to more traditional methods to assess antimicrobial activity, particularly for dressings on the market. It is quite common for the term “antimicrobial” to be used in many different ways and this is often interpreted as meaning that micro-organisms are killed. However, antimicrobial is simply a general term for agents which 227 © Woodhead Publishing Limited, 2011

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Table 10.1 Examples of commercially available antimicrobial dressings Dressing

Composition

Manufacturer

ActicoatTM

Three layers of polyethylene gauze coated with nanocrystalline silver Nylon dressing containing activated charcoal bound to silver Silver impregnated within a carboxymethylcellulose fibre dressing Polymer film containing silver phosphate Calcium alginate dressing containing silver Foam dressing containing silver Dressing impregnated with polyhexamethylene biguanide Surgical incise drapes with an iodophor impregnated adhesive Non-adherent dressing containing povidone iodine Cadexomer dressing with iodine Soft silicone foam dressing with silver Transparent adhesive dressing and an integrated gel pad containing chlorhexidine gluconate Silver suspended within a polyacrylate dressing Carboxymethylcellulose fibre dressing blended with a metallic silver-coated nylon fibre Polymeric fabric coated with metallic silver Hydrocolloid dressing impregnated with Vaseline and silver sulphadiazine

Smith & Nephew

ActisorbTM Silver 220 Aquacel® Ag

Arglaes® Askina® Calgitrol® Ag Biatain® Ag Kerlix AMDTM IobanTM Inadine® IodoflexTM MepliexTM Ag TegadermTM CHG

SilvaSorbTM SilvercelTM

Silverlon® Urgotul® SSD

Systagenix

ConvaTec

Medline B Braun Coloplast Tyco-Kendal Healthcare 3M Health Care Systagenix Smith & Nephew Mölnlycke Health Care 3M Health Care

Acrymed Systagenix

Argentum Medical Urgo Medical

The list provided here is not exhaustive for commercially available antimicrobial dressings nor the different actives or dressing formats.

kill, slow or inhibit the growth of micro-organisms and it is not always clear to those not skilled in the art of microbiology what each test is showing. Therefore, the aim of this chapter is to give some background information and explanation of antimicrobial tests to non-microbiologists in order to

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10.1 Agar plate with colonies.

aid the understanding and interpretation of data and by the end of this chapter, it should be clearer what each test demonstrates.

10.2

Log reduction testing

10.2.1 Background This test, described since USP 18 in 1970,1 challenges antimicrobial dressings with a known number of micro-organisms (e.g. bacteria), incubating them at defined temperatures, withdrawing samples at specified time points and counting the number of organisms remaining (Fig. 10.1 shows a typical agar plate with bacterial colonies generated from this test). The term ‘log reduction’ is used to describe the number of micro-organisms which have been killed relative to the starting concentration. It should also be noted that a number of different names can be used to describe log reduction tests, such as time-kill curve, challenge test, die-off test, antimicrobial effectiveness test and suspension kill test. A selection of test methods which are based on the log reduction test are shown in Table 10.2.

10.2.2 Principles of the log reduction test Gallent-Behm et al. have published a summary of the log reductions achieved by a range of antimicrobial wound dressings2 and although the

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Table 10.2 List of useful standards Reference number

Title

JIS Z 2801: 2000

Antimicrobial products – Test for antimicrobial activity and efficacy Chemical disinfectants and antiseptics. Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic, and institutional areas Antibacterial Activity Assessment of Textile Materials: Parallel Streak Method Evaluation of Antibacterial Finishes on Fabrics

EN1276

AATCC Test Method 147–1998 AATCC Test Method 100–1999 ASTM E2149-10

ASTM E1054-08 ASTM E2180-07

ASTM E2315-03(2008) ASTM E1326-08

Standard Test Method for Determining the Antimicrobial Activity of Immobilised Antimicrobial Agents Under Dynamic Contact Conditions Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents Standard Test Method for Determining the Activity of Incorporated Antimicrobial Agent(s) In Polymeric or Hydrophobic Materials Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure Standard Guide for Evaluating Nonconventional Microbiological Tests Used for Enumerating Bacteria

Note: these standards may describe slightly different calculations to that shown in Section 10.2.3, for example JIS Z 2801 calculates an ‘antimicrobial activity value’ using the 24 hour counts from test and control materials rather than a log reduction from the baseline initial test organism concentration added to the samples at the start of the test. Key: JIS = Japanese Industrial Standard ASTM = American Society for Testing and Materials AATCC = American Association of Textile Chemists and Colorists

specifics of the tests used may vary, the basic principles of the testing remain the same. A simple schematic for carrying out log reduction testing is given in Fig. 10.2. Potential variables to the test range from choice of test organism to incubation times and it is important to consider these prior to starting the test. A particular standard may dictate specific test parameters, such as the volume of test organism to be added to each sample, yet in practise tests may need to be custom designed based upon the intended use of the dressing. For example, highly absorbent dressings may require a larger volume of culture than a non-absorbent dressing in order to make the test more representative of its actual use in a clinical setting. The individual steps of the log reduction test method and the factors to be considered are shown in Table 10.3.

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Step 4

Serial dilutions (usually 1ml)

-1 -2 -3 -4 -5 -6 Step 3 Incubation at defined time/temp

Dressing sample placed into test vessel

Step 1

Test culture added

Diluent (usually 9 ml)

1st tube contains neutralising solution

Step 2

Samples (usually 1ml) from each dilution plated onto duplicate agar plates Agar plates incubated for at least 48 hrs before colonies counted

Step 5 Calculation to determine the LOG REDUCTION (number of bacteria killed relative to starting amount in control sample calculated in log10)

10.2 Schematic for log reduction testing.

The basic method for log reduction demonstrates a single challenge with a test culture; however, it is also possible to re-challenge the test samples at a designated time point, thus demonstrating the dressing’s ability to maintain antimicrobial activity over a longer period of time, as has been reported in the literature.3,4 This kind of repeated microbial insult to samples is highly challenging, but depending on the intended application and wear duration of the dressing, it may be a more relevant model for longevity than a transfer zone of inhibition test (see Section 10.3.3). The appropriate number of replicates and time points for the test should be planned for well in advance of starting testing. A range of time points, for example, 0, 1, 2, 4, 24, 48 hours can be considered and, as a minimum, testing carried out on triplicate samples of each dressing and control(s) under test. With any log reduction experiment, it is imperative that an appropriate negative control is used. This demonstrates that in the absence of an antimicrobial, the test organism will thrive. Wherever possible this negative control sample should be identical to the antimicrobial dressing under test except for the addition of the antimicrobial agent. A relevant positive control may also be used to demonstrate the test organism is susceptible to the antimicrobial under test.

10.2.3 Calculation and interpretation of results When calculating log reductions, a few basic principles must be followed. The most important of which are: (a) to convert plate counts to log10 values before carrying out manipulations (e.g. taking a mean of replicate plate

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Table 10.3 Individual steps of the log reduction test Step

Points to consider

1.

Sample preparation

• Choose an appropriate test vessel for the size and type of dressing under test, and the volume of culture suspension to be applied. • Decide on sample numbers based on the level of expected variability and whether statistical analysis is to be carried out, this number should never be less than 3. • Depending on the type of sampling used (i.e. from a suspension surrounding a dressing or directly/ destructively from the dressing sample) enough samples should be allowed for each time point to be tested.

2.

Test culture

• What is the dressing’s intended use? A range of micro-organisms should be considered including bacteria (aerobes and anaerobes), fungi and yeasts to demonstrate a broad range of antimicrobial efficacy. • What is the nature of the dressing under test? Highly absorbent dressings may require a larger volume, and even excess, of culture, whereas non-absorbent dressings may require a very small amount of test culture.

3.

Incubation time and temperature

• What is the intended duration use of the dressing? 24 hours up to several days may be appropriate. • Will it be used on infected wounds? If so, additional, shorter time points may be appropriate such as 30 minutes or 4 hours. • What is the most appropriate incubation temperature? Skin temperature may only be 32oC not 37oC. • Consider whether samples are to be incubated under static or shaking conditions; a particular standard or protocol may dictate which should be used.

4.

Sampling and serial dilution

• What antimicrobial does the dressing under test contain? When samples are taken at the various time points (e.g. 24 hours), the activity of the antimicrobial must be neutralised and a suitable neutraliser must be identified and validated against all the chosen organisms before the test is carried out. Failure to do this may render false negative results (i.e., antimicrobial continues to be active in the agar plates whilst being incubated and prevents colonies from growing, leading to a false conclusion that the sample has a high antimicrobial activity).

5.

Calculations

• Calculate log reduction results using the initial concentration in the test culture added at the start of the test. • A lower detection limit should be established for the test based on the volume of the sample added to the volume of neutraliser (note: unless the entire volume of the first dilution in neutraliser is counted there will always be a detection limit based on the volume of the neutraliser used).

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counts) and (b) to calculate reductions from the initial concentration used to inoculate the test samples. A simple log reduction calculation can be represented as shown below though percentage reductions can be used instead, where 99.9% represents a 3 log reduction:1 Log reduction in numbers = log10(a) − log10(b) (a) = mean plate count of starting concentration (b) = plate count after relevant incubation time (mean or individual replicate counts) For example, samples of an antimicrobial dressing are inoculated with 1 ml of test culture containing 1.00 × 106 CFU*/ml (a). After 24 hours incubation at 37oC, the remaining cells left in the sample are enumerated and found to be 2.56 × 102 CFU/ml (b). The log reduction is calculated to be 3.59. Log reduction in numbers = Log10 (1.00 × 106) − Log10 (2.56 × 102) = 6.00 − 2.41 = 3.59 The value used as the starting concentration, which acts as a baseline in calculations, should wherever possible be from a count recovered from the negative control sample immediately after the microbial culture is added to it (Time 0). When interpreting log reduction tests, a minimum 3 log reduction (or 99.9% kill) should be achieved to demonstrate adequate antimicrobial activity. If the data generated is to be used as part of a regulatory 510(k) submission, it is worth noting that the FDA may require 4 log reductions (99.99% kill) for antimicrobial dressings from a starting test culture of 1 × 106 CFU/ml.5 The guidance does not note that any statistical tests be performed on the data and it is recommended a suitably qualified statistician be consulted to aid with interpretation of data. The duration over which these log reductions are achieved may be determined by considering the type of dressing and its expected length of clinical use. Log reduction tests provide useful in vitro performance data for antimicrobial dressings and, whilst these tests alone may not be sufficient to fully predict clinical performance, they are designed to help demonstrate that an antimicrobial wound dressing is sufficiently efficacious against a range of micro-organisms, whether this is aimed at reducing the microbial load in the dressing or within the wound itself.

* CFU = colony forming units (in microbiological convention counted colonies from plates are treated as if they were formed from one initial cell, but due to the fact there may have been a clump of cells from which the colony formed they are termed as colony forming units).

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10.3

Zone of inhibition (ZOI)

10.3.1 Background There are several methods which can be employed to determine the minimum concentration of an antimicrobial required for it to be efficacious against a target organism. Such methodologies may either involve diffusion of the antimicrobial agent through an agar medium, or alternatively a dilution series of the antimicrobial in agar or broth. In the clinical microbiology laboratory, the requirement to understand the antibiotic susceptibility profile of a suspected infectious agent is necessary to determine the most appropriate antibiotic regime. The method of choice for screening several antimicrobials at once is the disc diffusion method6 (BSAC Working Party, 2010). This methodology has been adapted to create a ZOI test which is useful in determining antimicrobial efficaciousness and longevity of wound dressings and other antimicrobial devices.7,8

10.3.2 Principle of the ZOI test There are several variations that can be readily made to the ZOI test but all essentially rely upon the diffusion of the antimicrobial agent through agar. The choice of test medium must be made depending upon the needs of the test organisms. Mueller-Hinton agar is the most commonly used media for ZOI testing of non-fastidious organisms and was recommended by Bauer, Kirby, et al. in 1966 when they first described the disc diffusion antimicrobial susceptibility test on which the ZOI test is based.9 The agar is inoculated with the test organism either by spread plating across the surface of a pre-poured agar plate, or by incorporation into the molten agar prior to preparing the plates. With the latter incorporation method the definition of the ZOI edge can be increased by creating a layered agar plate in which the bottom layer consists of sterile agar and the top layer inoculated agar. The test plates can range in size from a suitably sized Petri-dish for a single test sample to a large bio-assay dish for multiple test samples. The aim of the inoculation stage is to create a confluent lawn of the test organism which can be used to assess the antimicrobial device. For each organism tested it may be required to establish the correct microbial concentration required to achieve this confluent lawn without over challenging the test product. There are a plethora of materials used for antimicrobial wound dressings which include, although not limited to, absorptive filler, alginates, foams, gauze, hydrocolloids, hydrogels and transparent films.10 Consideration of the dressing material must be made to determine the suitability of the ZOI test, and whether modifications to the methodology may be useful.

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Table 10.4 Examples of problems with zone of inhibition methodology and possible modifications Problem

Method modification

The test material fuses to agar preventing removal/transfer The test material breaks or dissolves preventing removal/transfer The zone edge is obscured due to staining of the agar with the antimicrobial

⎫ Sterile layer that allows transmission of active used as ‘interleave’ ⎪ ⎪ between the test dressing and agar ⎪ (e.g. filter paper) ⎬ ⎪ ⎪ ⎪ ⎭

Test dressing curls at edges and loses contact Uneven, poorly defined zone edge

⎫⎪ Use of weight on top of sample to maintain contact between test ⎬ ⎪⎭ dressing and agar

No zone is produced due to low moisture conditions of test

Pre-wetting of the test dressing to release the active

Lack of sharpness in zone edge due to bowing as diffusion gets vertically deeper into agar

Layered agar plate consisting of a sterile bottom layer and a thinner inoculated top layer

Examples of problems which may be encountered and useful method modifications can be found in Table 10.4. In antimicrobial testing of wound dressings and devices, the antimicrobial is contained within or coated on the test dressing, and once cut to an appropriate size, will act as an antimicrobial reservoir which can be placed directly onto the freshly inoculated agar. However, other antimicrobial reservoirs may be employed. These include removing a plug from the inoculated agar to create a well into which a liquid or cream can be placed. Further to that, paper discs impregnated with the antimicrobial can also be employed. The latter are often used by clinical microbiology laboratories as a convenient way to test several antimicrobials at once, especially as antibiotic impregnated paper discs can be readily purchased from commercial sources. Once the antimicrobial wound dressing, or active, has been applied to the lawn of test organisms, the zone plate is incubated, usually at 37°C for 18–24 hours. During the incubation period, the test organism growth and the diffusion of the antimicrobial through the agar reach a balance, creating what is seen as a ZOI. At the end of the incubation period a well-defined clear zone of agar can be seen, surrounded by a lawn of bacteria. A diagram and image demonstrating a ZOI can be seen in Fig. 10.3.

10.3.3 Time points The ZOI test can be used to asses the longevity of the antimicrobial activity of a wound dressing.8 Such information is useful in determining the wear time

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Bacterial lawn growing over 24 hours

Antimicrobial diffusing out

Petri dish, agar and bacteria

Antimicrobial dressing Edge of antimicrobial diffusion

10.3 A pictorial view of zone of inhibition development.

of the device. After the initial 18–24-hour incubation period, the wound dressing is transferred, in the same orientation, to a second freshly inoculated zone plate. Once again, the second zone plate is incubated using the same conditions as those of the previous plate. If the wound dressing still contains sufficient antimicrobial, a second ZOI will be achieved after the incubation period. The whole process can be repeated a number of times with the same dressing sample to determine the longevity of the antimicrobial product.

10.3.4 Calculation of results The interpretation of ZOI data may be as simple as recording the presence of a zone or not, but some researchers may find it useful to record the actual ZOI size either by area or diameter. An important consideration to make when determining the most suitable method of measurement is if (1) each of the dressing samples is the same size and (2) if the nature of the test material means the samples are likely to alter size either from moisture absorption or evaporation. A corrected ZOI measure can account for such factors by either measuring the zone from the test dressing footprint to the edge of the zone, or by measuring the sample size in both axes (if square) and subtracting this from the zone size.

10.3.5 Other considerations The zones which are produced are only indicative of microbial inhibition (bacteriostatic action), not microbial kill (bactericidal action). To gain more

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information on whether the test organism has been killed rather than just inhibited (i.e. in stasis), further investigation is required. On removal of the test dressing, swabs of the agar surface can be taken. These swabs can then be plated onto agar to allow any living micro-organisms to grow. If growth is present, this indicates the wound dressing only inhibited bacterial growth. If no growth is present, this would indicate the wound dressing killed the bacteria. To prevent the possibly that the diffused antimicrobial with the zone area may also be transferred during the swabbing procedure and inhibit subsequent bacterial growth, neutralisation of the antimicrobial should be considered. This can be achieved either by pre-moistening of the swab with a neutralising agent or by the plating of the swab onto a known neutralising agar. In the Kirkby-Bauer test (disk sensitivity assay) used for antibiotic sensitivity assays, the appearance of a zone is not sufficient to determine efficacy, zone size must meet certain determined standards dependent on whether the organism is to be classified as sensitive, tolerant/insensitive or resistant to the test antibiotic.11 Although ZOI quantification is perfectly acceptable for antibiotic compounds, literature surrounding the application of ZOI tests to silver compounds contradicts this type of relationship, as discussed by Gallant-Behm et al.2 Silver is a popular choice as the antimicrobial agent in wound dressings. Studies with silver demonstrate that zone size can reach a maximum measurement at low concentrations. Tests showed zone size was not proportional to silver concentration. Further tests investigating silver sulfadiazine efficacy show lack of correlation between zone size and MIC (minimum inhibitory concentration) values. Moreover, silver diffusion assays have demonstrated a 10-fold decrease in the activity of the silver for every 2 mm of diffusion through agar, indicating that the relationship between silver release and diffusion does not follow a linear pattern as with antibiotics, suggesting that the silver agent interacts with the test medium and affecting diffusion. Therefore the lack of a ZOI does not necessarily indicate that the antimicrobial activity of the test dressing has been lost.

10.4

Bacterial barrier testing

10.4.1 Background An important, but perhaps less considered, element in the performance of a wound dressing is its ability to impede or prevent the passage of bacteria through it. This is, of course, a potential property of a wound dressing that can be engineered for and tested on all wound dressings regardless of whether they are antimicrobial or not. The benefits of bacterial barrier properties in a dressing are twofold.

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Firstly, it protects the wound from bacterial contamination from the hospital environment, the patients own skin flora and other wounds and from the healthcare practitioner.12,13 This is most important in initially uninfected wounds such as surgical incisions as prevention of wound infection is obviously preferable to having to resort to treatment. Secondly, the barrier properties of a dressing may prevent the spread of bacteria from an infected wound protecting those giving care to the patient, any other wounds present on the patient, the healthcare environment and other patients in close proximity.18 This is of importance when considering infection control, particularly of antibiotic-resistant strains, in a healthcare environment. For any dressing with a film backing a trade off may be involved in deciding on the porosity of a film, to balance the permeability to moisture to aid wound healing, with the film’s ability to prevent the passage of bacteria. It is also worth noting that the methods referred to here are distinct from those that are used, for instance, in measuring the protective properties of surgical facemasks14 and medical device packaging15 which focus on the challenges of aerosols, splashes and soaking rather than the more complex two way barrier interaction faced by a wound dressing in the clinical setting. There are some reports in the literature of in vivo models which have been developed to investigate the bacterial properties of dressings and these can be perused for further information.12,13,16

10.4.2 Testing When selecting a method to test the bacterial barrier properties of a dressing it is important to consider several factors such as what product claims are required, the composition of the dressing both physically and chemically, and choice of test organism. However, the principle of the test is the same in all cases: a bacterial challenge is applied to one side of the dressing and, after an appropriate time, the other side of the dressing is sampled to in order to establish whether bacteria have penetrated through the dressing. Wherever possible, testing should be carried out on a finished dressing as any heating, stretching or pressure applied to the components of a dressing during its manufacture may affect the barrier properties of the materials. Whilst specific bacterial strains may be used in the test, such as to produce marketing literature demonstrating the product is a barrier to MRSA, for example, the range of organisms should always include one which provides the most difficult challenge to the product in order to sustain the robustness of the claim. The two major factors that influence an organism’s ability to penetrate through a dressing are the cell size and motility. The smaller and more motile the organism, the greater the challenge for the dressing. A good example organism for this type of testing is Serratia marcescens which is both relatively small in its cell size (0.5–0.8 μm in diameter and 0.9–2 μm

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in length17) and is highly motile. A further advantage of using S. marcescens is that on agar its colonies have a distinctive red/pink pigmentation, which easily allows contamination to be ruled out in cases where a dressing or material has failed the test, resulting in growth on the un-inoculated side of the dressing. All forms of barrier testing should be carried out in multiple replicates, preferably at least in triplicate. In order for a passing result to be achieved, all replicates must prevent the passage of bacteria. There are several variations of test method that are acceptable for demonstrating bacterial barrier properties and these are distinguished from each other by the nature of the contact with bacteria, these can be designated as: (a) Dry : dry bacterial barrier test (b) Wet : dry bacterial barrier test (c) Wet : wet bacterial barrier test. The simplest form of bacterial barrier testing, dry : dry, is carried out under low moisture conditions. This involves preparing an inoculated plate by streaking a recognisable shape, such as a cross, of the test organism on a contact agar plate and incubating this at the relevant temperature for the test organism for 24 hours. A sterile sample of the test dressing is then placed wound contact layer down over the inoculated plate and an uninoculated contact agar plate is placed on top of the dressing and a weight is placed on top of the whole plate-dressing-plate assembly. The whole assembly is then incubated at the relevant temperature for 24 hours or the desired time period for demonstration of barrier properties. The uninoculated plate is then removed and incubated for a further 24 hours at the same temperature. Whether the dressing passes or fails this test is then indicated by the presence or absence of growth on the un-inoculated plate. There are several advantages to dry : dry testing. Firstly this method is simple, cheap and easy to perform requiring little specialist equipment. Secondly this method can demonstrate the barrier properties of a dressing in its totality, including the effect of any antimicrobial compounds. However, this also means that the method has a converse disadvantage in that it is unable to distinguish whether the physical properties (e.g. top film) or the antimicrobial agent within the dressing is the cause of the impedance of bacterial movement. The other major disadvantage of this method is that it is a poor replication of the clinical setting in which the dressings are designed to be used. A variation on this method that provides a slightly greater challenge is a test under semi-wet conditions, known as a wet : dry test. In this form of the test the dressing is applied wound contact layer down to an un-inoculated agar plate and a broth culture is applied in small aliquots to various points

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on the external side of the dressing. The dressing and plate assembly is then incubated at the relevant temperature for the test organism for 24 hours, the excess culture and the dressing are then removed and the agar plate returned to the incubator for a further 24 hours. As with the previous variation of the method the dressing passes the test if there is an absence of growth on the agar plate after further incubation. The benefits of this method are largely the same as the dry : dry method, but it offers a more realistic and difficult challenge to the dressing due to the additional moisture in the inoculum suspension facilitating the movement of the motile cells through the dressing, which is more representative of an exuding wound. For both the dry : dry and wet : dry methods, neutralising agar plates may be required when releasing antimicrobial agents are present within the dressing. The most stringent and complex test of bacterial barrier capabilities can be achieved using a wet : wet method,18 so called due to the fact that the dressing is immersed in fluid on both its wound contact and external faces. This test is carried out by clamping the test dressing between two vessels which are then filled with a liquid culture medium such as Tryptone Soya Broth (a diagram demonstrating a test rig set up can be seen in Fig. 10.4). Bacteria are added to the media on the external side of the dressing and the testing rig is incubated at room temperature for a length of time appropriate to the intended wear time of the dressing, from a minimum of 24 hours. At the end of the incubation period the media on both sides of the dressing is sampled onto agar plates and these are incubated at the relevant temperature for the test organism. Growth from the inoculated side of the test rig is required to validate the result by demonstrating that the culture has remained viable throughout the test period. Growth from the uninoculated side of the test rig demonstrates that bacteria have penetrated through the dressing and so represents a failure. An absence of growth from the un-inoculated side of the dressing demonstrates that bacteria have been unable to pass through the dressing and as such represents a test pass as a bacterial barrier. It is also possible to sample at various points throughout the incubation period in order to track performance of the dressing over time, for instance on a daily basis over 7 days. If the tested dressing fails the test before the target time this allows at which point failure occurred to be established. It could be argued that the wet : wet method provides a challenge that is tougher than would be found in a clinical setting due to the total immersion of the dressing and the pressure that this exerts on it. However, the strength of the challenge provided by this method gives as close to absolute proof as can be reasonably achieved of a dressing being impervious to the transmission of bacteria over the test period. Another key advantage of this method is that it can be used to separate physical barrier properties from barrier

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Sample can be taken out for streak plate after required test duration Sample can be taken out for streak plate after required test duration

Bacterial culture added to apparatus half containing external face of dressing at start of testing

Dressing sandwiched between two halves of test apparatus and assembly filled with broth medium on both sides

10.4 An example of a typical test rig set up for wet to wet bacterial barrier testing.

properties provided through the addition of an antimicrobial agent, by using neutralisers in the media. Plates of neutralising agar should also be used in the sampling from an antimicrobial dressing to make sure any carried over agent doesn’t impede the growth of any bacteria that are present. The major disadvantage of the wet : wet test method is that it is unsuitable for dressings which disintegrate in any way when moistened. It is a requirement of the method that a seal can be maintained between the two portions of the test apparatus otherwise the liquid medium will leak away leaving nothing to test at the end of the incubation period. The method is therefore largely restricted to dressings that contain a film or membrane element. The wet : wet method was the proposed choice of a European attempt to standardise a method for bacterial barrier testing and formed part of a draft standard,19 but no final resolution to adopt it could be reached and the standard was never implemented in that form. The wet : wet method also lends itself to possible adaptation to test the viral barrier properties of a dressing, although this is highly specialised work and considered to be outside the scope of this chapter and will not be discussed further except to advise that specialist contract test houses are available to carry out viral barrier testing.

10.5

Other considerations

10.5.1 Choice of test method As has been alluded to throughout the method descriptions in this chapter, the variations in details of the method used for testing a dressing should be

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defined by several considerations. Moreover, the choice of which type of test should be used in the first place is defined by similar factors. The first and most important factor to consider is ‘what is the aim of the testing?’ If, for instance, a desired claim is that the dressing is effective at killing Staphylococcus aureus, then the most suitable test is a log reduction test using a suitable S. aureus strain. Whilst some degree of antimicrobial activity can be demonstrated in a ZOI test, this would not demonstrate kill, but rather only inhibition of growth (Section 10.3.5), and would not provide the quantitative data required to make a claim of bacterial kill (i.e., a reduction of at least 3 log CFU/sample). Beyond the choice of the type of test, it is vital to tailor the method to allow the information required to be generated. As discussed in earlier sections, the manner in which a dressing is exposed to bacteria, how bacteria are recovered and enumerated from the sample in a log reduction test, the test organisms to be used, the concentration of the test organism(s), the time points for test duration and incubation temperature used (amongst many other factors) should all be guided by the physical properties and intended clinical application of the final product. Further considerations that may alter all aspects of the planned tests are the requirements of a regulatory body. Bodies such as the FDA provide guidelines as to the packages of testing that are required to launch products or to make particular claims, which often specify the details of methods that they consider requirements for testing to be valid.20 However it is not wise to rely on these guidelines alone; there are often long periods between the issue of individual sets of guidelines and they are continually evolving so the actual requirements stipulated by bodies are often subject to change. These guidelines cover a range of points across the whole process of testing from required performance levels to the specifications of control materials. In order to avoid delays and unexpected extra testing burdens it is often better to meet with regulatory specialists and representatives of the regulatory bodies where possible, to review the intended test plan to ensure it will be acceptable for submission (note: the regulatory requirements for different classes of devices have not been discussed in this chapter but the device classification should also be considered when formulating a test plan).

10.5.2 Future trends Although the main scope of this chapter was to cover the more common antimicrobial test methods, there are certain trends in the area of evaluating antimicrobial wound dressings that are worth noting, such as biofilm testing and molecular-based methods. Both areas are not as commonly dealt with in development of antimicrobial methods, partially due to the aforementioned lack of standardisation and partially due to the industry in general

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being slow to adopt new techniques with a reticence to replace well-known traditional ones when it is not demanded of them. Firstly, there is the ever-increasing importance placed on the role of biofilm in wound healing and infection. The literature in the area of wound care paints a far more complex picture of the interactions and roles of different microbes than was initially conceived.21 This in turn brings into question the relevance and validity, with regard to the clinical situation, of the traditional methods that are based on free floating (planktonic) single species cultures, as have been discussed in this chapter. As such, methods have been developed to test the ability of antimicrobials to act on biofilms ranging from the qualitative/semi quantitative approach of using cell stains and confocal microscopy to visualise microbial communities in situ22,23 to more well-regulated and robust quantitative methods such as those based on the use of the MBEC AssayTM (initially known as the Calgary Biofilm device).24 Due to increasing interest in this area, standards are beginning to emerge25,26,27 and it is likely that in future there will be a more widespread adoption of testing against biofilm into the battery of routine testing applied to the development of antimicrobial wound dressings, continuing its transition from research tool to industrial test. The other area of research that may have an impact on antimicrobial dressing testing in coming years is the use of molecular biology methods, many of which are carried out as part of academic research. These methods are at the cutting edge of studying genetics and protein expression but in relation to routine testing of antimicrobial dressings they should be considered more as alternatives to current methodologies described previously in this chapter, rather than the biofilm methods which explore a new area of dressing performance. The use of polymerase chain reaction (PCR) technology is a good example.28 In this context PCR would replace the plating and incubation of recovered bacterial suspension in log reduction testing, allowing enumeration of cultures in a much reduced time compared to the traditional methods. Currently this kind of method is only used in clinical, environmental and food microbiology, but it could readily be adapted for wound dressing testing. Other than decreasing lead times of microbiology testing, due to the need to incubate agar plates for several days, molecular techniques offer several other advantages over these traditional plate counts. Firstly, there is the increased precision of these methods; cells that are damaged or stressed by exposure to an antimicrobial preventing them growing on agar would be enumerated by a molecular method. Secondly, there is a reduction in the amount of physical processing that samples require, for example the hundreds of serial dilutions required for log reduction testing would not be required. Thirdly, a huge amount of time would be saved from not needing

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to count colonies on agar plates (though it should noted that there are commercially available plate-counting systems which can significantly speed up colony counting on agar). In techniques such as PCR, counts would be generated directly from the number of copies of the DNA found in the sample (though validation ahead of the test using agar plates would likely be required in order to achieve this). Finally and most basically, there is a reduction in the amount of space that an experiment takes up in terms of media storage and plate incubation. The main factor preventing the adoption of molecular methods is the upfront investment in terms of time and resources required to ‘trailblaze’ these kinds of methods with regulatory bodies. Whilst there are no rules against replacing existing approved methods, they need to be validated and demonstrated to be equivalent or better than those already in existence. Once these kinds of methods are accepted by regulatory bodies it is likely that uptake will increase.

10.6

Sources of further information and advice

10.6.1 Websites American Society for Microbiology. http://www.asm.org/ Centre for Biofilm Engineering. http://www.biofilm.montana.edu/ Society for General Microbiology. http://www.microbiologyonline.org.uk/ World Wide Wounds – Understanding the effects of bacterial communities and biofilms on wound healing. http://www.worldwidewounds.com/2004/ july/Percival/Community-Interactions-Wounds.html Wounds UK. http://www.wounds-uk.com/

10.6.2 Books The Biofilm Primer, Costerton.29 This book provides a general introduction into microbial biofilms including those in the healthcare environment. Microbiology of Chronic Wounds, Percival and Cutting.30 This book provides a general introduction into the microbiology of wounds, wound healing and the treatment of chronic and acute wounds. Skin and Wound Infection: Investigation and Treatment in Practice, Hay, Middleton and Seal.31 This book provides a general introduction into both skin and wound infection and considers some tropical infections. Theory and Application of Microbiological Assay, Hewitt and Vincent.32 This book provides a general introduction into microbiological test methods.

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References

1. Prince Herbert N, Prince Daniel L. Antimicrobial silver in orthopedic and wound care products. Orthopedic Design & Technology, May 1 2008 2. Gallant-Behm CL, Yin HQ, Liu S, Heggers JP, Langford RE, Olson ME, Hart DA, Burrell RE. (2005). Comparison of in vitro disc diffusion and time killkinetic assays for the evaluation of antimicrobial wound dressing efficacy. Wound Rep Reg 13:412–421 3. Driffield K, Woodmansey E, Walter J, Floyd H. Determination of the microbiological performance of a nanocrystalline silver dressing and silver-containing hydrofiber in a 7 day repeat challenge assay. SAWC 2007 (poster) 4. Greenman J, Thorn RM, Saad S, Austin AJ. (2006). In vitro diffusion bed, 3-day repeat challenge ‘capacity’ test for antimicrobial wound dressings. Int Wound J Dec;3(4):322–329. 5. Class II Special Controls Guidance Document: Wound Dressing with Poly(diallyl dimethyl ammonium chloride) (pDADMAC) Additive. October 16, 2009 http:// www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/Guidance Documents/ucm186571.htm 6. BSAC Working Party. BSAC Methods for Antimicrobial Susceptibility Testing. Version 9.1 March 2010. www.bsac.org.uk 7. Basterzi Y, Ersoz G, Sarac G, Sari A, Demirkan F. (2010). In-vitro comparison of antimicrobial efficacy of various wound dressing materials. Wounds 7:165–170 8. Cavanagh MH, Burrell RE, Nadworn PL. (2010). Evaluating antimicrobial efficacy of new commercially available silver dressings. International Wound Journal 7(5):394–405 9. Bauer AW, Kirby WMM et al. (1966). Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45:493–496 10. Cuzzell J. (1997). Choosing a Wound Dressing. Geriatric Nursing 18(6):260–265 11. Performance Standards for Antimicrobial Disk Susceptibility Tests, 5th ed. M2-A5. 1993. Clinical Laboratory Standards Institute (CLSI – formerly NCCLS), Villanova, PA 12. Mertz PM, Marshall DA, Eaglstein WI. (1985). Occlusive wound dressings to prevent bacterial invasion and wound infection. J Am Acad Dermatol 12(4):662–668. 13. Mertz PM, Eaglstein WH. (1984). The effect of a semi-occlusive dressing on the microbial population in superficial wounds. Arch Surg 119:287–289. 14. ASTM F2101-07. Standard Test Method for Evaluating the Bacterial Filtration Efficiency (BFE) of Medical Face Mask Materials, Using a Biological Aerosol of Staphylococcus aureus 15. ASTM F1608-00 (2009). Standard Test Method for Microbial Ranking of Porous Packaging Materials (Exposure Chamber Method) 16. Mertz PM, et al. (2003). Barrier and Antibacterial Properties of 2-Octyl Cyanoacrylate-Derived Wound Treatment Films. J Cutan Med Surg 1–6 17. Holt J et al. (1994). Bergey’s Manual of Determinative Bacteriology: Ninth Edition, Baltimore MA: Williams and Wilkins 18. Ameen H, et al. (2000). Investigating the bacterial barrier properties of four contemporary wound dressings. J Wound Care Sep;9(8):385–388

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19. prEN 13726-5:2000, Draft European standard, Test methods for primary wound dressings – Part 5: Bacterial Barrier Properties draft standard for bacterial barrier. CEN (2000) 20. Guidance for Industry and FDA: Class II Special Controls Guidance Document: Antimicrobial Susceptibility Test (AST) Systems. Centre for Device and Radiological Health (2009) 21. Percival S and Bowler P. Biofilms and Their Potential Role in Wound Healing. Jul 10 2004. Wounds Research http://www.woundsresearch.com/article/2870 22. Percival et al. (2008). Assessing the effect of an antimicrobial wound dressing on biofilms, Wound Repair Regen Jan–Feb;16(1):52–57 23. Thorn RM, Austin AJ, Greenman J, Wilkins JP, Davis PJ. (2009). In vitro comparison of antimicrobial activity of iodine and silver dressings against biofilms. J Wound Care Aug;18(8):343–346. 24. Ceri H et al. (1999). The Calgary Biofilm Device: New Technology for Rapid Determination of Antibiotic Susceptibilities of Bacterial Biofilms, Journal of Clinical Microbiology June;37(6):1771–1776. 25. ASTM E2196-07, Standard Test Method for Quantification of a Pseudomonas aeruginosa Biofilm Grown with Shear and Continuous Flow Using a Rotating Disk Reactor 26. ASTM E2647-08, Standard Test Method for Quantification of a Pseudomonas aeruginosa Biofilm Grown Using a Drip Flow Biofilm Reactor with Low Shear and Continuous Flow 27. ASTM E2562-07, ASTM E2562-07 Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow using CDC Biofilm Reactor 28. Martin FE et al. (2002). Quantitative Microbiological Study of Human Carious Dentine by Culture and Real-Time PCR: Association of Anaerobes with Histopathological Changes in Chronic Pulpitis, Journal of Clinical Microbiology May;40(5):1698–1704 29. William Costerton. The Biofilm Primer. Springer; Novemeber 2010. ISBN-10: 3642087655 30. Steven Percival and Keith Cutting. Microbiology of Chronic Wounds. CRC Press; April 2010. ISBN-10: 1420079937 31. Roderick Hay, Keith Middleton and David Seal. Skin and Wound Infection: Investigation and Treatment in Practice. Informa Healthcare; November 2000. ISBN-10: 1853177350 32. William Hewitt and Stephen Vincent. Theory and Application of Microbiological Assay. Academic Pr; January 1989. ISBN-10: 0123464455

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11 Adhesives and interfacial phenomena in wound healing B. J. T I G H E and A. M A N N, Aston University, UK

Abstract: This chapter deals initially with the underlying principles of adhesion and adhesives and the understanding of interfacial behaviour. This provides a basis upon which to understand biological interactions (Chapter 12). The two broad types of adhesive materials encountered in wound healing are pressure-sensitive adhesives (PSA) and tissue sealants. The function of pressure-sensitive adhesives is to form an adhesive bond between tissue and biomaterial under the influence of pressure. Tissue sealants are liquids that convert to solid form at the tissue surface and in so doing form either an effective seal against fluid leakage or a bond between adjacent tissue surfaces. The different requirements and characteristics of these systems are discussed. Key words: pressure-sensitive adhesives, tissue sealants, tissue interfaces, hydrocolloids, hydrogels.

11.1

Principles of adhesion, adhesivity and interfacial behaviour

11.1.1 Interfacial Interactions Adhesion can be defined as the state in which two surfaces are held together by interfacial forces. The molecular concept of interfacial forces follows logically from the more familiar physicochemical ideas of wettability and surface tension. Wettability can be thought of as the formation of a continuous fluid film over a solid surface. More specifically it is defined as the adhesion of a liquid to a solid. The concepts of cohesion and adhesion need to be distinguished. Cohesion refers to the force of attraction between molecules of the same substance. It is these forces that hold a droplet of liquid together. In contrast, adhesion refers to forces acting between molecules of two different substances. Wetting can be thought of as adhesion between molecules of a wetting liquid and molecules of the surface over which it is to spread. Some liquids, when placed in contact with a solid surface, are mainly attracted to other molecules of liquid rather than molecules of the solid. It is this cohesive force that must be overcome if wetting of a solid surface is to be achieved. 247 © Woodhead Publishing Limited, 2011

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This leads on to the concept of surface tension. In a droplet of liquid, the molecules at the centre of the drop are surrounded by similar molecules. The cohesive forces of attraction between these similar molecules are the same in every direction, effectively cancelling each other out. This is not true, however, at the surface of the drop. There is no outward attraction to balance the cohesive forces pulling the molecules inwards. This creates the familiar spherical shape of the liquid droplet. As a result of this unopposed inward pull, the surface molecules have an excess amount of potential energy. The excess energy per unit area is known as the surface-free energy or surface tension. It is a force that resists attempts to deform the liquid surface. The stronger the cohesive forces between the molecules of a liquid, the higher the surface tension of that liquid will be. Surface tension is not limited to liquids; solids will also have a surface tension that arises in the same way. Because the bonding in a solid is much stronger, the inward pull of the surface molecules cannot change the solid shape but the same surface forces are there. This is an important point and underlines the fact that solids, just like liquids, exhibit a surface tension – usually referred to as surface-free energy. This results in interactive forces between two solids when they are bought into contact – which leads to the concept of interfacial tension. When a liquid wets a solid surface, one must take into consideration not just the unopposed inward attraction of the molecules at the surface of the liquid drop, but also the forces of attraction between the liquid molecules and the solid. Like and unlike forces come to equilibrium across the interface and produce a net energy difference between the surfaces – this value is referred to as the interfacial tension. The greater the commonality of surface forces between the liquid and the solid, the lower the interfacial tension will be and the more likely it is that the liquid will spread over the solid surface. It is convenient to simplify forces by dividing them into two categories – polar and non-polar. In the systems we are dealing with, hydrocarbon groups are non-polar and hydrophilic groups are polar – characterised by the possession of a marked dipole moment. All structures, even water, have a non-polar component, however, and it is necessary to measure, or calculate the values. The terminology is important. The symbol for total surface tension or surface energy is γ and is made up of separate polar (γp) and dispersive (γd) components. When the total surface tension or the separate polar and dispersive components refer to separate phases (such as a liquid, 1 and a solid, 2) subscripts are used to distinguish between them. This enables us to define the various contributing elements that contribute to the interfacial tension, γ1,2 between two phases (such as a liquid, 1 and a solid, 2) which is given by: γ1,2 = γ1 + γ2 − 2(γ1δγ2δ)1/2 − 2(γ1πγ2π)1/2

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where γ1d and γ2d are the non-polar (called dispersive) components of the liquid and solid phases, and γ1p and γ2p are the polar components of the liquid and solid phases. Two important consequences are made clear by this relationship: • •

The first is that interfacial tension is at its lowest if the polar and nonpolar forces in the two phases match each other. The second is that if the polar component of only one of the phases (such as the solid) falls to zero, the value of the expression 2(γ1p γ2p)1/2 also falls to zero and the value of γ1,2 (the interfacial tension) rises dramatically.

Thermodynamics rules natural phenomena and will always drive systems to their lowest energy state. This principle drives the value of interfacial tension down and as a result polymers undergo chain rotation to match their surface energy components to those of the phase with which they are in contact. This is particularly important for polymers with very flexible chains having a relatively low energy barrier to rotation such as hydrogels (Section 11.2.3 and Chapter 13). This qualitative discussion is underpinned by the quantitative expression given above. Water has a very high polar component (51.0 mNm−1) and a dispersive component (21.8 mNm−1) that is very similar to most low molecular weight organic compounds. Hydrocarbons and oils have dispersive components around 20 mNm−1 and virtually zero polar component. Consequently, the interfacial tension between water and hydrocarbon groups is around 50 mNm−1, which is very high considering that the interfacial tension between hydrocarbon groups and air is close to 20 mNm−1. The consequence is that conventional hydrophilic polymers and hydrogels undergo chain rotation when exposed to air or other hydrophobic domains such as lipids, thereby exposing the hydrocarbon groups of the hydrophilic polymer and reducing interfacial tension. This is an important factor in understanding both skin adhesion and wound fluid interactions of these materials. In summary, high interfacial tensions are likely to arise when synthetic polymers, which are for the most part dominated by hydrophobic (frequently carbon-carbon backbone sequences) domains, interact with aqueous environments such as exposed wound surfaces. Dry unbreached skin is much more hydrophobic and is therefore capable of forming low interfacial tensions with polymers that are similarly hydrophobic. This situation is exploited in pressure sensitive adhesives for dermal applications. In order to understand the principles of pressure-sensitive adhesive selection in wound healing and other dermal applications the principles of adhesion and wetting must be explored.

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γ1 (liquid) θ γ2 (substrate)

γ1,2 (interface)

11.1 Generalised liquid/solid wetting parameters.

11.1.2 Adhesion and adhesivity The importance of Equation 11.1 and of understanding the magnitude of the polar and dispersive components of material and substrate becomes apparent when the concept of work of adhesion is introduced. The work of adhesion (Wa) is given by the expression Wa = γ1 + γ2 − γ1,2

11.2

Thus when the interfacial tension is high, the force of adhesion is weak. A molecular principle of adhesive behaviour is that good wettability and low interfacial tension promote good adhesion. This general principle becomes more apparent in terms of a measurable physicochemical property if wettability is considered in terms of the contact angle of the liquid (1) on the solid substrate (2) together with the consequent interfacial tension, γ1,2 (Fig. 11.1). Ignoring the effect of the vapour phase, the work of adhesion is linked to the contact angle by the expression: Wa = γ(1 + Cos θ)

11.3

These underlying concepts provide the basis for understanding of adhesion phenomena with pressure-sensitive adhesives, tissue sealants and adhesives and the general principle of biocompatibility, where there is a general drive to reduce high interfacial tension between a biomaterial and the biological environment by ‘coating’ the material to reduce interfacial tension. The principle that perfect wetting will produce maximal intermolecular forces between two phases is sound. Furthermore we need the underlying knowledge of interfacial forces in order to address the current position and potential future prospects of adhesives and adhesive phenomena in wound healing. Despite this, the practical – as distinct from theoretical – situation presents additional challenges. Real surfaces are not perfectly smooth and

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in simplistic terms entrapped air and undulating surfaces reduce the area of contact and thus the effectiveness of the bond. This problem increases as the viscosity of the adhesive increases and will be potentially severe in a polymer-based pressure-sensitive adhesive. Additionally, the mechanical properties of the substrate are influential both in forming the interface and in determining the consequent bond strength. In consequence there have been many attempts to investigate the nature of, and enhance the strength of, adhesive bonds on the one hand, and to understand the particular problems of skin and other biological substrates on the other. In surface science, the term ‘adhesion’ almost always refers to dispersive adhesion resulting from the forces discussed above and involving van der Waals interactions or hydrogen bonding, but other mechanisms have been identified and can become important in different circumstances. It is useful to identify these mechanisms and to note their relevance to wound healing materials and devices: •

•

•

•

Mechanical adhesion refers to the situation in which adhesive materials fill the voids or pores of the surfaces and hold surfaces together by interlocking. In the context of wound repair the use of sutures provides an example of a large-scale mechanical bond. Chemical adhesion involves the formation of a chemical bond between the two interfaces or between the interface and the adhesive. These can be ionic or covalent bonds and involve the interaction of functional groups. The area of tissue adhesives or tissue sealants is an obvious example of an application in which such a strategy is important. Diffusive adhesion occurs as a result of molecular interpenetration or interdiffusion. This requires sufficient molecular mobility for one component at the interface to penetrate the surface of the other. The mechanism is enhanced if there is a degree of mutual solubility and a carrier solvent is often used in commercial systems to enable this to occur. It is also the mechanism involved in sintering of metals or ceramics and also the fusion of polytetrafluoroethylene (PTFE) particles. Wound healing is dependent upon diffusional adhesive processes since these underpin the necessary cell-cell interactions for wound repair. Diffusion and flow are inextricably linked to the function of both pressure-sensitive adhesives and tissue sealants. Electrostatic adhesion is thought of conventionally as arising from the electronic mobility in some conducting materials which leads to a difference in electrical charge at the join. This creates an electrostatic force of attraction between the materials. This is not of obvious importance in wound repair although some analogies exist with the behaviour of polyelectrolytes such as poly(acrylic acid) which are used in some types of skin adhesive bioelectrodes.

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11.1.3 Interfacial tension and biological interfaces The same interfacial phenomena that govern adhesive behaviour also influence biocompatibility. The need to promote chain flexibility and to maximise hydrophobic interactions with the substrate will become apparent in considering the structural requirements for pressure-sensitive adhesive behaviour; this will inevitably have consequences for the molecular response of such materials to biological fluids. The key fact is that although polymers may have hydrophilic groups capable of interacting with water, when presented with hydrophobic domains, such as lipids or air, chain rotation occurs in order to expose the hydrocarbon groups of the backbone and reduce interfacial tension. This is beneficial in the design of skin adhesives but it can lead to unintended interactions with wound fluid and the wound site. The physicochemical aspects of polymers that are involved in these processes are chain rotation and surface hysteresis. The wettability of a surface is usually measured by observing its contact angle behaviour – the angle formed between the surface and a tangent to the surface of the droplet placed on that surface (Fig. 11.1). An angle of zero implies complete wettability. A contact angle measured in this way can use a droplet either advancing over the surface, receding over the surface, or in equilibrium and at rest. The advancing angle is usually measured in air with the droplet advancing over a surface that has been exposed to air. The receding angle is measured when a droplet is withdrawn from a surface with which it has previously been in contact. The receding contact angle is often smaller than the advancing angle and the difference between the advancing and receding contact angles is known as contact angle hysteresis. The main reason for contact angle hysteresis in the context of the flexible polymer systems involved in wound dressings is the reorientation of polymer chains at the surface of the material. In many polymeric materials, especially hydrogels, polymer chains have some mobility, allowing them to rotate at the material surface. If a material is in contact with air (which is a very hydrophobic medium) or other hydrophobic surfaces, hydrophobic groups within the polymer will rotate towards the surface of the material creating a less wettable surface. In contrast, if the material surface is in contact with an aqueous liquid, the hydrophobic groups will rotate inwards and be replaced at the surface by more hydrophilic groups increasing the surface wettability. Thus when an advancing angle is measured, the wetting liquid encounters more of the surface hydrophobic groups and a larger contact angle is obtained. When a receding angle is measured, the previously wet surface expresses more of the hydrophilic groups at the surface and it is these groups that the wetting liquid encounters, resulting in a smaller contact angle. In conventional hydrophilic polymers, even those swollen with water such as hydrogels, the water-binding properties of the pendant hydrophilic

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groups (typically hydroxyl, carboxyl or amide) are inadequate to shield the hydrocarbon backbone. This is particularly true if the chain has a pendant methyl group such as poly(2-hydroxyethyl methacrylate), or polyHEMA. Although the advent of hydrogels, typified by polyHEMA, brought many advantages they do not overcome the problems that are reflected in chain rotation and contact angle hysteresis. Their longstanding use in the contact lens field, despite all the commercially well-funded attempts to overcome the problem, still manifest the problem of non-specific adsorbtion of proteins and lipids. The influence of key proteins on ocular compatibility is much more readily studied than is the influence of wound dressing biomaterials on the wound interface and specific protein interactions. There is no doubt, however, that the two situations are influenced by the same thermodynamic driving forces and interfacial phenomena. These points are explored in Chapters 12 and 13.

11.2

Bioadhesion: principles of adhesion applied to wound healing

A bioadhesive has reasonably been defined as a substance that has the ability to adhere to a biological material and is capable of being retained on the biological substrate for a protracted period. One distinctive feature of bioadhesion is that adhesion usually occurs in the presence of water. Three different types of bioadhesion have been identified (Park et al., 1986): • • •

Type I: adhesion between biological objects. Type II: adhesion of biological substances to an artificial substrate. Type III: adhesion of an artificial component to a biological substrate.

This approach avoids the unique categorisation of bioadhesion and allows for appropriate mechanisms to be used to describe and interpret the molecular processes that are involved in forming each type of interfacial bond. Various mechanistic interpretations have been advanced and are reviewed elsewhere (Park et al., 1986; Chickering and Mathiowitz, 1999). The importance of interfacial properties on the adhesion bond between an adhesive and a substrate has been discussed. Mechanical properties of the substrate also have a role to play and are particularly important in their influence on rheological behaviour at the interface. This is governed by the rheological properties of the adhesive (which dictate flow and elasticity) and the rheological behavior behaviour of the adherend substrate. It will be apparent therefore that in designing and selecting adhesives for dermal applications, adhesion data generated on a rigid, high energy substrates such as steel and glass will be of limited value.

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This chapter deals with two distinct ways in which adhesive bonds are formed in wound healing. The first is the use of a fluid that undergoes a chemical reaction – usually polymerisation – at the interface to form an adhesive bond. That is the domain of surgical adhesives and tissue sealants. The second is the use of an adhesive polymer which is placed in contact with a surface (usually unbreached skin) and forms a bond under the action of pressure. This is a so-called pressure-sensitive adhesive used in a range of applications including surgical tapes, ostomy products and wound dressings. The first attempt to quantify the rheological requirements for good adhesive behaviour arose from the work of Dahlquist (1969). He observed that a minimum level of compliance was required in order for a pressuresensitive adhesive to exhibit tack. This observation can also be interpreted as a maximum value of the modulus, the measure of the stiffness and resistance of the adhesive, and is known as the Dahlquist criterion. This can be broadly described as a need for the material to be deformable but not flow – that is it should be a viscoelastic material which will absorb energy rather than allowing it to propagate through crack formation. The Dahlquist criterion requires that the elastic modulus of the material should be below 105 N/m2 (Pa). Polymers undergo changes in molecular mobility corresponding to detectable phase changes as their temperature is raised. The most important transition for pressure-sensitive adhesive behaviour is the glass transition temperature (sometimes referred to more fully as the glass-rubber transition temperature). As the name implies, only if a polymer is above its glass transition temperature can it be rubbery, or elastic, and therefore able to meet the requirements of the Dahlquist criterion. This means that a minimum requirement for pressure-sensitive adhesive behaviour is that the material should have a glass transition temperature below room temperature. There are, however, two further considerations: the first is that the temperature at which the adhesive is stored will normally be appreciably lower than skin temperature and the second that the glass transition temperature reflects not an abrupt, but a gradual change in molecular mobility which extends over a temperature range of some thirty degrees. Consequently, the target glass transition temperature for a pressure-sensitive adhesive lies in the range −20°C to −60°C. This guide, together with the concept proposed by Dahlquist, addresses the need for adequate flow and resistance to crack propagation within the adhesive. The counterbalancing requirement is that the adhesive must have sufficient cohesive strength to maintain the bond once formed. Thus, cohesive failure (i.e. within the adhesive layer rather than at the interface) can be a consequence of brittle fracture or flow – sometimes called ‘legginess’. The modes of failure of PSAs are presented in Fig. 11.2.

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Cohesive failure Failure within substrate interface

11.2 Schematic representation of the four sites of cohesive/adhesive failure.

A wide range of studies have been carried out to determine the extent to which the substrate (backing) to which the adhesive is applied affects adhesive behaviour when applied to skin. Because of the extensive number of experimental parameters involved in such studies, not to mention the inherent patient-to-patient variability of skin, synthetic skin models are often used. An important conclusion is that the adhesive properties, and peeling behaviour in particular, depend strongly on the mechanical properties of the substrate as well as on the properties of the adhesive material (Chivers, 2001; Renvoise et al., 2007). In addition peeling rate has been shown to be infuential, not only in relation to peel strength but also in its effect on the mode of failure. Similarly, the mode of failure depends on both the rheological behaviour of the adhesive and the rheological behaviour of the substrate and these are in turn dependent upon the rate of peeling. Absorbtion of moisture, from sweat glands for example, brings a further complication since it has a progressive effect on rheological properties, peel strength and mode of failure (Renvoise et al., 2009). The background literature relating to the rheological requirements for good adhesive behaviour has been very usefully assembled by Renvoise et al. (2009), who has also collected available published data on the rheological and surface energy characteristics of skin. An additional valuable overview of the factors that guide selection and assessment of pressuresensitive adhesives for dermal applications has been compiled by Venkatraman and Gale (1998). The surface and rheological properties of skin depend on its location on the human body and in the case of surface properties the way in which the skin has been treated or washed. Useful guideline values (Table 11.1) can be extracted from the extensive range of studies which have been compiled and reviewed elsewhere (Kenney et al., 1992; Mavon et al., 1997; Venkatraman and Gale, 1998; Webster and West, 2001; Renvoise et al., 2009). This table emphasises the importance of removal of moisture and excess lipid, since they have a marked effect on skin properties. They increase the polar component of surface energy but in particular lead to

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Table 11.1 Representative values of human skin under different conditions*

Human Human Human Human Human

skin skin skin skin skin

(23°C, 34% RH) (36°C, 50% RH) (cleaned) (untreated) (high lipid)

Total surface energy (mNm−1)

Estimated polar component (mNm−1)

38 57 25.3 38.9 42

5 22 2.5 9.3 26

*Data from (Kenney et al., 1992; Mavon et al., 1997; Venkatraman and Gale, 1998; Webster and West, 2001; Renvoise et al., 2009).

the occurrence of adhesive failure within the skin interface (Fig. 11.2). It should be noted that there is considerable error associated with contact angle measurements made in different laboratories and error limits of up to ±50% are reported for some quoted values of polar components. The nature of the dermal surface presents unique problems for PSAs in the fact that human skin is an extremely variable substrate. Human skin is an organ and consequently dynamic and, although it is a barrier, it needs to breathe, stretch and perspire. The fact that transdermal delivery devices are growing in importance indicates that skin has absorbtive capability. The texture of skin, which also has an impact on PSA adhesion, is variable: elderly patients may have rough skin, whereas babies may have very smooth skin. Additionally, lipid and moisture content have a huge effect in modifying the surface properties of skin and these – as with all other factors mentioned here – show very significant patient-to-patient variation. It is quite clear, then, that there are several levels of additional complexity to be accommodated in moving from dry rigid inorganic substrates to composite organic tissue structures which generate moisture. For application onto unbreached skin these include: • • • • • • • •

appropriate surface energy to wet and satisfy minimum work of adhesion requirements appropriate rheological properties and glass transition temperature to provide compliance and avoid cohesive failure maintain adhesion to the skin for required period (1 to 7 days) absence of toxicity be comfortable to wear and not cause unacceptable irritation allow removal without excessive trauma to skin leave no residue on skin upon removal have adequate moisture transmission properties.

As with all medical devices safety and efficacy are of prime importance. Skin adhesives used in bandages, wound healing, and in transdermal systems

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must meet the overarching toxicological requirement since no PSA would be acceptable if it were to induce skin reactions. Three major types of assessment are important: a) b) c)

Primary skin irritation (PSI). Skin sensitization, measured by a repeat patch insult (RPI) test. Cytotoxicity of water-soluble extractants.

Details of required safety testing of adhesives for use on human skin are contained in FDA guidelines and ISO 10993 standards. The efficacy of skin adhesives is assessed with two broad types of test: a) b)

Peel and tack measurements which measure adhesive properties. Shear strength and viscoelastic behaviour which reflect cohesive properties.

For the adhesive to adhere to a substrate it must satisfy thermodynamic and kinetic conditions. Thermodynamically its measured surface energy must be equal to or less than that of the adherend, which will in principle produce a contact angle of zero (Fig. 11.1) and allow effective wetting of the substrate. A useful working parameter used to define this condition is the ‘critical surface tension’ of the substrate, which is the surface tension of the liquid that would just produce a contact angle of zero (Shafrin and Zisman, 1967). It approximates to the dispersive component of surface energy – or the total surface energy in the case of non-polar substrates. The working parameters for skin adhesion can now be summarised. The surface energy of clean and dry human skin is in the region of 30–40 mNm−1 (Table 11.1). Kinetically, the adhesive must flow sufficiently to promote intimate contact between the adhesive and the adherend. The Dahlquist criterion states that its elastic modulus should be <105 N/m2 (Pa), ideally between 103 and 104 N/m2 (Pa). This can only be achieved by polymers well above their glass transition temperature, which limits the choice to polymers having glass transition temperatures substantially below zero. The principles described in previous sections can now be applied to the design and selection of adhesives and sealants for wound healing applications.

11.3

Adhesives in wound healing: materials overview

There are many compilations of wound healing materials detailing types of dressing, characteristics, commercial products and clinical performance. Materials aspects of wound dressings are described particularly well by Ovington (e.g., Ovington and Pierce, 2001; Ovington, 2007). Similarly, Webster presents excellent accounts of the design and selection of medical adhesives (e.g., Webster, 1997; Webster and West, 2001). It is not the purpose of this chapter to review commercial products or simply to

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repeat compilations of dressing type and function. The focus here is on comparative adhesive and interfacial characteristics of the types of materials found in wound healing in the context of the underpinning principles involved and their relevance to the broader questions of biocompatibility discussed in Chapters 12 and 13. The principles of interfacial behaviour provide a useful basis upon which to categorise the wound healing materials to be considered: •

•

•

First, there are the conventional pressure-sensitive adhesives and their applications. They are designed primarily for contact with unbreached skin and usually would be expected have no adhesive or interactive function with the wound bed. Medical tapes and adhesive borders around island dressings serve as examples. There is, however, the unusual family of hydrocolloid materials that employ pressure-sensitive adhesives but in the form of a composite material that does not behave like a conventional PSA. Secondly, there are wound dressing materials that are used directly in contact with the wound, which do have some level of adhesive capability. Examples include the thin adhesive layer found on some transparent polyurethane films and hydrogel and hydrocolloid materials. Finally, there is the wide-ranging area of tissue adhesives and tissue sealants. These are quite different from the other two chasses – they are liquids which on solidification, gelation or polymerisation must form a good interface with the tissue with which they are brought into contact in order to produce a seal against fluid leakage or a bond between adjacent tissue surfaces.

These different categories of adhesive behaviour are summarised in Table 11.2.

11.3.1 Hydrophobic pressure-sensitive skin adhesives: structure and properties Pressure-sensitive adhesives (PSAs) have been described and defined in several ways: they are dry rather than wet, have a distinct surface tack, are pre-coated onto a carrier base such as a film, tape or bandage and form an adhesive bond with a variety of substrates when applied with pressure. They do not require activation by heat or removal of solvents and have sufficient cohesive strength to maintain the bond until removed, usually by peeling. The bonding is produced primarily by dispersive interactions but in some systems (skin adhesive hydrogels, for example) polar interactions play an important part. Pressure is required to achieve sufficient flow (hence the Dahlquist criterion) to enable the adhesive to ‘wet’ the surface in order to

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Table 11.2 Surface property criteria for PSA selection: typical critical surface tension values of polymer and copolymer systems (Shafrin and Zisman, 1967) Polymer

γC (mN/m)

Poly(dimethyl siloxane) Polyethylene Cis-polyisoprene Polyisobutylene Polystyrene Polyvinyl alcohol Polyvinyl chloride Polyvinylidene chloride Polymethyl methacrylate Polyhexamethylene adipamide (6,6 Nylon) ABS (acrylonitrile-butadiene-styrene rubber) Butyl rubber (polyisobutylene-co-isoprene) SBR (styrene butadiene rubber) SBR (styrene-isoprene-styrene rubber) Acrylic PSA

22 31 31 31 33 37 39 40 42 46 35 32 30 30 28

produce adequate adhesion. The principles involved are those described in Section 11.1. The essential requirements for PSAs intended for medical applications have been listed in Section 11.2. Of these, the need to stick firmly but also to be easily and cleanly removed are paramount. The families of materials that have proved to be successful as hydrophobic pressure-sensitive adhesives for use on skin fit the wetting and flow characteristics, defined in Section 11.2, in terms of their surface energy and glass transition temperatures (Tg). One of the first materials to be used as a pressure-sensitive adhesive for medical use was natural rubber or cispolyisoprene. Its structure, shown in Fig. 11.3a, provides a useful starting point in classifying PSAs used in wound healing. Cis-polyisoprene (whether of natural or synthetic origin) has a Tg of ca −70°C and a surface energy (expressed as its critical surface tension) of 30–32 mNm−1. In order to maintain the requirement of low Tg it is important that the potential effect of any structural changes in moving from the structure of cis-polyisoprene are understood. There are a small number of key structural guidelines: •

The ‘stiffness’ of the polymer backbone linkages decreases along the sequence: —C—C— > —C—O—C— > —Si—O—Si—

•

The Tg will increase if polar substituents are introduced into the backbone (hydrogen bonding and dipole effects).

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CH3

H C

CH2

CH3

C

H C

CH2 CH2

CH3

C

H C

CH2 PICH2

C CH2

Region of high crystallinity Amorphous region

(a)

(b)

(c)

11.3 Representations of polymer structure and organisation: (a) polyisoprene; (b) an isolated chain; (c) regions of crystalline order and amorphous disorder.

• •

The Tg will increase if bulky substituents are introduced close to the backbone (steric effects). In contrast the Tg will decrease if long flexible non-polar substituents are increased (‘internal plasticisation’).

There is one additional important point. Polymer chains that are regular and symmetrical will tend to pack into crystalline regions, which means that individual chains (Fig. 11.3b) pack into inflexible crystalline regions instead of being randomly entangled. These cannot flow at room temperature and thus destroy adhesive behaviour. These simple rules explain why some polymers have evolved as successful pressure-sensitive adhesives while others, with apparently similar structural credentials, are ineffective as PSAs. The first simple modification is to remove the methyl (CH3) groups from the cis-polyisoprene structure shown in Fig. 11.3a. This produces cispolybutadiene which has a Tg of ca −110°C. The reduction in Tg is a direct consequence of reduction in the small degree of steric hindrance in the backbone caused by the methyl groups. Some other potential candidate materials are shown in Fig. 11.4. Although polyethylene (Tg −90°C) would seem to be an obvious candidate it has a very regular structure, without the disorder caused by the cis double bonds that is obvious from Fig. 11.3a. This means that it very readily forms the crystalline regions shown in Fig. 11.3c.

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[CH2 – CH2]n

261

[Tg –90oC]

CH3 Polyisobutylene:

[ CH 2

C ]n

[Tg –90oC]

CH3 CH3 Polydimethyl siloxane:

[Si–O]n

[Tg –125oC]

CH3 Polyvinyl alcohol:

[ CH2

CH ]

[Tg 80oC]

OH

Polyvinyl chloride:

[ CH2

CH ]

[Tg 80oC]

CI

Polyvinyl methyl ether:

[ CH2

CH ]

[Tg –10oC]

OCH3

11.4 Selection of candidate PSAs: effect of structure on Tg.

Thus polyethylene is a thermoplastic not an elastomer. Similarly the introduction of a hydroxyl group or a chlorine atom inevitably introduces hydrogen bonding – poly(vinyl alcohol) – or a marked dipole moment – poly(vinyl chloride). Each of these polymers has a Tg of around 80°C and is thus clearly not a PSA candidate. Figure 11.4 does contain three strong potential candidates, however, which illustrate the subtlety of the effects involved: •

Simply ‘capping’ the hydrogen bonding of poly(vinyl alcohol) with a methyl group (e.g. poly(vinyl methyl ether) reduces the Tg from 80°C to −10°C. • Introducing two methyl groups on alternate backbone carbons of polyethylene – poly(isobutylene) – inhibits crystalline packing at the expense of some rise in Tg from −90°C to ca −60°C. • Retaining the two methyl groups of poly(isobutylene) but replacing the —C—C— backbone with the readily rotatable —Si—O—Si— backbone produces silicone rubber – poly(dimethyl siloxane) – which has a very low Tg of −125°C.

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OR

Poly alkyl methyl ethers

Polyvinyl methyl ether:

[Tg –10oC]

Polyvinyl ethyl ether:

[Tg –17oC]

Polyvinyl n-propyl ether:

[Tg –27oC]

Polyvinyl n-butyl ether:

[Tg –32oC]

11.5 Polyvinyl ethers: effect of chain length on Tg.

It is apparent from Section 11.1 that some small but controllable degree of polarity would be of help in matching the polar and dispersive components of a substrate such as skin. Figures 11.5 and 11.6 show how the balance of steric hindrance and the phenomenon of (‘internal plasticisation’) by long, flexible, non-polar substituents are employed to achieve this balance. Figure 11.5 is quite simple; it illustrates that by simply increasing the length of the hydrocarbon substituent in the poly(vinyl ether) family, the Tg is progressively reduced. Figure 11.6 shows this principle at work in the acrylate family. The consequent versatility has meant that acrylic PSAs are of major importance in a wide range of applications. The first point to note is the marked effect of the backbone methyl group in the methacrylate family compared to the acrylates. Poly(methyl methacrylate) having a Tg over 100°C is a glass-like material and not a promising starting point for PSA design. Removal of the backbone methyl has a dramatic effect on the steric hindrance of the chain – a point that is more readily appreciated from three-dimensional models than two-dimensional formulae. When this is overlaid with the internal plasticisation effect consequent upon moving to poly(butyl acrylate) the glass transition drops to ca −54°C. By use of a longer eight carbon atom substituent with a branch point well removed from the polymer backbone such as poly(2-ethylhexyl acrylate), the Tg is reduced further to ca −70°C. The great advantage of the polyacrylate family is that the use of a mixture of monomers (copolymerisation) enables the use of more polar monomers such as vinyl acetate and

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[ CH2

CH3 C ]n C

O

H C ]n C

O

O R

O R

Poly alkyl methacrylates

Poly alkyl acrylates

CH3 [ CH2

[ CH2

C ]n C O O

[ CH2

CH3

CH3

C ]n

[ CH2 C ]n

C O O

C O O

263

CH3 [ CH2

C ]n C O O

CH2 CH2 CH2 CH3 Poly(methyl methacrylate) CH2 CH3 CH2 Tg = 100–120 oC Poly(ethyl methacrylate) CH3 CH2 Tg = 65 oC [Acrylate Tg 3oC] Poly(propyl methacrylate) CH3 [Acrylate Tg –20oC] Tg = 35 oC Poly(butyl methacrylate) [Acrylate Tg –45oC] Tg = 20 oC [Acrylate Tg –54oC]

11.6 Poly acrylates and methacrylates: effect of chain length on Tg.

acrylic acid together with monomers such as butyl acrylate and 2-ethyl acrylate to manipulate the Tg. Surface energy considerations for the candidate materials that have emerged in this discussion are important. The lowest surface energy is found in the siloxane family, the silicone elastomers. Poly(dimethyl siloxane) has a critical surface tension of 22 mNm−1 with very little polar component. Polyisobutylene together with polyisoprene and polybutadiene have critical surface tensions in the region of 30–32 mNm−1. The polyacrylates, which are a more diverse family because of the range of co-monomers that can be employed in PSA manufacture, have critical surface tension values ranging from 27 to 40 mNm−1. There is therefore a wide range of PSAs available for medical applications. It is now a mature field and, because of the structural and physicochemical requirements described in this chapter, there is no immediate prospect of dramatic developments. There is one additional aspect of PSA design that must be mentioned. It will be apparent from inspection of Figs 11.3b and 11.3c that the mobility of bonds in a chain is likely to be greatest at the chain end – and that is the case. The presence of chain ends contributes to surface tack or ‘grab’. In order to increase chain end concentration two strategies are regularly

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employed. The first is the admixture of lower molecular weight polymer and higher molecular weight polymer (higher molecular weight polymer contributes more effectively to cohesive strength and the lower molecular weight increases tack because of the increased chain end concentration). The second approach involves addition of a so-called tackifying resin. This again is a lower molecular weight material with high chain end concentration. Since the tackifier will not have the same molecular structure as the base polymer, questions of compatibility are extremely important. In consequence the range of potential tackifying resins is large and the choice of material and concentration is very application-dependent, but it does provide a valuable route to final refinement of properties. Applications for PSAs in health care include wound coverings and closures, ostomy adhesives, electrocardiograph (ECG) bioelectrodes, electrosurgical grounding pads and transdermal drug delivery. There has been a considerable amount of cross-fertilisation and both materials such as hydrocolloids (originally used in ostomy and related applications) and hydrogels (used in ECG electrode assemblies) have found application as wound dressing materials. Those materials are dealt with in subsequent sections whereas here the emphasis is PSAs in contact with unbreached skin, although the application may involve wound closure. Such products include wound closure strips or tapes, which are useful as an adjunct to, or a substitute for, other wound closure materials. They are typically used for minor wounds that are small, nonexudative and under minimal tension, but also to reinforce a wound after the removal of sutures or staples. In terms of volume of use, the largest dermal application of hydrophobic PSAs is in wound protection, encompassing the wide range of bandages, tapes and dressings. Bioelectrodes, transdermal drug delivery and ostomy devices are smaller market areas but still extremely important. There are several commonly used techniques available to convert the PSA material, whether rubber-based, acrylic, poly(vinyl ether) or silicone adhesives, to a form in which it can be used. These involve application of the PSA polymer to supports, such as fabric, film, paper or foams. The more common technologies for doing this include solvent-based coating, hot-melt coating or deposition from an emulsion. The polymer preparation process dictates the application method. In solvent-based fabrication, the adhesive ingredients are polymerised in solvent, coated onto the substrate and the solvent removed to leave a coating of the adhesive. If the monomer components have been polymerised as a water-based emulsion the emulsion is directly applied to the substrate and then dried. If the polymer has been prepared in the absence of a solvent, or isolated from the polymerisation medium, hot melt application techniques can be used. Because the coating process involves elevated temperature antioxidants, together with formulation additives such as tackifying resins are incorporated. No carrier solvent

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is required but the coated rollstock needs time to cool. In all these processes the surface of the coated adhesive is covered by a release liner. Conversion to the final packaged product, for example an island dressing, typically requires several subsequent conversion steps. The types of polymer structures that are most useful for formulating medical PSAs have been discussed; critical surface tension data for these and comparator materials are summarised in Table 11.2. A brief summary presented here of the features for each class will augment that information. Rubber-based polymers were the earliest form of adhesive used, in particular natural rubber (cis-polyisoprene), polybutadiene and polyisobutylene. Their high peel strength, good cohesion and ease of acceptance by skin tissue are still relevant as is their relatively low cost. Synthetic variants such as the thermoplastic elastomers based on styrene have led to more versatility and improved performance within this group of PSAs. Thermoplastic elastomers typically have an ABA form; for example, long chains with an elastomer such as polyisoprene (I) in the centre flanked by two thermoplastic styrene (S) segments. This means that at elevated temperature this S-I-S block copolymer will flow like a thermoplastic, whereas at room temperature it takes on the characteristics of the central elastomer segment. These materials are then compounded with tackifying resins, oils and antioxidants and can be applied by hot melt processing techniques. Commercial rubberbased adhesives have extremely good initial tack together with excellent adhesion to skin and good shear strength (cohesion) and are readily formulated for specific applications. Acrylic adhesives have great versatility because of the wide range of constituent monomers that can be co-polymerised to produce tailor-made products. They are readily prepared using emulsion techniques, which obviates the need for solvent processing. Acrylic PSAs share the many advantages of acrylic polymers in general including very reasonable stability to thermal and photoxidatative degradation. This means that they provide very good long-term ageing and environmental resistance. They are in general more expensive than rubber-based varieties and have less good initial tack for a given level of cohesive strength. Because of their synthetic versatility, however, their properties can be varied over a wide range. This fact, together with their (non-yellowing) colour retention means that they constitute the majority of adhesives suitable for use on human skin. As with rubber-based PSAs, the acrylics can be formulated with components such as tackifiers. Although this improves initial tack and adhesion, it is a tradeoff that leads to a reduction in cohesion and temperature resistance. Poly(vinyl ether) based PSAs have inherently higher moisture vapour transmission rates (MVTR) than unmodified acrylics or rubber-based adhesives. This is an important property for medical PSAs since high MVTRs, the rate at which moisture permeates a dressing measured in grams per

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square metre per day, reduce skin lesions caused by moisture accumulation. Additionally, the adhesive will also have better resistance to the loss in adhesion caused by patient perspiration and adhere better to skin surfaces even if the patient sweats. The importance of MVTR has meant that other types of PSA, particularly acrylics have been formulated to enhance moisture permeation, thus reducing the wider use of the poly(vinyl ether) PSAs which have a relatively specialist range of application in moisture permeable skin patches. Silicone adhesives fill a small but important niche in the PSA market. They are more expensive than other classes of PSAs and are generally reserved for very demanding applications in which, for example, their excellent dielectric properties and resistance to temperature and chemical or biological degradation are important. Additionally, given that silicone release liners are an important part of PSA processing because of their low adhesivity, it is perhaps surprising to find that silicone adhesives have any role as dermal PSAs. Their primary role at the skin surface is in transdermal drug delivery systems, rather than wound healing applications. In this application the high cost and modest adhesion of the silicones are counterbalanced by their outstanding dissolution and diffusional characteristics when makes them extremely effective in drug uptake and release. Silicone adhesives are based on poly(dimethyl siloxane). The unique chemical composition produces a soft gel-like feel – rather like a non-aqueous hydrogel. Although tacky to the touch, they give easy trauma-free removal and do not leave adhesive residue. Because of the very low surface tension and mobility silicone adhesives conform to the rugous skin surface and maximise contact area. One common problem in dermal applications of PSAs is skin contamination. The silicones, the acrylates and the rubber-based adhesives satisfy the thermodynamic criterion for adhesion to clean, dry skin. The influence of lipid and moisture on surface energy of skin has been shown in Table 11.1. Lipid modified, wet or unclean skin invariably has a higher surface energy than dry skin with a higher polar component. These data emphasise the need to ensure that a PSA-coated device is applied to prepared (i.e. clean and dry) skin surface. Although the presence of surface contamination such as skin oils and moisture increases surface energy, that is not the major problem. Indeed using the criteria described in Section 11.1, a liquid of a given surface tension will more easily wet a higher energy surface than a lower energy surface. There are two problems, however, as previously explained; the critical surface tension is only an empirical parameter that reflects primarily the dispersive component of surface energy. In order to maximise work of adhesion the adhesive must match the individual dispersive and polar components of the surface energy of the adherend. Thus a rise in polar component

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of surface energy of a substrate, when substrate and adhesive were reasonably matched, can only increase interfacial tension and reduce the work of adhesion, or bond strength. The second problem is that the skin contaminant is a low molecular weight material not itself firmly adhered to the skin substrate. The resulting bond failure in that case will be within the substrate – rather than between the adhesive and skin substrate. Loosely attached contaminants or debris result in poor bond strength, and results in an adhesive that will have a contaminated surface with low tack after removal. One of the important research and development activities for all PSA types is the design and formulation of adhesives that will stick well to wet and/or greasy skin whilst maintaining the ability to remove the adhesive from the patient without trauma.

11.3.2 Hydrocolloid dressing materials: structure and properties Hydrocolloids are a unique family of materials in several senses. Even the name hydrocolloid belies their nature and certainly their properties. It is widely recognised that the use of the term to describe these products is not strictly correct but since it has been in place for several decades it would be inappropriate to become pedantic now. Hydrocolloid wound dressings are, in essence, composed of a compounded mixture of natural polymer particles (typically gelatin, pectin, and sodium carboxymethylcellulose) dispersed in a matrix of a hydrophobic pressure-sensitive adhesive polymer, such as polyisobutylene. They contain no water and the particulates are not colloidally dispersed. On exposure to aqueous fluid, however, the hydrophilic natural polymers absorb moisture. The most active component in this respect is carboxymethyl cellulose (CMC). As the moisture absorbtion increases, the phase structure inverts and a continuous aqueous phase is formed in which the hydrophobic polyisobutylene becomes dispersed. Even at very high levels of water absorbtion, beyond those that are desirable in clinical use, it is debatable whether a colloidal structure is formed. That point is of little importance in terms of the practical performance of the dressing. The purpose of this chapter is to examine interfacial and adhesive behaviour not to discuss clinical performance, but there are points at which the two approaches overlap. The simple composition described above was devised some thirty years ago and it has stood the test of time. Modifications have been explored in terms of the hydrophilic particles and a range of naturally occurring carbohydrate-based polymers has been examined. The functionality of CMC together with pectin and gelatine still provide,

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however, an adequate basis to describe and explain hydrocolloid behaviour. The functionality of polyisobutylene also describes adequately the requirements of the continuous phase although the alternative strategies involved in PSA design, the use of mixed polymers, thermoplastic elastomers and tackifiers, for example, have all been employed in the search for product optimisation. Most of those variations were envisaged in, or are logical extensions of the key 1985 patent (Doyle and Freeman, 1985). Excellent reviews of progress in hydrocolloid properties and performance are compiled by Thomas (e.g. Thomas, 2008). The key feature of hydrocolloids is that they are responsive. The property requirements of PSA behaviour for application to clean dry skin described in Section 11.2 are adequately met. Incorporation of a loading of some 30% of the compounded particulates increases the stiffness of the isobutylene, which means that it is beneficial to allow time for the adhesive to rise in temperature and flow effectively on contact with the skin surface. The presence of the dispersed hydrophilic polymers does give the hydrocolloid adhesive adequate absorbent capability to ensure that normal skin moisture loss does not lead to bond failure at the skin-adhesive interface. Similarly, when applied to an exuding wound, the hydrocolloid components absorb exudate and eventually formed a soft mobile gel. As this happens, the hydrophobic polyisobutylene surface in contact with the wound then converts from a dispersion of hydrophilic granules in an adhesive mass to an aqueous CMC hydrogel containing a dispersion of the adhesive. This is extremely significant in terms of the biological interaction at the wound-dressing interface. The extremely high interfacial tension between polyisobutylene and wound fluid reduces dramatically. As a result hydrocolloids have a wound interface compatibility that would not be predicted from their appearance in the dry state. Hydrocolloids can be left in place for several days and on removal there is little or no wound trauma because of the responsiveness of the hydrocolloid system to the wound environment. Consequently, hydrocolloid dressings are able to provide a moist environment and additionally to avoid the potential problems that a hydrophobic dressing surface would cause. An early study of the hydrocolloid effect on wound fluid confirmed that the potentially disadvantageous effect of the hydrophobic polyisobutylene on wound healing was not observed (Chen et al., 1992). Hydrocolloid dressings have performed well in a number of clinical studies since their introduction. Although their favourable performance might not be intuitively predictable from the initial state and composition of the dressing, it is the surface responsiveness that provides an adequate explanation. Inspection of the surface parameters of the type of binding polymer used in hydrocolloids together with those of clean dry skin (Tables 11.1 and 11.3) against the requirements of Equations 11.1–11.3 indicate a good match of surface energy parameters. The same

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Table 11.3 Summary of pressure-sensitive adhesives, wound dressings and tissue sealants: subsets and properties Hydrophobic pressure-sensitive adhesives (primarily for unbreached skin) Group

Adhesive material/polymer

Adhesive property

Rubber-based elastomers

Cis-1,4-polyisoprene (natural rubber) Polybutadiene Polyisobutylene (butyl rubber) Thermoplastic elastomer block copolymers, e.g. styrene isoprene styrene (SIS) Copolymers of alkyl acrylates with comonomers (e.g. vinyl acetate, acrylic acid)

Medium to high tack, adhesion and shear strength, readily enhanced by tackifiers

Acrylics

Specialist adhesives

Silicone gels Polyvinyl ethers

Low to medium tack, good adhesion, high shear strength Versatile formulation control Low tack and adhesion Good adhesion and peel strength

Wound dressing materials Group

Adhesive material/polymer

Adhesive property

Hydrocolloids

Hydrophobic PSAs (butyl rubber or SIS block copolymers, etc.) compounded with hydrophilic particulates Fully hydrated conventional hydrogels (e.g. cross-linked polyethylene glycol) Partially hydrated – ionic monomer – based on ecg electrode gels Polyurethane films with a thin layer of acrylic PSA or foams coated with adhesive hydrogel

Good adhesion to unbreached skin surface

Conventional sheet hydrogels Skin-adhesive hydrogels Coated films and foams

Low adhesion to unbreached skin Adhesion controlled by hydration level and monomer formulation Dictated by nature and thickness of adhesive coating

Tissue sealants and adhesives Group

Adhesive material/polymer

Adhesive property

Synthetic products

Cyanoacrylates

High adhesion and strength low flexibility Excellent sealant properties Long cure times to maximum strength Low adhesion good flexibility and resorption Properties dependent upon activation system Good bond strength in aqueous environments

Functionalised PEGs Polyurethanes Biological products

Fibrin Mammalian protein Marine proteins and synthetic/ GE analogues

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Surface-free energy (mN/m)

40

30

γd

20

10

0

10

20

30

40

50

60

Equilibrium water content (%)

11.7 Changes in polar and dispersive components of hydrogel surface energy as a function of equilibrium water content.

binding polymer, in contact with an aqueous fluid which, as indicated in Section 11.1.1, has a large polar component of surface energy, will produce a very high interfacial tension (in excess of 20 mNm−1). Because of the compounded CMC, however, the hydrocolloid in contact with the wound bed takes up wound fluid and becomes a hydrogel. As the discussion of water uptake will show (section 11.3.3 and Fig. 11.7) this will reduce the interfacial tension dramatically which is desirable (Chapters 12 and 13) for good wound bed compatibility.

11.3.3 Hydrogel dressing materials: structure and properties There is no precise and limiting definition of the term hydrogel but perhaps the most useful description is that hydrogels are water-swollen polymer networks, of either natural or synthetic origin. Of these it is the cross-linked, covalently bonded, synthetic hydrogels whose use in biomedical applications including wound dressings has grown most dramatically in recent years. The special position that hydrogels occupy in the biomedical field can be illustrated by comparing their properties with those of more established polymers (such as shown in Table 11.3) that are used in medicine, and with

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natural tissue The feature that characterises non-hydrogel polymers such as poly(ethylene), poly(isobutylene), silicone rubber and poly(vinyl chloride) – all of which have important biomaterials applications – is their relative hydrophobicity. Even the more polar materials, such as poly(methyl methacrylate) and poly(hexamethylene diamine), have polar components of surface energy that are much lower in magnitude than the dispersive, or non-polar, component of the polymer. In contrast, water has a surface energy dominated by its polar component. The behaviour of water at the surfaces of these relatively non-polar polymers is necessarily dominated by hydrophobic interactions. In contrast, the cell surface and extracellular matrix is greatly influenced by more hydrophilic groups such as oligosaccharide and glycosaminoglycan units. Soft tissue interfaces in the body therefore interact with water in a quite different way from conventional synthetic hydrophobic polymers. It is this aspect of behaviour that sets the class of polymeric materials, known collectively as hydrogels, apart from conventional synthetic polymers. These ideas date back to the pioneering work of Otto Wichterle, who was not only the ‘father’ of hydrogels but also an early advocate of the principles of biomimesis. He recognised quite clearly the importance of attempting to match mechanical properties, allow diffusion of metabolites and achieve a compatible interface with biological fluids. To achieve these ends he attempted to harness water as a component of the biomaterial and together with his co-worker Drahoslav Lim he demonstrated the usefulness of lightly crosslinked polymers of 2-hydroxyethyl methacrylate (usually referred to simply as HEMA) for biological applications (Wichterle and Lim, 1960). The single most important property of a hydrogel is undoubtedly the water content, because this, in turn, influences several other properties. The water in a hydrogel acts as: • • • •

a transport medium for dissolved species, allowing diffusion of metabolites a surface energy ‘bridge’ between hydrogel and the biological host a plasticiser, allowing hydrogel modulus to be matched to host tissue a lubricant, influencing the coefficient of friction and lubricity.

Thus, the permeability, the mechanical properties and the surface properties of the hydrogel and its resultant behaviour at biological interfaces are all a direct consequence of the amount and nature of water held in the matrix and all are relevant to wound dressing function. In the context of adhesion and interfacial behaviour quantitative information relating to surface properties is the most important requirement. The importance of water stems from its ubiquitous presence in biological systems. The uniquely hydrogen bonded structure in turn produces unique surface properties. Of the total surface energy (or surface tension) of

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72.8 mNm−1 the dispersive component is unexceptional (21.8 mNm−1) whereas the polar component makes the dominant contribution (51.0 mNm−1). For the interfacial tension to reach zero the polar and dispersive components of both surfaces of an interface must match. Similarly, if one surface has a polar component of zero the interfacial tension between water and that surface will be very significant. Hydrophobic PSAs, oils and tackifiers for example, have polar components that are very small and as a result show interfacial tensions with water of around 50 mNm−1. In order to predict the interfacial behaviour of hydrogels and to understand the responsive behaviour of hydrocolloids we need to know more precisely how water content affects their surface energy components. Figure 11.7 shows changes in polar (γp) and dispersive (γd) components of hydrogel surface energy as a function of changing water content in the gel. The hatched boxes encompass results for many variations in monomer structure. Values for the interfacial tensions between water and hydrogels can be derived from Equation 11.1 in conjunction with the information contained in Fig. 11.7. The greatest change in polar component occurs during the incorporation of the first 20% of water by weight and the interfacial tension has already become very low (around 1.5 mNm−1) at this point. Some modification is necessary because biological fluids such as wound fluid are not simply water and contain naturally occurring surface-active molecules, but the principle remains. Hydrogels do not suffer deficiencies in terms of inherent wettability. These data, and particularly the form of Fig. 11.7, have significance for hydrocolloid behaviour. As hydrocolloids begin to take up wound fluid their polar component of surface energy rises rapidly and their interfacial tension with the wound surface will fall. It is this simple observation that helps to explain the clinically observed compatibility of hydrocolloids – and of course hydrogels. This does not mean that these systems are perfectly compatible with wound fluid – or any other complex biological fluid. The polymer chains are able to rotate rapidly in response to a changed interface. In contact with water, hydrophilic groups rotate to the surface, whereas in contact with more hydrophobic structures at the interface, such as air or lipids, the hydrophilic groups ‘bury’ themselves within the gel and more hydrophobic structures are exposed. Chain rotation is a dynamic process and molecular processes such as protein deposition and denaturation are well able to respond to such processes, which is why biological interfaces present such a challenging environment for molecular design. The other surface property of hydrogels affected by water is coefficient of friction or lubricity. This can be thought of as a ‘molecular ball bearings’ effect – while there is a hydrodynamic fluid layer at the surface of a sheet hydrogel there is good lubricity, a low coefficient of friction and negligible adhesive capability. On the other hand, if the interfacial layer of water is

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removed, the sheet hydrogel has the capability to become a pressure-sensitive adhesive – provided that the other criteria for PSA behaviour are met. These two extremes of behaviour are reflected in two hydrogel applications. The first is the hydrogel contact lens – an application in which the continuous eyelid movement over the hydrogel surface requires a lubricious non-adhesive surface with a low coefficient of friction. The other extreme is the hydrogel ECG bioelectrode gel, which requires good pressure-sensitive adhesive properties and the absence of an interfacial water layer – no ‘molecular ball bearings’. There are three requirements for good skin adhesive behaviour in a hydrogel: • • •

a high residual ‘appetite’ for water viscous behaviour at low shear rates (application) and elastic behaviour at high shear rates (removal) chain rotation allowing adequate hydrophobic bonding.

Only the first is unique to hydrogels and it is achieved by preparation of the gel in a partially hydrated state and incorporation of repeat units with high hydration capability – such as anionic monomers. Since the level of residual hydration capacity is controlled at the time of fabrication it is a relatively easy matter to formulate sheet hydrogels that have wound fluid absorbtion capability and some adhesive capability to unbreached skin. In recent years commercial wound dressing products of this type have begun to appear – usually from small specialist manufacturers. The wound dressing market now contains two types of sheet hydrogel: •

•

The first is the long established non-adhesive materials that have high (>70%) water content, little or no fluid uptake capacity and fabricated from lightly (often radiation) cross-linked neutral hydrophilic polymers such as polyethylene glycol and polyvinyl pyrollidone. The second is the sheet hydrogel with low to moderate skin adhesion, medium (ca 50%) water content, good fluid uptake capability and fabricated from lightly cross-linked copolymers usually containing ionic monomers.

11.4

Surgical adhesives and tissue sealants: structure and properties

11.4.1 Introduction Unlike the mature areas of medical adhesive technology such as hydrophobic pressure-sensitive adhesives and hydrocolloids, the field of tissue adhesives is still in a state of relatively rapid development. Similarly, the chemistry

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involved is much more complex and diverse, ranging from structurally simple synthetic species to organisationally complex biological or genetically engineered peptide sequences. Tissue adhesives and sealants contrast with preformed PSAs in that the polymerisation takes place at the wound site and seeks to induce chemical bonding of some type with the tissue substrate. The polymerisation must either hold tissues together or serve as a barrier to leakage, hence the encompassing description ‘adhesives and sealants’. The field is both diverse and detailed and since the first exploratory work on cyanoacrylates in the 1960s it has expanded to encompass the distinctly different families of tissues sealant materials shown in Table 11.3. Polymerisable tissue sealants are not only common in many different surgical specialties including ophthalmic, cardiac, thoracic, vascular, general, plastic, and neurologic in addition to trauma surgery and general topical wound closure, but can also be used in a variety of experimental applications such as drug delivery and tissue engineering. The field now includes cyanoacrylates, bovine gelatin and human thrombin, native and engineered protein products, functionalised poly(ethylene glycol) polymers, polyurethanes and fibrin-based products. Fibrin sealants were the first to receive US FDA approval just over a decade ago and there are over 3000 articles published relating to fibrin sealants alone. There are several excellent reviews and compilations concerning the relevant chemistry, available products and clinical uses of tissue sealants and adhesives (Reece et al., 2001; Webster and West, 2001; Quinn, 2005; Spotnitz, 1996; Spotnitz, 2008; Spotnitz, 2010). Webster and West provide a particularly detailed account of the underlying chemistry of the competing systems. The aim of this chapter is to provide an overview of the families of tissue adhesives in the context of the broader topic of adhesives and adhesion and to identify areas of potential activity for future developments.

11.4.2 Ideality and practicality The substance must hold tissue in place to allow the healing of cut or separated areas long enough for the wound to hold without further support. The optimal adhesive should break down so that no foreign body remains. But adhesion alone is not sufficient. It must, at the very least, not hinder the progress of the natural healing process. The range of adhesives, both experimental and commercially available, differs in the way that they function at the sequential stages of healing. There are three areas of use for tissue adhesives. The first is haemostasis. Indeed the concept of tissue adhesives developed from haemostasis through improvement of focal coagulation, and one family of adhesives aims to

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parallel the mechanisms of in vivo coagulation systems. The second use of tissue adhesives is for tissue bonding and sealing. The adhesion prevents leaks of air and lymphatic fluids, for example, and this requires more than augmentation of the existing clotting system. The third and least documented area of use of tissue adhesives is local delivery of active substances including drugs, growth factors, etc. The specific clinical application dictates which sealant or adhesive will be most appropriate.

11.4.3 Cyanoacrylates Cyanoacrylate tissue adhesives are the most closely related to the chemistry involved in PSA production. Cyanoacrylate monomers are structurally related to methacrylate monomers (Fig. 11.4), the only difference is that the methyl (CH3) group directly attached to the double bond in methacrylates is replaced by a cyano, or nitrile group (CN) in the cyanoacrylates. As with the methacrylates the nature of the side chain affects the properties of the monomer and resultant polymer. The most significant and technologically valuable effect of introduction of the CN group into the monomer is that the electron withdrawing power of the group makes the monomer very susceptible to anionic polymerisation. Even the hydroxyl anion of water is able to initiate rapid polymerisation, which produces the well-known ability of ‘super glue’ to polymerise instantaneously in the presence of a trace of water – at the skin surface, for example. The nature of the alkyl side group affects the physical properties of the monomer – the methyl ester is volatile and lachrymatory – and of the resultant polymer. As with the methacrylates the methyl substituted monomer produces a rigid polymer but the flexibility of the product increases with the length of the side chain. With increasing size of the alkyl group, monomer volatility, polymerisation rate and toxicity decrease. The histotoxicity of the polymer is a rather more complicated matter, although earlier studies suggested a simple association between increasing length of the alkyl ester side chain and a decrease in histotoxicity. The rate of polymer degradation is an issue because although the polymers clear or degrade more slowly than is desirable, the degradation products are potentially toxic, depending on the degradation environment. Cyanoacrylate degradation occurs by the breakdown of the polymer backbone and the presence of water can induce cyanoacrylate hydrolysis producing formaldehyde and alkyl cyanoacetate. For this reason, although approval for topical use of cyanoacrylates for wound closure is established, there is still contention over their suitability for wider use, particularly in the US (Mattamal, 2009). As a result of the observed structure-property trends in the cyanoacrylate family, research for clinical applications has focused on the higher

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homologues. The two clinically established members of the series are the butyl and the octyl derivative. There is an extensive literature reporting clinical success at many body sites and for many procedures provided for example by Sierra and Saltz 1996; Reece et al, 2001; Chan and Boisjoly, 2004; Pursifull and Morey 2007; Yoo, 2007. The advantages claimed by clinicians include reduced inflammatory reaction and a faster healing process compared to sutures together with less scarring. Surgeons who regularly use cyanoacrylate sealants claim it is a much faster procedure when compared to the use of sutures, and a far more acceptable cosmetic effect. Although cyanoacrylate adhesives in their current form have achieved widespread use, improvement will increase their acceptability and versatility. Toxicity issues, more rapid clearance from the wound site and improvement in their degradation rate, are the most widely raised areas for improvement. Cyanoacrylate adhesion to skin involves two of the classical adhesion mechanisms outlined in Section 11.1: mechanical interlocking and covalent chemical bonding. In the first case the relatively low viscosity of the monomer allows the surface to be wetted (Fig. 11.1), so that when polymerisation occurs, the fact that undulations and imperfections of the rugous substrate have been penetrated forms the basis for excellent mechanical interlocking of the poly(cyanocrylate) and the substrate. There are many functional groups present within biological surfaces capable of bonding, particularly the nucleophilic amine groups present in protein structures.

11.4.4 Fibrin sealants Fibrin sealants are quite different in function from cyanoacrylate adhesives. It exploits a key element of the blood clotting cascade – the conversion of the soluble protein fibrinogen to fibrin monomer, which in turn converts fibrin dimers into an insoluble fibrin mesh. The key activating agent is thrombin, although factor XIII and calcium act as catalysts and the rate of fibrin mesh formation is influenced by pH, fibronectin, and temperature. Although earlier work on haemostasis had been carried out, the use of fibrinogen as an adhesive was attributed to Young and Medawar (1940). Fibrin sealants were available in the 1980s but it was not until 1998 that an FDA-approved commercial product was available (Spotnitz, 2010). There are several advantages to the use of the fibrin cascade in this way. In terms of the adhesive process, the underlying energetic criteria are met, since both polar and dispersive components are well matched. Since the polar component is very high (51.0 mNm−1) it is the driving force for surface energy mis-match between many synthetic-biological material pairings. The fibrin sealant starts as a liquid which enables maximisation of contact area through good interfacial wetting. The fibrin clot caused by the sealant ultimately degrades in the physiological environment which leads to optimal

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tissue reformation (Lontz et al., 1996). The compositions of the commercial products have underlying similarities. They are essentially two-pack systems introduced to the dry wound site with a dual syringe. The components of the reacting system are typically fibrinogen, thrombin, factor XIII and a calcium salt (normally chloride). The attractions of fibrin sealant – it consists of biologic components of the natural clotting system – have also given rise to concerns. The adhesive is manufactured from blood, and this raises the possibility of viral bloodborne-disease transmission. Developments involving a variety of viral sterilisation techniques have led to the successful use of large pools of human plasma as the fibrinogen source. Thrombin isolation has also been the subject of extensive development. Bovine sources were initially used, although the possibility of an IgE-mediated allergic reaction, as well as risk of transmission of bovine spongiform encephalitis, hepatitis A, B, and C and HIV, is widely accepted. The safety of source materials has been an important part of the fibrin success story, together with detailed aspects of formulation development involving, for example, fibrinolytic inhibitors to stabilise the fibrin clot.

11.4.5 Protein-based adhesives The adhesive principles of a low viscosity fluid that can effectively wet the skin substrate and has the ability to match the surface energy parameters of the wound interface with a naturally occurring precursor are satisfied, as with fibrin systems, by protein-based adhesives. The absence of the hydrophobic carbon backbone that produces a dominant dispersive surface energy component is the key in so many synthetic systems. The effectiveness of this strategy is reflected in the large and growing number of proteinbased systems. These range from well-known proteins such as gelatin, collagen and albumin to less familiar proteins with properties that appear to be uniquely relevant to tissue repair, such as mussel adhesive protein. More recently, genetically engineered proteins have emerged in which key amino acid sequences have been replicated. This is an area of great activity and promise but the discussion here is necessarily limited to the general theme of adhesion phenomena.

11.4.6 Collagen, gelatin and albumin-based adhesives Gelatin is a protein obtained by a controlled hydrolysis and extraction from collagen, which itself is a fibrous insoluble protein widely found in nature, being the major constituent of skin, bones and connective tissue. Gelatin is particularly rich in glycine, proline and hydroxyproline which occur in the gelatin structure as repeating sequences of glycine-X-Y triplets, where X

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and Y are frequently proline and hydroxyproline. These sequences are responsible for the triple helical structure of gelatin which drives its ability to form aqueous gels. The molecular weight of gelatin is quite large, ranging from a few thousand up to several hundred thousand daltons. It is by control of the molecular weight and molecular weight distribution of gelatin that the physical properties of gelatin and particularly viscosity and gel strength are controlled. So-called gelatin-resorcinol-formaldehyde (GRF) adhesive was developed in the 1960s and is formed from gelatin, resorcinol, and water in the presence of formaldehyde, glutaraldehyde, and gentle heat. This resultant reaction produces a polymer that is an effective adhesive. The most effective clinical applications of this agent have been reported to be in cardiovascular surgery. Although functional and cost effective, this product is not widely accepted, in part because of toxicity concerns. In order to address this a commercially available modification – GR-DIAL glue – has been developed by removing the formaldehyde component and replacing it with less toxic aldehydes, pentane-l,5-dial and ethanedial. An alternative approach involving gelatin is composed of thrombin and a gelatin matrix manufactured by extracting collagen from bovine corneal tissue. The collagen undergoes gelatinisation, glutaraldehyde cross-linking and is powdered. The thrombin component, a sterile freeze-dried powder, is reconstituted in saline and mixed with the gelatin matrix just before use. The components work together promoting clot formation whilst the gel particles swell, reducing bleeding and providing a matrix on which the clot can form. Uniquely, this FDA-approved product requires the presence of blood for activation. Because collagen is the starting point for gelatin extraction these are sometimes referred to as collagen-based adhesives. More recently, a versatile light-activated gelatin polymerisation has been used to produce very elastic and adhesive cross-linked gelatins that are resistant to swelling in the presence of aqueous fluids, retaining elasticity and high adhesive strength. The adhesive polymerises rapidly at the wound site on photoactivation and when tested in sheep lung in vivo, the adhesive effectively sealed a wound in lung tissue from blood and air leakage, was not cytotoxic and did not produce an inflammatory response. Although still in the early stages of development, the approach offers several unique features and behavioural aspects (Elvin et al., 2010). A related group of adhesives are based on the combination of albumin and adhesion compounds. They are related to, and sometimes referred to, as gelatin-resorcinol-formaldehyde-glutaraldehyde (GRFG) glues. Whereas limited FDA approval has been obtained for use of a combination of bovine albumin and glutaraldehyde, formaldehyde based glues are regarded as presenting greater problems. Bovine serum albumin-gluataraldehyde adhesive was found to perform comparably with both fibrin and gelatin

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matrix-thrombin sealants as a homeostatic agent in urological applications (Pursifull and Morey, 2007).

11.4.7 Specialised protein structures: mussel adhesives and genetic engineering This is a very interesting and active research field. The difference, in terms of adhesion principles, between common mussel adhesive protein and the mammalian proteins considered above, is that this protein appears to form a strong adhesive bond in the presence of water. Although cyanoacrylate polymerisation is initiated by water, the resultant bond strength is poor in the presence of interfacial water. There has naturally been tremendous curiosity about the specific structural features that promote this adhesion – so evident in the ability of the mollusk to adhere to rocks or other substrates in turbulent waters. The natural protein has been well characterised but that does not necessarily unlock the key to replicating its adhesive characteristics away from the natural environment. The 120 kDa molecular weight mussel structure is rich in lysine, proline, hydroxyproline, threonine, tyrosine and 3,4-dihydroxyphenylalanine. It contains a repeated decapeptide sequence in addition to essential cross-linking sites provided by the lysine, tyrosine and 3,4-dihydroxyphenylalanine or DOPA residues. The repeated decapeptide sequence is not typical of mammalian connective tissue, having essentially a complete absence of glycine, together with an unusually frequent appearance of proline or hydroxyproline residues. The fact that other mollusks have quite different sequences in which glycine is abundant indicates that there is not one unique biological solution to the problem of underwater adhesion. Although extraction of the native protein has been very effectively carried out and useful products made, the low yield mitigates against commercial use of natural sources. There have been significant and successful research efforts using established synthetic routes to polypeptides. More recently genetically engineered products have been obtained and successfully used in adhesive products (Wang et al., 2010). This is clearly an area that will expand and in principle should form an effective base for purposedesign of tissue sealants and adhesives for the environmental demands of specific body sites.

11.4.8 Synthetic adhesives Polyethylene glycols (PEGs) form the basis for a series of adhesive materials. They are water-soluble polymers with low toxicity with hydroxyl groups at each end of the chain. These terminal groups can be converted to more

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reactive groups that enable the PEG chains to undergo polymerisation and network formation or, in principle to co-polymerise with other reactive species. The fact that PEGs are well tolerated in the body makes them ideal for in situ polymerisation at the required site. By incorporating hydrolysable segments of, for example lactic acid, into the PEG backbone and capping with reactive acrylate groups, a photopolymerisable macromer can be prepared. The presence of the polylactic acid segments will enable the resultant polymer to degrade into soluble metabolisable low molecular weight end products. Photopolymerisation at the site of use was the route initially favoured but this is not a necessary requirement of this type of PEG macromer, and products employing other activation mechanisms have been developed and approved (Reece et al., 2001; Glickman et al., 2002; Barnard and Millner, 2009). Polyurethane adhesives are also prepared as pre-polymers with active groups. There are many synthetic variants on the polyurethane backbone but the use of oligomeric diols in conjunction with di-isocyanates to form chains with repeating urethane links is commonplace. If both ends of this prepolymer are capped with isocyanate groups, the material will rapidly undergo gel formation in contact with water or moist tissue. Such a prepolymer may be suitable to act as a tissue adhesive, providing that the gel itself and the products of biodegradation are nontoxic. Polyurethane prepolymers of this sort have been used experimentally for tissue bonding for many years and did result in products that came to market. The limiting factors in their success have been slow and somewhat unreliable rates of polymerisation of the isocyanate, on one hand (because of variations in the local environment), and reported adverse tissue response, on the other. More work will be required in order to provide a sound basis for the development of safe and efficacious products (Kobayashi et al., 1991; Webster and West, 2001).

11.5

Conclusions

Adhesion and adhesive behaviour are important topics in the area of wound healing. They represent, however, only one aspect of interfacial behaviour, the other important aspect is the biochemical and biological response of the wound with the biomaterial in the form of the dressing. These two aspects of interfacial phenomena are both influenced by the molecular interactions occurring at the biomaterials interface. This chapter has focused on the two major categories of adhesive materials encountered in wound healing and the particular requirements that drive materials selection and development. The function of pressure-sensitive adhesives is to form some sort of adhesive bond between tissue and a biomaterial under the influence of

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pressure – which is necessary to ensure an adequate degree of flow to ensure good ‘wetting’ of the substrate. The pressure-sensitive adhesive bond may be very weak, as is the case if a layer of compliant polyurethane film is applied to moist skin, or much stronger, as in the case of a hydrogel-based bioelectrode, or a hydrocolloid-based ostomy adhesive. The role of tissue adhesives and sealants is rather different. They must function under the adverse moist conditions of the surgical and traumatic wound. They are liquids which on solidification, gelation or polymerisation must form a good interface with the tissue with which they are brought into contact. Their role is then either to produce a seal, acting as a permanent barrier to fluid leakage or to form a bond between adjacent tissue surfaces. They are not physically removed so must be resorbable. In both cases good molecular contact is required and, when that occurs, either by liquid flow or pressure-induced flow of a viscoelastic solid, interfacial forces dominate the interaction between the two phases. An important aspect of this chapter is the basis that it forms to extend understanding of the interfacial phenomena that govern and modulate adhesive behaviour into another arena – the longer-term interaction between the wound healing biomaterial and the wound fluid. This chapter forms a basis for the understanding of the interfacial driving forces for the biological and biochemical changes that always arise when a biomaterial is introduced to a host biological environment. In that situation the wound fluid can readily flow over the dressing biomaterial surface allowing wetting and intimate molecular contact. As a consequence, we can see the commonality of importance of intermolecular forces that influence adhesion, on one hand, and biointeractions on the other. This topic is extended in subsequent Chapters (12 and 13). The development of understanding in this area is being increasingly recognised as an important aspect of the development of new and more biologically responsive or active wound healing biomaterials.

11.6

References

Barnard J and Millner R (2009), ‘A review of topical hemostatic agents for use in cardiac surgery’, Annals Thoracic Surg, 88, 1377–1383. Chan S M and Boisjoly H (2004), ‘Advances in the use of adhesives in ophthalmology’, Curr Opin Ophthalmol, 15, 305–310. Chen W Y J, Rogers A A and Lydon M J (1992), ‘Characterization of biologic properties of wound fluid collected during early stages of wound healing’, J Invest Dermatol, 99, 559–564. Chickering D E and Mathiowitz E (1999), ‘Definitions, mechanisms and theories of bioadhesion’, in Mathiowitz E, Chickering III D E and Lehr C-M, (eds), Bioadhesive Drug Delivery Systems Fundamentals, Novel Approaches and Development, Marcel Dekker, Inc, New York, pp. 1–10.

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Chivers R A (2001), ‘Easy removal of pressure sensitive adhesives for skin applications’, Int J Adhes Adhes, 21, 381–388. Dahlquist C A (1969), in R L Patrick, (ed), Treatise on Adhesion and Adhesives, 2, Marcel Dekker New York, pp. 219–260. Doyle A and Freeman F M (1985), ‘Adhesive composition resistant to biological fluids’, US Patent 4551490, 06/567012. Elvin C M, Vuocolo T, Brownlee A G, Sando L, Mickey G, Huson M G, Liyou N E, Stockwell P R, Lyons R E, Kim M, Edwards G A, Johnson G, McFarland G A, Ramshaw J A M and Werkmeister J A (2010), ‘A highly elastic tissue sealant based on photopolymerised gelatin’, Biomaterials, 31 8323–8331. Glickman M, Gheissari A, Money S, Martin J and Ballard J L (2002), ‘A polymeric sealant inhibits anastomotic suture hole bleeding more rapidly than gelfoam/ thrombin’, Arch Surg, 137, 326–331. Kenney J F, Haddock T H, Sun R L and Parreira H C (1992), ‘Medical-grade acrylic adhesives for skin contact’, J Appl Poly Sci, 45, 355–361. Kobayashi H, Hyon S-H and Ikada Y (1991), ‘Water-curable and biodegradable prepolymers’, J Biomed Mater Res, 25, 1481–1494. Lontz J F, Verderamo J M, Camac J, Arikan I, Ankan D and Lemole G M (1996), ‘Assessment of restored tissue elasticity in prolonged in vivo animal tissue healing: comparing fibrin sealant to suturing’, in Sierra D and Saltz R, (eds), Surgical Adhesives and Sealants, Technomic, Lancaster, Pa US pp. 79–90. Mattamal G J (2009), ‘U.S. FDA perspective on the regulations of cyanoacrylate polymer tissue adhesives in clinical applications’, Mat Sci Forum, 638–642, 624–628. Mavon A, Zahouani H, Redoules D, Agache P, Gall Y and Humbert Ph (1997), ‘Sebum and stratum corneum lipids increase human skin surface free energy as determined from contact angle measurements: A study on two anatomical sites’, Colloids and Surfaces B: Biointerfaces, 8, 147–155. Ovington L (2007), ‘Advances in wound dressings’ Clinics in Dermatology, 25, 33– 38. Ovington L, Pierce B (2001), ‘Wound dressings: form, function, feasibility, and facts’, in: Krasner D L, Rodeheaver G T, Sibbald R G (eds). Chronic Wound Care: A Clinical Source Book for Healthcare Professionals, Wayne, PA. HMP Communications, pp 311–319. Park K, Cooper S L and Robinson J R (1986), ‘Bioadhesive hydrogels’, in Peppas N A, (ed), Hydrogels in Medicine and Pharmacy, Vol. 3, Properties and Applications, CRC Press, Boca Ranton, Florida, pp. 151–175. Pursifull N F and Morey A F (2007), ‘Tissue glues and non-suturing techniques’, Curr Opin Urol, 17, 396–401. Quinn J V (2005), ‘Overview of tissue adhesives’, in Quinn J V (ed), Tissue Adhesives in Clinical Medicine (2nd ed.), BC Decker Inc, Hamilton, pp. 1–13. Reece T B, Maxey T S and Kron I L (2001), ‘A prospectus on tissue adhesives’, Am J Surg, 182, 40S–44S. Renvoise J, Burlot D, Marin G and Derail C, (2007), ‘Peeling of PSAs on viscoelastic substrates: A failure criterion’, J Adhes, 83, 403–416. Renvoise J, Burlot D, Marin G and Derail C (2009), ‘Adherence performances of pressure sensitive adhesives on a model viscoelastic synthetic film: A tool for the understanding of adhesion on the human skin’, Int J Pharmaceutics, 368, 83–88.

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Shafrin E G and Zisman W A (1967), ‘Critical surface tension for spreading on a liquid substrate’, J Phys Chem, 71, 1309–1316. Sierra D and Saltz R (1996), ‘Surgical Adhesives and Sealants, Current Technology and Applications’, Technomic, Lancaster, Pa US. Spotnitz W D (1996), ‘History of Tissue Adhesives’, in Sierra D and Saltz R, (eds), Surgical Adhesives and Sealants, Technomic, Lancaster, Pa US pp. 3–11. Spotnitz W D (2008), ‘Tissue adhesives: Science, products and clinical use, musculoskeletal tissue regeneration’, Ortho Biol Med, 4, 531–546. Spotnitz W D (2010), ‘Fibrin sealant: Past, present, and future: A Brief Review’, World J Surg, 34, 632–634. Thomas S (2008), ‘Hydrocolloid dressings in the management of acute wounds: a review of the literature’, Int Wound J, 5, 602–613. Venkatraman S and Gale R (1998), ‘Skin adhesives and skin adhesion I. Transdermal drug delivery systems’, Biomaterials, 19, 1119–1136. Wang M, Mattson M S, Tirrell D A and Kornfield J A (2010), ‘Tissue adhesive using engineered proteins’, US Patent Application 20100261652, 12/749507. Webster I (1997), ‘Recent developments in pressure-sensitive adhesives for medical applications’, Int J Adhes Adhes, 17, 69–73. Webster I and West P J (2001), ‘Adhesives for medical applications’, in Dumitriu S, (ed), Polymeric Biomaterials (2nd Edn) CRC Press Boca Raton, pp. 703–737. Wichterle O and Lim D (1960), ‘Hydrophilic gels for biological use’, Nature, 185, 117–118. Yoo J (2007), ‘Clinical application of tissue adhesives in soft tissue surgery of the head and neck’, Curr Opin Otolaryngology, 16, 312–317. Young J Z and Medwar P B (1940), ‘Fibrin suture of peripheral nerves’, Lancet, 275, 126–132.

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12 Wound healing studies and interfacial phenomena: use and relevance of the corneal model A. M A N N and B. J. T I G H E, Aston University, UK

Abstract: The interaction of the wound dressing as a biomaterial with the wound bed is the central issue of this chapter. The interfacial phenomenon that encompasses the biological and biochemical consequences that arise when a biomaterial is introduced to a host biological environment is discussed. A great deal can be learned from observations arising from the behaviour of biomaterials at other body sites; one particularly relevant body site in the context of wound healing is the anterior eye. The cornea, tear film and posterior surface of the contact lens provide an informative model of the parallel interface that exists between the chronic wound bed, wound fluid and the dressing biomaterial. Key words: biomarkers, contact lens, tear film, wound fluid, ocular surfaces.

12.1

Wound dressing biomaterials: interfacial aspects of compatibility and wound response

The chronic wound can be characterised as an environment with excessive proteolytic activity and low protease inhibition capability, resulting in the degradation of many potentially pro-healing protein components, including growth factors and adhesion molecules. This non-healing wound is associated with an imbalance in matrix metalloproteinase (MMP) activity, which is thought to degrade newly formed extracellular matrix and interfere with the pro-healing activities controlled by growth factors and cytokines. A major factor in understanding the host response in wound healing is the presence of a dressing and the consequent interactions that will occur, particularly at the interface. Ideally, wound bed management should not impede on the normal pathways of wound healing and the biomaterial dressing should further the progression of the healing wound. Indeed, wound bed management must work hand in hand with the actual dressing choice and design, for example efficiently managing levels of oxygenation and bacterial load. The presence of a dressing will alter the biochemical wound bed dynamics to varying extents, dependent on the design and 284 © Woodhead Publishing Limited, 2011

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composition of the material. This interaction may be detrimental or beneficial but will ultimately induce some change in the wound site dynamic. The first step towards achieving a compatible wound dressing biomaterial lies in understanding the components and pathways required for complete healing. Only when the distinguishing mechanisms of the healing wound and the non-healing wound are fully understood will the functional requirements of wound dressings at a molecular level be clear and thus the design requirements become apparent.

12.1.1 Biomaterial aspects In terms of the dressing response, the principles that underpin adhesion also drive biological and biochemical interaction in the wound bed environment and are key to the understanding of interfacial interaction between the dressing and the host environment. The participation of the many and varied components in wound healing, and more specifically the proteinaceous content, in the host defence mechanism is of particular interest due to their specific involvement in, and regulation of, the immune and healing response. When a protein solution contacts another phase (a solid, liquid, or a gas) with which it is ‘immiscible’, protein molecules tend to accumulate at the interface between the two phases. On the other hand, protein adsorbed onto the surface and into the matrix of the contacting biomaterial will initially be denuded from the interfacial microenvironment. This tendency has a great effect on various natural and technological processes not least because adsorption of proteins takes place almost instantaneously when a solid surface comes into contact with most biological fluids (Wahlgren and Arnebrant, 1991). Importantly, the initially formed protein film is subject to subsequent change (Vroman, 1962) and can then act, for example, as a substratum for subsequent adhesion of other components such as eukaryotic cells or microorganisms. Protein adsorption has an impact on many processes and is itself driven by various types of interaction between the different components present in the system. These include the sorbent surface, protein structure and the nature and composition of the solution and its component electrolytes and organic solutes. The surface of proteins is complex, having a precise distribution of hydrophobic, hydrophilic and charged domains. The fact that synthetic surfaces are molecularly heterogeneous, taken together with the complex nature of the protein surface, makes it difficult to predict how different proteins will interact with specific surfaces. One major factor influencing protein adsorption is the surface energy of the contacting surface. Within a specific group of materials it has been shown that hydrophobic surfaces adsorb more protein than hydrophilic ones (Norde, 1986) but this does not mean that the correlation between hydrophobicity and protein

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deposition is a universally applicable paradigm. Neither is prediction of the effect of surface charge on protein adsorption straightforward. Proteins with a similar net charge to the surface may still bind through local domains of opposite charges. It has been suggested that the driving force may be an increase in entropy, due to conformational changes of the protein resulting in the loss of secondary structure (Norde, 1986). This study also points out that exposed groups at the surface of a protein are most likely to interact with a solid surface, although interior groups might be exposed through conformational changes. In the case of proteins with strong internal coherence – ‘hard’ proteins – structural rearrangements do not significantly contribute to the adsorption process. The ‘soft’ proteins, which have lower structural stability, will adsorb, even under unfavourable conditions, due to the consequent structural rearrangements. Binding of ions and small molecules such as fatty acids ions to specific sites in proteins can change the adsorption behaviour of proteins (Pitt and Cooper, 1988). Extending these concepts to the complexity of the healing process and the influence of the dressing biomaterial, we know that for complete repair to occur, a balance between protein degradation pathways and cellular and protein synthesis pathways must be obtained. Ideally, the presence of the dressing should not disturb or accentuate the normal immunoregulatory balance. Conversely, with the non-healing wound it should ideally encourage or provide a means for the wound site to switch from the cycle of a non-healing dynamic to a pro-healing environment. In this respect it is important to recognise the key individual multifunctional species required for normal wound healing and to maintain or allow their bioavailability at the wound site. There is a great need in this field for studies that address the specific influence of the dressing biomaterial on the wound bed milieu. One excellent review (Palfreyman et al., 2007) assesses available information on individual wound dressing performance from published studies. He examined the influence of the nature of the dressing against one wound type (venous leg ulcers) by underytaking a meta-analysis of the reported wound response to an array of dressings. This analysis of published data from 254 studies found that none of the dressing comparisons showed conclusive statistically valid evidence that one dressing type allowed a greater healing prospect than another. Importantly the review points out that most of the studies assessed had a small sample size (mean = 76). This can limit clinical information comparisons because of the many other variables; not least differences in wound bed management prior to the study. It may, however, reflect the fact that the presentation of hydrophobic domains at the wound interface is a characteristic of the carbon-backbone polymers that dominate current wound dressing materials, a point highlighted in both Chapters 11 and 13.

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12.1.2 Biological aspects Under the normal healing response a wide number of factors control the proteolytic activity in the wound. An in-depth description of the biological or biochemical aspects of wound healing is beyond the scope of this section, which is to simply highlight the interactive mechanisms involved between the dressing and the wound bed, together with the potential limiting outcomes. Thus, when investigating any biochemical response in the healing pathway, the specific effect that a dressing biomaterial may have on the underlying wound bed site must be considered carefully. For example, the family of zinc-dependent endopeptidases, the matrix metalloproteinases (MMPs), have the ability to digest most extracellular components and are integral to the wound repair process. They are regulated by a series of enzyme inhibitors called tissue inhibitors of matrix metalloproteinases (TIMPs), and non-specifically by α2-macroglobulin. In addition stringent regulation of repair processes occurs at transcription, post-transcriptional modulation and protein secretion levels (Opdenakker and Van Damme, 1994). It is widely accepted that in the non-healing wound the balance is tipped in favour of the MMPs (Bullen et al., 1995; Trengove et al., 1999). Consequently TIMPs are fundamental to the regulation of MMP related repair particularly in cell migration and wound contraction (Mirastschijski et al, 2004). A limited number of current wound-healing products claim to provide growth factor therapies at the wound. Regranex Gel (Systagenix), for example, when placed under moistened gauze dressing, aims specifically to deliver platelet derived growth factor. Whilst growth factors are known to complement a healing wound, the gel has not proved to be as successful as hoped. This is thought to be due to the high levels of MMPs and other proteases in the chronic wound (Falanga, 1992; Cullen et al., 2002a). Growth factor therapies, in general, have so far been proven to be disappointing even when used at high doses. Consequently the move has been towards the incorporation or addition of protease inhibitors to the wound bed. Many synthetic MMP inhibitors have been identified such as tetracyclines, tetracycline derivatives, and bisphosphonates (Yager and Nwomeh, 1999; Valleala et al., 2003). Collagen and collagen/oxidised regenerated cellulose dressings have been used as a competitive substrate for MMPs. Promogran (Systagenix) a freeze-dried sponge prepared from bovine collagen and oxidised regenerated cellulose (ORC) was observed, in a small study with nine patients, to inactivate key proteases elevated in chronic wound fluid of diabetic foot ulcers (Cullen et al., 2002b). The proposition was that Promogran could bind and inactivate any existing excess proteases in addition to binding and protect growth factors, allowing them to be re-delivered in their active form once the product was re-absorbed. More recently

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Promogran was shown to be effective in granulation tissue formation, especially in wounds which were difficult to heal (Tausche and Sebastian, 2005). Suprasorb C (Lohmann & Rauscher GmbH & Co. KG, Rengsdorf, Germany) a bovine collagen type I sponge consisting exclusively of collagen is another example of a wound dressing using this technology. It should be stated that while the aim of these and other dressings in the chronic wound is to limit MMPs and improve healing, there is a great deal of evidence highlighting the need for both MMPs and TIMPs in the wound healing process. Therefore the availability, presence and importantly the optimum levels of these and other regulatory proteins is paramount and the presence of a dressing should not impinge on their normal function and interactive processes. Given the early expectation that these ‘bioactive’ products would exert a marked effect on the wound healing pathway, the fact that these approaches have not had a more dominant role in the development of wound healing therapies is significant and emphasises the complex role of the wound dressing biomaterial at the wound interface. Specific individual protein component regulators are also fundamental to the pro-healing wound process. One such example is the ubiquitous glycoprotein, vitronectin, which plays a role in most stages of wound repair. Vitronectin is a single-chain glycoprotein present normally in the extracellular matrix and also present in platelet α-granules and circulating in plasma. It is known to bind a wide range of ligands, through which it participates in a variety of regulatory processes. Initially after injury vitronectin can interact with platelets where it may contribute to physiological, and conversely, pathophysiological events associated with thrombosis and haemostasis (Thiagarajan and Kelly, 1988; Asch and Podack, 1990). During inflammation it serves to limit and control fibrinolysis through its interaction with, and stabilisation of, plasmin activator inhibitor-1 (PAI-1) (Declerck et al., 1988). It also functions in cell migration and attachment via its RGD sequence (Pytela et al., 1985) and it can limit bystander cell proteolysis through the inhibition of the membrane attack complex of complement (Preissner and Seiffert, 1998). Additionally, it can act as a pain suppressor though βendorphin binding (Hildebrand et al., 1989). Not surprisingly then, it has been shown that its absence from the provisional wound matrix leads to microvascular haemorrhage which thus delays the healing process (Jang et al., 2000). The denuding effects that a dressing may have on the wound bed interface can be highlighted by the interaction of this glycoprotein with synthetic surfaces. Vitronectin, a sticky adhesion protein known for its avidity for glass, plastic and a host of synthetic surfaces, is very likely to interact and bind strongly with the overlying dressing. It has an important pro-active role in modulating the conversion of plasminogen to plasmin. The plasminogenplasmin activation process is kept in equilibrium via the antagonistic activities

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of the plasminogen activator system and plasminogen activator inhibitor (PAI) system. The most significant plasminogen activator inhibitor in cellular systems is PAI-1 which binds to vitronectin, thus localising PAI-1 to the pericellular compartment where active plasminogen activator is generated (Vaheri et al., 1992). If vitronectin is localised on a (synthetic) surface adjacent to the cellular site (such as a chronic wound), it removes plasminogen activator inhibitor from the reaction by fixing it, creating an imbalance in favour of the plasminogen activator and production of active plasmin. This results in a local upregulation of plasmin formation, thereby controlling an important regulatory mechanism in wound repair: an increase in degradative proteolysis which may then result in a state of non-healing and excessive inflammation. The creation of a local milieu of vitronectin on polymeric surfaces has been demonstrated on a variety of contact lens materials, where the micro-climate is influenced by lens material and in particular its anionic nature (Tighe et al., 2001). The unique conformational flexibility and multidomain structure of vitronectin, which allows it to bind to a large repertoire of ligands, makes its potential interactions with biomaterials all the more intriguing. The versatile binding capabilities and receptor functions of vitronectin could, in the future, be used in the characterisation of structurefunction properties and importantly in the design of new biomaterials. This is just one example of the way that an individual component in the complex wound healing environment can greatly affect the normal wound repair process. Deviation from optimum levels of any of a host of biomolecules, resulting in an increase or decrease in levels of certain key components, can adversely affect the healing pathway resulting in a non-healing wound. The factors affecting these changes remain poorly understood but one factor contributing to a change in the wound fluid dynamics is that of the contacting wound dressing. Ideally the dressing would denude the wound site of anti-wound healing factors like the recognised detrimental proteases while allowing the critical pro-healing factors (e.g. growth factors) to move freely in the wound site. In addition to facilitating the bioavailability of pro-healing factors, the added capacity for the dressing to deliver other additional factors to trigger or enhance the pro-healing response of the host would be desirable. However, even if a dressing could be engineered to denude and add specific components to the wound site at will, we are, as yet, unsure of all the elements required to achieve a balanced wound response. Until we fully understand the ideal wound fluid composition the goal of the optimal dressing design remains elusive.

12.1.3 Functional aspects of wound dressing design In considering potential factors that may lead to the improvement of the wound dressing as a biomaterial the established functional requirements

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for optimal wound dressing performance provide an important baseline. The importance of taking into consideration the nature and phase of the wound and the progression of the wound response when introducing a dressing to the wound site are clear. Features relating to functional aspects of the wound dressing are of equal importance in terms of interfacial compatibility and healing response. A brief summary of each of these aspects as currently understood is provided below with a few focal examples of their potential influence on the host response. In all of these functional requirements of dressings, the status of the wound bed and the phase of healing are of fundamental importance. Wound fluid management Originally dressings were employed to cover the wound from the external environment and bacterial contamination but modern-day dressings are also used to maintain a moist wound setting. It is generally accepted that wound healing requires, and is enhanced by, a moist environment, and effective epithelial cell movement across the wound edge to cover and close the wound site depends upon this. The basis of a good dressing ultimately lies in good moisture balance, since too much moisture slows epithelial cell migration and can cause wound margin maceration, whereas too little moisture can result in dehydrated, dead tissue (a scab) which is a barrier to cell movement and wound healing. The moist environment has been shown to advantageously decrease dehydration, necrotic skin formation and inflammation whilst additionally providing an exudate rich environment complete with epithelialisation promoting growth factors. Debridement of the wound is also more effective when performed on a moist occluded site. Not unexpectedly, however, the moist wound environment is also associated with an increase in bacterial count. The observed increase in bacterial proliferation does not, however, manifest itself as a prerequisite for an increase in observed infection or distress (Mertz et al., 1985). Conversely, clinical studies have shown that wounds maintained in a moist environment have lower infection rates and heal more quickly (Telfer and Moy, 1993). The exudate absorbers make up a large proportion of dressings on the market and their role is important in the overall management of the wound. Temperature The loss of moisture through evaporation is also known to result in a temperature decrease and a cooling effect. The use of a dressing, and particularly a cooling absorbing hydrogel, on the wound can greatly affect the temperature of the wound site. A cooling wound bed can result in vasoconstriction thereby restricting the influx of mediators into the wound site.

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Reduced leucocyte mobility, hypoxia and phagocytic function are all cited as problems arising from a decreased wound temperature resulting in wound healing deterioration. However a decrease in temperature is obviously of significant importance in burn wounds where beneficial effects of cooling have been demonstrated (Venter et al., 2007). Biochemically, the effect of changing temperature on protein adsorption is not always predictable given that both increases and decreases in their adsorption with increasing temperature have been reported (Dillman and Miller, 1973; Norde and Lyklema, 1978). There are numerous conflicting reports on the detrimental effects of temperature fluctuations on wounds, and the choice and nature of the dressing will influence this parameter. Cellular interaction The surface chemistry of the biomaterial will influence the ability and potential of a cell to attach and move on the biomaterial. The movement and interaction of the cells will also be dependent on the presence of serumderived proteins at the surface and the nature of the receptors presented to the cell. It is widely recognised that cellular interaction with an artificial surface can affect the cell response. One study of interest (Sundaram et al., 1996) investigated the interaction of neutrophils on polyurethanes with different ionic groups. Sulphonate groups (anionic) and quarternary amine groups (cationic) were compared to the base polyurethane (which is commonly used for a variety of blood-contacting biomaterials). The results demonstrated that not only were the numbers of adherent neutrophils on the sulphonated polyurethane higher compared to the base polyurethane, but these cells exhibited greater surface spreading – which would suggest a ‘happier’ cell. In parallel, the workers assessed integrin expression and in particular Mac-1 expression. Mac-1 is involved in many of the key cellular functions including adhesion, spreading, chemotaxis and phagocytosis. The results demonstrated a significantly higher expression of Mac-1 by the adherent cells on the sulphonated polyurethane compared to the other surfaces. This is particularly significant in the context of studies that have shown that patients with leucocyte adhesion deficiency (which involves deficient expression of three related leukocyte adhesion glycoproteins including Mac-1) are more susceptible to bacterial infection (Kuijpers et al., 1997; Sergio et al., 2004). It is reasonable to assume a material that enhances or maintains integrin expression is extremely advantageous and will allow natural neutrophil function to be maintained against the constraints of bacterial phagocytosis and normal cellular migration. Another study investigated the influence and ability of four different wound dressings to bind polymorphonuclear elastase from enzyme solutions and in chronic wound fluid. It was demonstrated that the in vitro binding capacities and

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the ability to inactivate polymorphonuclear elastase of the investigated dressings varied considerably from dressing to dressing (Schönfelder et al., 2005). Control of infection Control of infection and microbial contamination is of paramount importance in chronic wounds where the presence of a large bacterial (and sometimes fungal) bioburden is commonplace. The nature and diversity of the microorganism colonisation is important but it has been suggested that a figure of 106 organisms per gram of tissue, or greater, can be correlated with impaired healing (Falanga, 2004) irrespective of the specific colonies present. It is accepted that the reduction and control of the numbers of microorganisms present at the wound interface is beneficial. Antibiotics delivered locally or systemically or via dressings containing topical antimicrobial are commonly used to control the infection of wounds. More recently, the presence of bacteria in their own protective biofilms, with a greater capacity to evade antimicrobials, has been specifically acknowledged in chronic wounds (rare in acute wounds) (James et al., 2008). The recognition of biofilm formation within 10 hours of wounding has highlighted the importance of improved wound dressings early in wound care that can offer controlled release of active agents and antimicrobial compounds such as iodine or most frequently silver ions. However, in the case of silver-impregnated dressings, the benefit of these dressings with regard to the wound healing process is still under debate as evidenced by a recent meta-analysis review (Carter et al., 2009). Cost, in addition to possible side effects to the patient, have limited their potential. Equally a recent review of iodine suggested that while it is an effective antiseptic agent that does not adversely affect the wound-healing process, and is comparable with other antiseptic agents in this respect, its actual promotion of the wound healing process has not yet been adequately demonstrated (Vermeulen et al., 2010). Another antimicrobial agent of growing potential in the wound care industry, a member of the biaguanide family of antimicrobials, is polyhexamethylene biguanide (PHMB). PHMB is commonly used in swimming pool treatment and contact lens care solutions. Kerlix AMD99, Excilon AMD99, and Telfa AMD99 (all Tyco Health Care Kendall, Mansfield, MA) and XCellAE Cellulose Wound Dressing Antimicrobial (Xylos Corp, Langhorne, Pa) are newer dressings to the market containing PHMB. Most of the literature, albeit positive, relates to the antimicrobial activity of PHMB in contact lens care solution, with little information available concerning its clinical outcomes in wound care. However, initial reports indicate that PHMB-containing dressings do appear to provide an effective barrier against bacterial colonisation and experiments indicate that foam dressings impregnated

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with PHMB have been shown to lower bacterial counts (Davis et al., 2009); however, its future in wound healing has yet to be established. Cell senescence The biocompatibility of wound dressings and in particular adhesive dressings must address the subject of cell senescence. Cell senescence refers to the ageing of cells after a finite number of cell divisions (dependent on cell type) whereby each cell loses its ability to proliferate and ultimately presents with impaired function (Martin et al., 1970; Schneider and Mutsui, 1976). It has been suggested that the presence of these ageing cells at the wound periphery may be detrimental to the wound responsiveness (Shai and Maibach, 2005). Senescent cells are thought to be cells with growth arrest that develop aberrant behaviour and other phenotypic changes as a result of altered gene expression. It has also been demonstrated that senescent fibroblasts can develop a proinflammatory phenotype as evidenced by an increase in matrix metalloproteinase production and a reduction in matrix metalloproteinase inhibitor production (Shelton et al., 1999). These cellular changes may also influence the growth and behaviour of neighbouring cells. This phenomenon is considered to be a causative aspect in the maintenance and continuance of chronic wounds (Muller, 2009) and senescent cells have been implicated in a variety of pathological conditions. The use of adhesive dressings can and will accelerate this process since they can denude the peripheral wound of cells or artificially increase the rate of cell turnover, thereby causing premature and undesirable cell senescence. Pain relief Pain management is a critical part of the treatment of wounds and in burns injuries it is the most distressing and urgent symptom to be addressed. Although pain is a protective response to insult, in some cases it is the pain, and not the injury itself, which can cause the greatest discomfort. Whilst it is advantageous that a dressing may provide pain relief, it is important that the dressing does not itself cause undue distress during wear or in the process of dressing change, which can take up to thirty minutes to complete. Pain and discomfort suffered at wound dressing change can largely be put down to the forces at the adhesive edge of the dressing. The peel strength of an individual dressing at the time of removal is critical to the pain response – which is an important factor in the design of pressure sensitive adhesives for medical use (Chapter 11). This process is not entirely governed by the dressing itself, patient influence and their dermal characteristics represent a significant variable. Some types of hydrogel dressings (Chapter 11) offer key advantages in pain management. In general the

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cooling properties of hydrogel dressings by virtue of their high water content alone are able to alleviate pain, and their ease of removal is also an important consideration in this respect. An interesting approach to pain management involves an absorbent foam dressing that releases ibuprofen locally and is indicated for moderately exudative wounds. Biatain-Ibu (Coloplast), has been shown to reduce wound pain in chronic non healing wounds (Gottrup et al., 2007).

12.2

The corneal model in wound healing and biomaterial studies

Although wound healing carries the obvious primary connotation of dermal related wounds; wound healing does not lie solely in the domain of the dermal response. Mucosal surfaces and specifically the cornea, present other important examples of wound healing sites. The cornea, like skin, is at the anterior surface of the body and acts as a barrier against the external environment. Cross-comparison of the literature between ocular and dermal sites should, and does, prove mutually beneficial. Advances in the understanding, and ultimately treatment of certain specific human disorders, can on occasion be achieved through the application of information derived from other, apparently distinctly different, host environments and body sites. This is particularly compelling in the case of the anterior eye and specifically the corneal surface and its potential parallel with the moist wound bed environment. There are in fact several similarities between the ocular environment and wound bed with irresistible comparisons to be drawn between the configurations of: wound bed/wound fluid/dressing on one hand, and cornea/tear film/contact lens on the other (Fig. 12.1). The cornea is a more vibrant and dynamic environment but if and when the overlying tear film begins to fail, as is the case in chronic dry eye, the cornea keratinises and essentially will eventually turn to a skin-like dry surface. Moreover, although the cornea and the secreted tear film may have outward similarities to the exuding dermal wound, they are dissimilar in the fact that the former is healthy and the latter is not. Likewise, the functionally damaged dry keratinised cornea is physically similar to intact skin but the former represents the non-healing cornea at the end stage of chronic disease and the latter is indicative of a healthy functional dermal barrier. Although polymeric biomaterials have been widely used in both ocular and wound-related applications, little exchange of knowledge exists between these two fields. This is even true within the field of ocular applications, where the materials employed as therapeutic bandage lenses are simply those designed for use in cosmetic contact lens applications. In this case,

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Electrolytes Metabolites Lipids Mucins Proteins

12.1 Analogy between cornea and dermal wounds; the contact lens and the posterior tear film versus the wound dressing and wound fluid.

however, there has been extensive commercially driven research to develop materials with optimal compatibility with the elements of the ocular environment – cornea, tear film and eyelid. There are clear analogies between the design of a successful contact lens and a successful wound dressing. With the contact lens, adverse interaction with the tear film and consequent deposition can affect the visual performance of the lens and therefore shorten the life span of the lens. There are two aspects to this interaction, however. Not only can the lenses become contaminated with denatured or degraded tear constituents, including lipids and proteins, but this absorption by the lens may denude the tears of important antibacterial or immunoregulatory proteins. Characteristic differences in ocular response to materials of different structures have been observed and classified. Knowledge of these parameters is now fundamental to contact lens material design and development. Understanding tear film/biomaterial interaction can provide important and relevant information pertaining to the interaction of biological species with the ever increasing range of wound dressing biomaterials on the market and their effects on the wound response.

12.2.1 Corneal wound healing The aim of this section is not to cover, in detail, the vast and extensive body of information currently available in the complex area of ocular healing but to simply give an overview of some of the distinctive healing properties of the highly innervated moist cornea. This locale provides an attractive and advantageous site to study wound healing with the ease of non-invasive analysis. Ocular injury, corneal ulceration and chronic dry eye represent a large proportion of the causes of corneal blindness. The cornea itself is a particularly sensitive organ and it is not an uncommon site of injury due to its external setting. The cornea provides a physiologic barrier against external insult and ocular infection, while also maintaining optimum vision through its ability to remain transparent and provide precise refractive property.

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Advanced wound repair therapies Structure of the Cornea Glycocalyx Epithelial cells

Bowman’s layer

Stroma

Descemet’s membrane Endothelial cells Anterior chamber

12.2 Illustration of the corneal anatomical structure.

Maintenance of a normal intact cornea is multifaceted with the added complexities that the cornea itself is avascular and requires a smooth refracting anterior surface for optical acuity. The human cornea is anatomically split into three distinct cellular layers comprising the outermost epithelial cells, a middle stromal section and an inner endothelial cell layer as shown in Fig. 12.2. A collagenous Bowman’s layer lies between the epithelium and the stroma and likewise the Descemet’s membrane lies between the stroma and the endothelium. The key to corneal integrity is the preservation of the intact epithelium and the tight junctions. Cell-to-cell contact, as well as, adhesion to the basement membrane (cell to extracellular matrix contact) are integral to the mechanical integrity of the epithelium. The cornea succeeds in part due to a constant turnover of the epithelial surface. Epithelial cells are shed on a continual basis from the surface of the cornea into the overlying tear film and cleared via lacrimal drainage system. At the same time cells move centrally from the peripheral limbus and anteriorly upwards from the basal epithelium layers (Thoft and Friend, 1983). The limbal region (see Fig. 12.3) at the interface between the cornea and sclera consists of a band of cells approximately 0.5–1.0 mm wide (Pfister, 1994). It has certain analogies to the wound edge in that it is the source of new basal cells at the periphery of the cornea (Cotsarelis et al., 1989). The limbus contains both stem cells, which differentiate into basal cells and migrate into the cornea, and progenitor cells that regenerate corneal epithelium (Schermer et al., 1986). Blood vessels enter and leave the sclera in

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Cornea Anterior Conjunctiva Limbus

Limbal stem cells Posterior

12.3 The corneal limbus.

this region, small vascular loops develop from the vessels and extend approximately 1 mm into the cornea, and they represent the closest blood supply to the cornea. In cases of severe trauma, such as that associated with chemical burns, where there is complete epithelial cell loss and limbal disruption, ulceration will occur and the long-term prospect for healing is not good. The fact that limbal stem cells disruption affects the wound healing response is due to the fact that the re-epithelisation process of the cornea is damaged. It thus struggles to cover the wound and heal adequately. When the limbus is damaged, conjunctival epithelial migration onto the cornea can occur, which is normally impeded by the limbal barrier (Huang and Tseng, 1991). This conjunctival epithelial in growth may be accompanied by unwanted corneal vascularisation. The cornea is a highly specialised organ which presents with unique features required to achieve its specific functions. The ocular environment is immunologically unique due to the fact that there is no lymphatic drainage, apart from at the conjunctiva, and the normal cornea like the brain and testes, is an immunologically privileged site. This privilege arises due to the presence of a special microenvironment and immunoregulation mechanism. Corneal avascularity is essential for optical clarity and due to the absence of blood vessels, protection of the eye is provided by proteins in the tear film, blinking and migrating lymphoid cells, macrophage and immune effector cells derived from the limbus region. The eye participates in all aspects of immune responses like any other tissue but the response is modulated by the cells and tissues of the eye; this privilege is lost when the cornea becomes vascularised. The significantly distinctive characteristic of the cornea is its avascular nature. Another major distinguishing feature that sets it apart from other body sites is the unusual diurnal variation brought about by the dramatic environmental shift from an open eye to an overnight closed eye stagnant setting. The unique features of the cornea distinguishing it from other body sites are as follows:

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avascular blink action overlying anti-microbial tear film moist-air interface diurnal variation • closed vs. open eye • stagnant vs. dynamic tear • changing oxygen supply.

After corneal injury the pattern of healing is not dissimilar generally to those observed at other body sites. Epithelisation is a three-stage process involving epithelial cell migration and proliferation followed by cell differentiation and reconstruction of adhesion bodies and cell adhesion (Crosson et al., 1986; Kuwabara et al., 1976; Gipson, 1992; Dua and Forrester, 1987). Corneal epithelium cells are central to repairing the cornea but, in common with epithelial cells at other sites, they are also an integral source of growth factors in the wound healing response (Imanishi et al., 2000; Yu et al., 2010). Upon injury or insult to the cornea, cytokines are released from the injured epithelium and the epithelial basement membrane. These include the pleiotropic factor interleukin-1, tumor necrosis factor-alpha (Wilson et al., 1999), epidermal growth factor and platelet derived growth factor (Tuominen et al., 2001). Keratocyte apoptosis occurs and is followed by cell necrosis (Wilson et al., 2001). After approximately twelve hours, the remaining keratocytes undergo proliferation and migration, which in turn, it has been suggested, results in cell differentiation, proliferation and migration into the depleted stroma (Fini, 1999). Also, in the first 24 hours of injury a variety of pro-inflammatory cytokines from the epithelium, and/or from keratocytes, initiate the movement of antigen presenting cells, phagocytes and T cells from the limbal blood supply (and possibly the tear film) into the stroma. Myofibroblasts which differentiate from the stromal keratocyte cells play a key role in the wound healing cascade. They appear to function in the organisation and deposition of extracellular matrix in corneal wounds and play a role in corneal wound contraction (Jester et al., 1999). The normal functioning cornea and its natural continual renewal process could be thought of as a body site in a state of continual healing.

12.2.2 The non-keratinised cornea: a wound bed mimic The normal healthy cornea is a non-keratinised, avascular tissue which, together with the overlying tear film, provide an interesting parallel with the moist wound environment. Conversely, at the extreme end of an inappropriate reaction or a disruption of the normal ocular wound repair

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response, keratinisation of the cornea can occur where the tissue becomes dry and skin like, which ultimately is the desired outcome in dermal wound healing. Keratinisation of the cornea occurs as a result of dry eye, where the quantity and quality of tears is reduced due to gland secretion dysfunction as a result of inflammation or other ocular disorders. This property alone highlights the importance in healing of the efficient control of hydration of the wound. Ultimately, the tear film is integral to the health and maintenance of the ocular epithelial surfaces. Any deviation from the normal state of equilibrium will affect the underlying ocular surfaces as evidenced by chronic dry eye and tear film dysfunctional disorders. The overlying tear film is in many ways unique. The functions of the tear film include: lubrication, maintenance of a smooth refracting layer for clear vision and support of the innate and acquired immune ocular defence. It is the participation of tears, and more specifically the proteinaceous content of tears, in the ocular host defence mechanism that is of particular interest in the context of wound healing due to their specific involvement in, and regulation of, the immune response and ocular protection. A great and varying number of proteins have been identified in tears, and in a recent study the presence of nearly five hundred different proteins has been detected (deSouza et al., 2006). Some of the proteins present in tears are indigenously produced tear-specific proteins (e.g. tear lipocalin, lactoferrin and lysozyme) secreted by the glands of Kraus and Wolfring and the lacrimal gland. Others detected in the aqueous tear are plasma derived (e.g. albumin and IgG) and their concentration in tears varies depending on the intactness and stability of the blood-tear barrier. This barrier can be affected by a number of factors including inflammation and overnight eye closure during diurnal variation (Zavaro et al., 1980; Sack et al., 1992). Even the normal uncompromised eye can, however, exhibit some levels of plasma proteins leakage and influx. Other proteins present in tears may be synthesised locally by epithelial cells, e.g. the secretory component involved in secretory IgA transport. The main indigenous proteins in tears serve to protect the anterior eye from a host of potential microbial pathogens. It has long been demonstrated that tears contain anti-bacterial substances. Lysozyme was the first anti-bacterial protein to be recognised in tears owing its activity to the fact that it degrades the NAM-NAG backbone structure of peptidoglycan and disrupts the bacterial cell wall of gram positive bacteria. The protein lactoferrin is also involved in the defence of the eye against bacterial invasion (Broekhuyse, 1974). Its proposed mode of action involves the inhibition of bacterial growth and colonisation by the uptake of iron (an essential mineral for the growth of bacteria) from the environment, in order to deprive the microorganism of this essential bacterial nutrient (Kijlstra, 1990). Other theories regarding its functionality have been put forward suggesting roles in the enhancement of natural killer cell function (Nishiya

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and Horwitz, 1982), suppression of primary antibody responses (Duncan and McArthur, 1981) and exhibition of anti-complementary effects (Kijlstra and Jeurissen, 1982) amongst others. The importance of the antibacterial nature of the tear film can not be underestimated particularly at the interface between the external ‘pathogen teeming’ environment and the ocular surfaces. The analogies with antibacterial components in wound fluid are obvious in principle but certainly worthy of more detailed exploration.

12.2.3 Corneal wound healing biomarkers The use of ocular-based studies and biomarkers to understand the importance of specific regulators in wound fluid environment studies can be evidenced by specific examples. Vitronectin, described in more detail earlier, which is a central component in the wound healing response, has also been detected in tears (Sack et al., 1993) where it is a prominent inflammatory regulatory protein and adhesion molecule. The most significant plasminogen activator inhibitor in cellular systems is plasminogen activator inhibitor-1 (PAI-1) which is known to bind to vitronectin and has been suggested as being a key feature in serving to localise PAI-1 to the pericellular compartment where active PA is generated. As mentioned earlier, if vitronectin is localised on a surface adjacent to the cellular site, it removes PAI-1 from the reaction by fixing it, creating an imbalance in favour of the plasminogen activator and a production of active plasmin. High plasminogen activator levels peripheral to injury sites are implicated in the pathogenesis of persistent corneal defects. To parallel this observation in the eye, chronic wound fluid from venous ulcers often shows complete degradation of vitronectin and fibronectin into smaller peptides that prevents local cell adhesion to the wound bed, preventing closure. In rabbits, fibronectin has been shown to be present at corneal wound sites but it is not detectable in the normal cornea (Suda et al., 1981–1982), suggesting therefore that detectable levels of fibronectin during wound healing dissipate when the normal cornea is restored. Fibronectin binding to fibrin from the blood may be important in the initial stages of wound healing in the damaged cornea, enabling corneal cells at the periphery of an injury to migrate across the defect. A thin fibronectin and fibrinogen film can form across the corneal wound and the epithelial cells migrate into the area. Additionally, the phenomenon of plasmin upregulation has been recognised in relation to corneal wound healing and in conventional cosmetic lens wear for several years. Tear plasmin activity has also been observed in the eye where corneal disease has been detected (Tervo et al., 1988), and in particular in corneal ulcers where a dramatic increase in plasmin levels has been demonstrated (Salonen et al., 1987) leading to the implication that elevated plasmin levels are important in the pathogenesis of ocular infection and disease. The signifi-

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cance of elevated plasmin has been noted by many researchers and the view is that it is an important potential trigger of additional events, leading in extreme cases to pathology in an otherwise healthy tissue. Plasmin was also been detected in tears of patients during contact lens wear (Vannas et al., 1992), and is suspected to contribute to the development of corneal epithelial pathologies associated with contact lens wear. It has been stated that ‘Plasminogen is synthesised in the cornea and can be activated to plasmin by plasminogen activator’ and ‘In turn, plasmin is able to activate latent collagenase. This system could lead to the collagen degradation of corneal ulceration’ (Farooqui et al., 2008). The conclusion specifically in relation to hydrogel wound dressings seems clear and is supported by experience in studies of contact lens-cornea interactions. The presence of hydrophobic domains within neutral and carboxyl-containing hydrogels leads to irreversible deposition of vitronectin and its consequent depletion in the tissue bed. Since this depletes active PAI-1 there is a consequent upregulation of plasmin, and ultimately collagenase. Another cascade of importance but largely ignored in the wound healing process is that of the kinin family of immunoregulatory proteins. Bradykinin, the end product of a kinin response, functions amongst others to induce pain and discomfort and cause inflammation. Its importance in relation to biomaterials can be highlighted by the simple fact that the kinins are generated on contact with an activating surface and in particular they have a strong affinity for negatively charged surfaces. This is a necessary parameter to consider in terms of a dressing or biomaterial and its potential interaction at the biological interface with this contact activation system. In the ocular environment some important characteristics of the kinin response have been determined. Kinin activity is not normally found in the open eye basal tear; however, in marked contrast, a contact lens induced kinin influx has been clearly observed. This has pointed to the generation of the kinin response, as a result of or in association with, contact lens wear emphasising a host-surface interaction related event (Mann and Tighe, 2002). This phenomenon was found primarily to be material related (and secondarily patient dependent) with certain materials determined to show a high propensity for kinin influx. Ultimately a material which does not enhance or allow the build up of kinin components would intuitively be expected to be the most favourable. However, the persistent presence of high molecular weight kininogen (as a marker for kinin influx) with specific lens materials did not greatly affect the subjective comfort levels achieved compared with those obtained for the non kinin up-regulating materials. The lens may somehow decrease the bioavailability of the kinins, and ultimately the pain inducing nonapeptide bradykinin, preventing its required interaction with the cell expressed bradykinin receptors. This emphasises the potential of a material to directly or indirectly become the causative agent of an adverse

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response. Conversely it may denude the contacting microenvironment, through absorption, of potential pro-inflammatory or anti-healing species thereby limiting their negative influence on the wound healing dynamics. For example, a wound dressing which would denude and retain the bradykinin component from the wound bed, for example, could help to reduce pain and inflammation. However, one which enhanced its accumulation and bioavailability may be detrimental to the normal wound healing process.

12.3

Interfacial phenomena in ocular surface contact lens studies

The development of new biocompatible biomaterials at the interface between the host and the external environment relies on understanding of the chemical and physical nature of the interfacial fluid and the contacting surface environment. The two key interfaces in the context of this discussion are, on one hand, wound exudate and the wound bed in contact with the wound dressing and, on the other, tears and the cornea in contact with a contact lens. A technique has been developed that enables the study of the tear fluid, and specifically in this case the proteinaceous content of the tear film, that is in intimate contact with the lens material at the time of removal. This interfacial fluid has become known as the ‘tear envelope’ (Peach et al., 2002; Mann and Tighe, 2004), this ‘envelope’ can be thought of as being the tear film that is attached by surface forces to the contact lens. Examination of the envelope allows us to investigate the interfacial interactions occurring between the tear film and the overlying contact lens material. Its analysis enables the instantaneous and longer term effects of a lens surface on tear film to be observed. Numerous lens materials in contact with a variety of tear film states (healthy, dry or diseased eyes) can be easily studied. This film and its composition are quite distinct from tear products deposited on the lens and from the tear volume held in the tear menisci. A detailed account explaining how the tear envelope is collected and its analysis by Lab-on-a-Chip microfluidic protein sizing separation technology can be found elsewhere (Mann and Tighe, 2007). In essence, the tear envelope protein analytes are separated electrophoretically, detected by their fluorescence [FU] (670–700 nm) and the data is then translated into individual electropherograms as seen in Figs 12.4 and 12.5. Each LabChip kit contains a standard molecular weight ladder well with a lower limit marker at 4.5 kDa and an upper limit marker at 240 kDa for internal calibration. Figure 12.4 shows the difference between a tear envelope collected after one minute wear and then the same patient’s tear envelope after thirtyminute wearing time with the same lens material (lotrafilcon B (O2OptixCibaVision)). Overall the tear envelope profile for both envelopes appear

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[FU] 250 200 150 100

1 minute

50

30 minutes

0 15

20

25

35

30

40

45

[s]

12.4 Interfacial tear envelope dynamics: influence of material over time, 1 minute vs. 30 minutes wear, lotrafilcon B (O2Optix – CibaVision).

7: Galyfilcon A 30mins TE

Sc Lotrafilcon A 30mins TE [FU] 200 150

lotrafilcon A

100 50

galyfilcon A

0 4.5

7

15

28

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95

150

240

[KDa]

12.5 Interfacial tear envelope dynamics: material influence, lotrafilcon A (Focus Night&Day – CibaVision) vs. galyfilcon A (Acuvue Advance – Johnson & Johnson).

similar but subtle changes in tear protein dynamics following lens insertion and the longer wear time are apparent. The one-minute wear sample was found to contain a higher overall concentration of protein compared with the thirty-minute wear sample. The initial build up of protein upon insertion represents the immiscibility of the protein for the silicone based material; however, over the longer time the build up at the interface is not as apparent demonstrating some measure of deposition and biofilm spoilation of the lens material. These changes in tear protein dynamics reflect the alteration and accommodation of tears as a consequence of the presence of the newly inserted lens.

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The protein dynamics of the surface interacting tears was also material dependent; this material to material variation is evident in the electropherogram in Fig. 12.5. The discrete differences are noticeable by the unique peak protein profiles representing the differential tear response to the two different materials. Differences between a tear envelope sample and a lens-wearing tear sample, the difference between the composition of the fluid at the interface between material and contacting surface and the overlying ‘circulating’ fluid, are also apparent. From a range of experiments analysing many different contact lens materials, from conventional hydrophilic poly(2hydroxyethyl methacrylate) (polyHEMA) based lenses to the more complex silicone-hydrogel hybrid materials, it is clear that distinct material to material differences in the tear envelope are readily detected and interestingly small changes over time can also be noted. Discrete distinctions can be determined between the lens interfacial tear envelope, the dynamic lens wearing tear and the normal non-lens wearing tear. The tear envelope is a discrete composition tear fluid which is intimately dependent on the lens material and contact period which highlights the effects of the inserted lens on normal tear dynamics. Due to ethical issues and the impracticalities involved in dermal wound studies, it would not be feasible to place and investigate a variety of dressing materials on the wound bed (this would cause more damage than good). However, our studies at the easily accessible site of the anterior eye can give us some insight into the unique and complex interfacial interactions occurring between biomaterial and host, which can ultimately affect the fate of wound healing. In ocular studies, tear envelope investigation has helped us to understand the interfacial dynamics which has ultimately assisted in contact lens material development optimisation. These studies highlight the importance of recognising that interfacial dynamics influenced by the material are crucial to material design at any body site. The understanding of how tears interact with contact lenses is both important and complex. Advantageously the contact lens can act as a valuable biomaterial probe enabling the interaction of new materials with a multifarious biological fluid to be studied.

12.4

Wound fluid and the tear film collection

In order to understand the effects a dressing material can have on a wound’s response, the levels of interaction between the wound and the dressing must be determined. This involves the individual investigation of both the wound exudate and the absorbed species in the dressing matrix. Examination of wound fluid exudate can be crucial in defining the biochemical response and this in turn can be used to investigate the potential effects an overlying

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material can have on the healing process. As we have shown throughout this chapter the cross examination of corneal and dermal literature is extremely useful and the same can be said in the area of biological fluid collection. Tear collection is a relatively straightforward technique that is minimally invasive and essentially discomfort free. Additionally, and more significantly, tear fluid collection is also feasible during contact lens wear to determine biomaterial interfacial interaction. This allows the researcher to study the effects of tears on the inserted biomaterial and significantly the biomaterial influence on the contacting biological fluid. While tear fluid secretion and wound exudate release are both obviously dramatically separate entities, which are controlled by distinct neurological mechanisms, lessons realised from tear fluid collection and analysis can aid the understanding of wound exudate analysis.

12.4.1 Tear film collection Even with many years of excellent research in ocular compatibility studies, tear collection is, however, by no means an exact science; various and numerous methods have been employed over the years to carefully extract tear samples from patients and volunteers. Tear fluid collection is performed most commonly using microcapillaries. This collection method uses blunt-ended glass narrow bore microcapillary pipettes to collect 1–7 μl of tears from the lateral canthus and/or inferior marginal strip. It is a relatively time-consuming method but there is little conjunctival irritation and collection in this manner demonstrates a ‘truer tear’. An alternatlie method, the Schirmer strip approach, used routinely to assess tear flow rate, uses filter paper to collect tears after which the tears and desired analytes have to be extracted. In short, a strip of filter paper usually 35 mm long and 5 mm wide is inserted into the lower conjunctival sac and the tears are adsorbed by the strip wetting approximately 5–6 mm. It is a simple and inexpensive test and is reliable in itself but ultimately it is a harsh procedure and will result in the production of unwanted reflex tears. The greatest disadvantage is the need to elute the analytes from the strip. Many methods are used both physical and chemical but no uniform method has prevailed and none can claim to be one hundred percent efficient. Another popular method of choice is the microsponge technique which, as the name suggests, uses an absorbent triangular cellulose sponge, 1.5 cm in length, to collect tears from the inferior marginal strip to the medial canthus. However, like the Schirmer strip, it is somewhat invasive and also requires an elution step to extract the analytes from the sponge. Other collection methods utilised include the use of porous polyester rods and filter paper circles, again all requiring some method of elution. The consequence of having these and other tools for collection is that there are many tear statistics that cannot be directly correlated with each

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Table 12.1 Summary of the variables involved in biological wound fluid collection Biological fluid collection variables

Comments

Collection tool employed?

E.g. microcapillary, syringe and needle, cellulose sponge, etc. Flow rate, stimulated or basal fluid collected. How efficient is the extraction method, what is left behind? E.g. methanol extraction from cellulose sponges required to extract lipid content. In contact with biomaterial? Was the fluid stimulated or expressed? Normal vs. healthy (related or unrelated). Immediate use or fridge/freezer storage.

Sample collection rate and volume? Extraction from collecting tool required? If yes what extraction protocol used? Fluid ‘state’ at time of collection? Patient’s health? Storage conditions prior to analysis? Sample pooling? Dilution required? Analysis assay employed? Sample preparation? Biomaterial material (if applicable)? Biomaterial wear/contact time (if applicable)?

One patient’s fluid pooled or a mixture of patients’ fluids? Nature of diluent? HPLC, MS, ELISA – assay limitations. Reducing agent used or methylation, etc? Nature of material if in contact with fluid at time of collection? Duration of wear/contact time prior to collection, minutes vs. weeks?

other, and today there still remains no universal method of sampling. An example of how tear sampling can affect protein composition is provided by a study back in 1984 where the Schirmer strip method was shown to present with higher concentrations of albumin and IgG (the plasma derived proteins-signifying vascular permeability at the ocular surfaces) than the less irritating microcapillary method (Stuchell et al., 1984). These inconsistencies and problems have been widely reported and the fact that tear fluid collection techniques greatly influence tear fluid composition has been extensively highlighted and scrutinised (Berta A, 1986; Fullard and Tucker, 1991; Esmaeelpour, 2004). Tear collection is just one variable in a series of other variables that can affect the nature and fate of the analytes under investigation. Table 12.1 summarises some of the many variables encountered and acknowledged in the tear fluid studies. These factors affecting biological fluid analysis are universal and can be representative of any other biological fluid collection and analysis, irrespective of body site. Individually and combined the variables involved can have a huge bearing on the fluid compositional profile determined and recorded.

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12.4.2 Wound exudate collection It is important to understand that exudate is crucial to the wound healing process and a full understanding of its role and influence in wound healing is critical. Exudate has been defined as a ‘fluid deposited in tissues (as a result of inflammation or infection) that has high viscosity and high protein (>30 gm/L)’ (Trenglove et al., 1996). Wound exudate or wound fluid is produced predominantly in the inflammatory and proliferative phases, and contains a variety of components including water, electrolytes, white cells, numerous inflammatory components, growth factors, enzymes and nutrients and waste products. What may be thought of as the simple task of collecting wound fluid (or indeed any biological fluid) could not be further from reality, as this step in protocol alone can markedly influence the outcome of the investigation. Wound fluid exudate analysis is an emerging discipline, made difficult due to the necessary constraints imposed by ethical demands. Tear film collection and analysis is better established and does not fall under the same ethical constraints imposed in dermal wound. Our understanding of the collection of tears and equally interfacial tears has exposed the difficulties concerning fluid analysis particularly in relation to a biomaterial interaction. Wound exudate collection is not vastly different from that of tear collection in principle. Wound fluid is also sampled by a number of similar methods including the glass microcapillary and sponge but also by syringe and needle, lavages underneath bandages or filters with the most common method involving the aspiration of the fluid from beneath occlusive transparent dressings. Wound exudates differ in the same way that we recognise that tear film differs; while the reasons for such are distinctly different, it is imperative to take into account the different states of each. For example, tear fluid analysis and collection relies on the understanding that tears are dynamic and their composition can vary dependent on a variety of parameters including, gender, age, ocular and systemic health, diurnal variation, emotion (psychogenic lacrimation) and stimulation (basal or reflex tearing) amongst others. Each of these factors alone naturally will affect the outcome of the results determined. Wound fluid collection is no different and perhaps is even more complex with the many key variables involved, encompassing the likes of wound size, volume of exudate, wound type, origin of wound, location of the wound, extent of the wound and the phase of healing. All these must be considered when investigating wound fluid exudate. Recording all these variables at the time of collection is fundamental to understanding the processes involved in all stages and presentations of wounds. There is an excellent discussion (Drinkwater et al., 2002) on the need for standardisation of techniques to sample and study wound fluid. However, even though this important paper was written in 2002, there has been little

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change or improvement in cross methodology standardisation. Tear film collection also relies on the ability to be able to collect and sample baseline tears, one not in contact with a lens material, in addition to baseline samples taken over the course of a study to identify ‘normal’ individual levels. This is more complex in wound fluid studies simply because a baseline sample in the true sense of the word does not exist; that being fluid from an acute healing wound from the same patient, in the same body site under similar conditions! Another important variable commonly overlooked in wound exudate analysis is the recognition of the wound bed management history prior to collection and analysis, i.e. what dressings were in contact with the wound bed and for how long. Wound exudate analysis invariably is assessed in the presence of a wound dressing but it is not always the case that the exudate is analysed in the presence of the ‘regular’ wound dressing, thus the characteristics of the wound bed are compromised. Very few studies address this variable in terms of the history of dressings the patient wore prior to the individual study; moreover, in some studies information pertaining to the dressing in contact with the exudate at the time of collection is not always provided. Each dressing in contact with the wound bed will, to varying degrees, influence the wound fluid composition and dynamics. The nature and composition of wound fluid will be influenced by the contacting biomaterial dressing particularly at the interface. Finally, one of the central factors in choosing the right tool for fluid collection is suitability, because ultimately it must be able to reproducibly collect the desired analytes and be compatible with the analytical technique used to detect these analytes. Parallel studies on biological fluids associated with wound dressings and contact lens materials show interesting biochemical analogies. Studies on wound fluid and the etiology of the non-healing wound are extremely important and to understand and modulate wound healing, specific mediators have to be identified and functionally characterised. From a clinical perspective the nature and effects of the wound dressing on the wound are of extreme importance; however, there is a limited amount of research afforded to the actual biochemical effects of the dressing on the wound bed and fluid. Information relating to the pro- and anti-healing factors present at the wound bed can help us to understand the optimum parameters required for the ideal wound care system; however, caution must be taken in the use of this information which, on occasion, can be contradictory.

12.4.3 Wound fluid models Biological fluid models provide a useful way to investigate and understand the behaviour of biomaterials in the appropriate bodily environment. Early theories suggested that wound fluid was an unwanted and superfluous by-

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product of wound healing but it has since been established as a vital component of the healing process. The design of an ‘ideal’ wound fluid, which would enable predictions relating to the effect of a range of materials on the state of healing using a standard wound fluid would be hugely beneficial and enable progression of our knowledge of wound healing. The complications of achieving a meaningful wound fluid model are numerous. Although simple electrolyte-based models are used for simple dressing uptake studies, none of them mimic the active components involved in the healing process. It is widely accepted there are differences in the composition of acute wound fluid and chronic wound fluid. A model for both of these states of the healing/non-healing wound would be desirable, since both regimes have an effect on the state and, ultimately, the fate of a wound. The nature and composition of the acute wound exudates, for example, would be expected to indicate a desirable composition for a healing wound. A clinical review by Cutting provides an excellent overview of the components and functions of acute and chronic exudates (Cutting, 2003). Many studies use fluids obtained from Jackson-Pratt or Portavac drains as standard practice to correspond to acute wound fluid. Surgical drainage and blister fluid have also been used as acute fluid models, amongst others. Chronic fluid is widely obtained by means of aspiration directly under occlusive dressings or from the dressing itself. However, neither is ideal because of the numerous variables involved from patient to patient and wound to wound, highlighting the need for a standard wound fluid.

12.5

Biomaterials in mucosal wound healing

Most of the literature on wound healing relates to dermal wounds. Mucosal wound healing is also an important subset of healing investigation, however, and an area which presents with some uniquely different properties in contrast to the dermal environment. Under normal conditions the mucosal surfaces present a moist mucin-rich environment, a stark contrast to the normal dry waterproof environment of the dermal barrier. This variable alone points to the underlying divergent contrast in physiological behaviour between the surfaces. Another point of note is that mucosal membranes are the first portal of entry and attachment for many microorganisms and can ultimately be more susceptible to invasion than the normal intact dry dermal surface. In terms of biomaterial design, mucosal tissue is characteristically delicate and difficult to bandage. The dry versus moist contrast between the dermal and mucosal surfaces respectively has the greatest bearing on dressing choice for the two sites. Mucosal tissue must remain moist after healing in order to carry out its normal function, in contrast to the need for the skin to eventually return to its dry state to complete the healing process. The main mucosal surfaces are as follows:

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Buccal Nasal Ocular Oesophageal Gastrointestinal Colonic Pulmonary Bronchial Uterine Urogenital.

The recognised differences between dermal and mucosal wound healing can be summarised as follows: •

• • • •

Mucosal surfaces are characterised by the presence of an overlying mucosal fluid, for example: saliva, tears, nasal, gastric, cervical and bronchial mucus, the functions of which include to supply and deliver an array of immunoregulatory and pro-healing species including growth factors, antimicrobial proteins and immunoglobulins. A summary of the roles of mucosal fluid at these surfaces is as follows: • Availability of antimicrobial proteins • Lubrication • Nutritional maintenance for underlying epithelium • Waste clearance • Flushing of foreign bodies/irritants • Serves as a delivery vehicle for the influx of healing agents during injury, e.g. polymorphonucleocytes and vitronectin • Regulation of hydration • Supports the immune response • Mucosal tears additionally facilitate the supply of oxygen to the cornea, allow the formation of a smooth layer over the irregular cornea and enhance corneal wettability. Epithelial turnover occurs at a faster rate at mucosal sites. Mucosal tissues are generally more vascularised (with the exception of the corneal surface, which is avascular). As the mucosal surfaces are highly vascularised the response time of inflammatory cells following insult is faster. Lower levels of neutrophils, macrophages and consequently their respective cytokines have been reported in mucosal wounds (Szpaderska et al., 2003).

The mucosa and healing is a vast topic covering a range of divergent mucosal surfaces, each requiring unique and specific attention primarily as

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a function of their locale; the aim of this section is to highlight some of the variations encountered and summarise the contrast to dermal wound biomaterials. Two distinctly different examples of mucosal sites, the nasal mucosa and the anterior ocular surfaces, are discussed below in more detail, with examples of the variety of biomaterials finding application in these areas of mucosal wound healing.

12.5.1 Anterior ocular surfaces and wound healing biomaterials The anterior ocular surfaces include the cornea, conjunctiva, sclera and lids of the eye. In the anterior eye one of the most commonly used biomaterials is the soft contact lens. In the anterior healing eye a bandage contact lens can be used to protect an injured or diseased cornea from external factors thereby improving its ability to heal. Such lenses are referred to as therapeutic, as distinct from cosmetic, lenses and if no refractive correction is required plano lenses are used. Therapeutic contact lenses are used in a variety of conditions including dry eye, allergy, ulcers and persistent epithelial defects. One of the few attempts to ascertain the frequency of use of bandage lenses (BLs) in North America revealed that the most common uses of BLs were for corneal wound healing and post-operative complications, being prescribed by 72% of the ophthalmic practitioners who answered the survey (Karlgard et al., 2002). Of the patients treated with BLs 81% were prescribed additionally with antibiotics and anti inflammatory drugs. Etafilcon A (Acuvue, Johnson & Johnson) and nefilcon A (Focus Night & Day, CibaVision, Novartis) lenses are the most widely used BLs. The potential value of exploiting the therapeutic lens as a drug delivery vehicle rather than simply as a protective barrier has been discussed elsewhere (e.g. Mahomed et al., 2003; Mahomed and Tighe, 2006). An important aspect of bandage lens dressing design lies in matching the base curve of the lens to the radius of curvature of the cornea – referred to as lens fit. A poorly fitting lens will impact on the underlying epithelium especially during the blink which may cause further epithelial erosion. Significantly, no lens has been specifically designed as an actual BL, they are simply cosmetic lenses used for a healing benefit. This is, in large part, a consequence of the extensive ocular compatibility studies that have underpinned the development of commercial contact lens materials. The published information in this area contrasts with the limited available information on biomolecular aspects of wound dressing-wound bed interactions. The advent and popularity of elective refractive laser surgery has opened up a whole new area in ocular wound healing and has allowed researchers

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to study corneal insult and biomaterial interaction more closely. Laser in situ keratomileusis (LASIK), laser in situ epithelial keratomileusis (LASEK) and photorefractive keratectomy (PRK) are the most commonly performed procedures. Laser eye surgery involves excimer laser ablation of the underlying cornea accessed by folding back a hinged corneal circular flap which is created by means of a microkeratome or by laser. Following refractive surgery soft contact lenses are commonly used as bandage lenses to protect the cornea, relieve pain and assisting in corneal flap healing. In the early days of LASIK a bandage lens would be worn for weeks but with advances in the technology the bandage lens is now generally only worn for a day. One Canadian study suggested that bandage contact lenses were not necessary for LASIK post-operatively, but they conceded that in the cases studied no post-operative complications were encountered rendering use of the lenses unnecessary (Ahmed and Breslin, 2001). There are many conflicting reports relating to the use of bandage lenses and in particular the net benefit to the patient. Tissue sealants such as fibrin-hydrogel glues have also been used to treat postoperative LASIK to prevent epithelial ingrowth and flap dislocation. Whilst the reported results look favourable, it is a relatively newer procedure and has been used very infrequently, which means that longer-term effects have yet to be adequately established. In surgically induced ocular wounds the standard procedure is to use nylon sutures, but these can cause secondary complications including extra trauma to the patient and infection. Consequently, new developments involving polymeric adhesives and sealants are gaining momentum in this area. Cyanoacrylates and fibrin sealants (Chapter 11) were initially the obvious choice and most widely used but more recently hydrogel sealants have become a viable option. The area of ocular surface reconstruction also involves a range of biomaterials which can be incorporated into the anterior ocular surface to encourage epithelial cell re-growth or to replace the ocular surfaces completely. In severe ‘end stage’ cases synthetic devices, known as keratoprostheses (KPro), can be used in place of corneal and conjunctival tissue. Comprehensive overviews detailing the biomaterials employed in the many KPros designs are provided in two separate chapters by both Evans and Princz in 2010 (Evans and Sweeney, 2010; Princz et al., 2010). Bandage contact lenses are also frequently used to aid healing after some keratoprostheses placement procedures.

12.5.2 Nasal wound healing biomaterials Nasal dressings are commonly used to assist healing after a variety of surgical procedures and epistaxis. Some of the many and varied options available in nasal wound care are as follows:

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Gelita-Spon® Gelatin Sponge and Film USP (Invotec International) PVA+TM Sponge Packing (Invotec International) MeroGel®, an absorbable unique esterified form of hyaluronic acid (Medtronic Xomed) Merocel®, a non-absorbable packing foam polymer of hydroxylated polyvinyl acetal sponge (Medtronic Xomed) JelonetTM Paraffin Gauze Dressing (Smith & Nephew) (Harb and Pandya, 2010) Nasopore®, a bio-degradable fragmentable polyurethane foam, the main material – soft segment – is based on lactide-caprolactone polymers (Polyganics BV) Gelfoam®, an absorbable gelatin sponge (Pharmacia and Upjohn Co.) Surgicel, an absorbable, cellulose-based material (Johnson & Johnson Co.) Telfa pads, absorbent nonadherent cotton (Kendall Healthcare Co.) DoyleTM nasal packs, absorbent sponges (Donald Doyle, MD, FACS) FloSeal® Matrix Hemostatic Sealant (Baxter Healthcare Corporation Fremont, CA) The Rapid Rhino®, a carboxymethylcellulose pack (ArthroCare Corporation) The Rocket Rhino®, compressed foam polymer tampon made with polyvinyl alcohol (Shippert Medical Technologies Corporation)

For nasal dressings the correct application and ability to place it correctly is crucial to the success of the dressing. Biomaterials that are too large for the small confines of the nasal anatomy can put pressure on the surrounding tissues and blood vessels and can result in necrosis and ischemia, thereby impeding normal function and healing. Improper application can also lead to all manner of complications and may impede wound healing. Additionally some nasal dressings need to be removed after a number of days; removal can not only cause patient discomfort but may also cause the wound to reopen. Acknowledging that a moist wound environment is beneficial to wound healing, nasal packing materials may be impregnated with hydrating additives in order to stop drying and possible crusting of the mucosa and importantly to minimise discomfort. There are two main categories of nasal biomaterials: those that need to be removed and those that do not, the latter having the ability to be bio-resorbable or to dissolve, negating the need for removal. While it is generally accepted that absorbable dressing packs are effective for haemostasis, their influence on the healing process is largely uncertain. In the case of nasal epistaxis the main aim of the biomaterial is to provide coagulation and haemostasis rather than to promote healing per se, calcium alginate meshes or swabs are commonly used for this application due to

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their high absorption capacity. Nasal dressings used after surgical procedures need to be absorbable and they should also promote healing and haemostasis. Currently no dressing is available that allows all three to occur. A study which assessed two absorbable biomaterials (MeroGel and FloSeal (see page 313)) on the healing process in the maxillary sinuses of rabbits found evidence to suggest that they may even impair mucosal healing and observed incomplete resorption of both materials which resulted in a degree of lymphocytic infiltration (MacCabee et al., 2003). A more recent paper suggests that ‘recent research shows promise with microporous polysaccharide hemospheres and chitosan gel having promising effects on haemostasis, and chitosan gel showing a significant adhesion prevention effect’ (Valentine and Wormald, 2010). Hyaluronic acid as a cream has been used to good affect during the first week of healing post surgery where it has been observed to prevent extensive crust formation which is important in allowing the airways to remain open (Soldati et al., 1999). Additionally, autologous fibrin tissue has been used post operatively in nasal or sinus cavities as an alternative to packing products and has demonstrated some wound healing properties (Gleich et al., 1995). Nasal wound care while relatively small compared to the dermal wound care industry has a vast range of biomaterials available for its specific and unique requirements.

12.6

Conclusions

An important aspect of this chapter is the extension of the interfacial phenomena that govern and modulate adhesive behaviour to encompass the biological and biochemical consequences that always arise when a biomaterial is introduced to a host biological environment. The development of understanding in this area has been recognised as an important aspect of the development of new and more biologically responsive or active wound healing biomaterials. Reviews which provide indications for specific dressings and their ‘proposed best use’ are invaluable in providing assistance in choosing the optimum dressing(s) for a wound. One example from the vetinary field (Stashak et al., in 2004) gives an overview of the indications of best use of dressings in equine practice, and provides a valuable resource for dressing comparisons in terms of their indications and contraindications. Others, which address and compare specific dressing functionality by meta-analysis, are extremely important in order to assess the efficacy and future usefulness of specific treatments. For example, the review by Carter et al., in 2009, which assessed the use of silver treatment in leg wounds and ulcers, found that although silver still remains a popular choice, there was little evidence to suggest it was beneficial to complete healing or healing rates. Reviews like these also highlight the need for better quality clinical studies in general

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and in particular longer follow up times. Biomaterials studies in wound healing would be made easier if a standard wound fluid model were available but the challenges facing this realisation are also great. A great deal can be learned from observations arising from the behaviour of biomaterials at other body sites; one particularly relevant body site in the context of wound healing is the anterior eye. The cornea, tear film and posterior surface of the contact lens provide an informative model of the parallel interface that exists between the chronic wound bed, wound fluid and the dressing biomaterial. The parallels that exist between these two situations have been highlighted, together with conclusions that can be drawn from work on tear models and tear fluid analysis. These have been related to the potential of wound fluid models and wound fluid analysis in furthering the understanding of the effects of the wound dressing biomaterial in the biological environment of the wound bed. This chapter is underpinned by the logic of considering integrated physicochemical, biochemical and biological aspects of biocompatibility and biomaterial design as they relate to current and future development of potential new materials.

12.7

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13 Sulphonated biomaterials as glycosaminoglycan mimics in wound healing B. J. T I G H E and A. M A N N, Aston University, UK

Abstract: This chapter considers the available evidence and underlying physicochemical principles that support the proposition that a biomimetic wound dressing based on glycosaminoglycan models offers a potential means of influencing wound bioactivity. Available evidence showing advantages in wound healing for experimental proteoglycanbased dressing materials is described, together with an overview of the bioactive role of sulphated macromolecules. This leads to an assessment of the analogies between the sulphonate group and the sulphate group and an explanation of their unique water binding behaviour. The available information suggests the desirability of an integrated physicochemical, biochemical and biological approach to the design and synthesis of new wound healing biomaterials. Key words: biomimesis, sulphate, sulphonate, hydrogel, proteoglycan, interfacial tension.

13.1

Introduction

13.1.1 Biomimetic biomaterials: current thinking There has been growing interest in recent years in the concept that synthetic polymers or polymers that have been assembled from fragments of natural polymers can have biological responsiveness. The general principle has been endorsed by several prominent scientists. Around nine years ago Tirrell wrote ‘Materials employed in biomedical technology are increasingly being designed to have specific, desirable biological interactions with their surroundings, rather than the older common practice of trying to adapt traditional materials to biomedical applications’ (Tirrell et al., 2002). This view has been progressively expanded by others who have reviewed developments in the emerging field of biomimetic polymeric biomaterials, which signal to cells via biologically active entities (Drotleff et al., 2004). This approach is increasingly employed in the field of tissue engineering where, as Robert Langer, one of the pioneers in this area has written ‘The approach does not seek to reproduce all the complexities involved in development, but rather seeks to promote an environment which permits the native capacity of cells to integrate, differentiate, and develop new tissues’ 321 © Woodhead Publishing Limited, 2011

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(Lavik and Langer, 2004). It is on this basis that we should examine the underlying principles that support the initial evidence of promise associated with sulphonate-based wound dressings. First of all we should acknowledge the conceptual similarity between tissue engineering and tissue regeneration that biomimetic wound dressings would seek to achieve. Within the tissue engineering field we see clear evidence that the idea of using proteoglycan fragments to enhance cell responsiveness of membranes is well recognised. Poly (vinyl alcohol) (PVA)-chondroitin sulphate (CS) hydrogels cross-linked with glutaraldehyde have been shown to combine the advantages of both materials as scaffolds in tissue engineering applications. They promote both cell attachment and cell growth. Similarly, bilayer gelatin-chondroitin sulphate-hyaluronic acid membranes have been prepared with the specific intention of producing a material that would mimic skin composition and create an appropriate microenvironment for cell proliferation, differentiation, and migration (Wang et al., 2006). This was able to produce a high degree of differentiation between the upper and lower layers. When the upper layer was seeded with keratinocytes it developed into an epidermis-like structure, while the lower part, which was seeded with dermal fibroblasts, developed into a dermis-like structure. Importantly, it was shown that keratinocytes maintain their phenotype, and stratified epidermis layers were formed within three weeks. The use of proteoglycan fragments to produce a biological dermal analogue and epidermal structure is impressive and suggests that similar approaches might be successful in wound healing. Evidence for this is not difficult to find.

13.1.2 Biomimetic biomaterials: proteoglycan as models in wound healing applications There are several reports going back to the 1990s of proteoglycan fragments being incorporated into hydrogel wound treatment structures accompanied by evidence of enhanced epithelialisation. The most significant recent studies appear to be those of Prestwich. He has aimed to produce simple and effective biocompatible materials that mimic the natural extracellular matrix (ECM). These were not directed solely, or even mainly at wound care but rather for a variety of uses in regenerative medicine. These synthetic ECMs were designed to encompass the minimal composition required to obtain functional ECMs. Three natural ECM macromonomeric building blocks were employed: hyaluronic acid, chondroitin sulphate and gelatin. The compounds were chemically modified to give crosslinking sites suitable for use with polyethylene glycol diacrylate. The aim was to combine biologically appropriate mechanical properties with bioerosion rates to meet the requirements of a given clinical application.

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The significant results in the context of this discussion were those concerned with wound healing. The balance of properties led to the expectation that a hyaluronic acid-based gel would be most suitable – a chondroitin sulphate dressing and a conventional dressing were used as controls. The studies involved trials with mice and showed markedly more re-epithelialisation for the proteoglycan-based dressing materials. The researchers reported that the biggest surprise of the trials was the efficacy of the chondroitin sulphate hydrogel, which was described as having been used as a placebo, but performed significantly better than non-sulphate containing proteoglycan equivalents. They used the phrase: ‘We jump-started the healing process’ (Prestwich, 2002; Gilbert et al., 2004; Kirker et al., 2004; Shu et al., 2004; Shu et al., 2006). There are, similarly, several accounts of synthetic sulphonate-based wound dressing materials. Not all report clinical results but those that do inevitably report success. Additionally there are several interesting reports of sulphonated materials that are claimed to inhibit protease activity. Three are chosen for inclusion here. The first involves low molecular weight, sulphated polysaccharide derivatives from native marine polysaccharides prepared by radical depolymerisation and sulphonation of the products. These sulphonated products have been shown to promote reconstruction and remodelling of injured tissues, inhibit secretion of proinflammatory cytokines (especially interleukin-1 beta and tissue necrosis factor alpha) and matrix metalloproteases (MMPs) (especially gelatinase A and stromelysin 1) by fibroblasts and to promote selective cell proliferation (Karim, 2005). The second is an active wound dressing material in which discrete particles of a strong cation exchange resin, such as sulphopropyl dextrans and sulphopropyl agaroses, are dispersed in a liquid-permeable matrix. The strong cation exchange materials are claimed to selectively remove elastase from wound fluid (Essler and Nisbet, 2007). The third describes low molecular weight, non-peptide inhibitors of matrix metalloproteinases for the treatment inter alia of abnormal wound healing. The interesting point is that the inhibitors are exclusively sulphur-containing compounds described as N-hydroxy-2- (alkyl, aryl, or heteroaryl sulphanyl, sulphinyl, or sulphonyl)3-substituted alkyl, aryl or heteroarylamides. This description does not specifically include the sulphonated monomers employed in 2-(acrylamido)2-methyl propane sulphonates (AMPS)-based technology, but the structural similarity is such that they would be expected to show equal efficacy (Venkatesan and Baker, 2002). Taking these three (and they are only representative) reports together we begin to see that there is compelling circumstantial evidence to suggest that a high water content hydrogel based substantially exclusively on sulphonated monomers could reasonably be expected to show some degree of matrix metalloprotease inhibition.

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This expectation receives further support from the extensive work of Vachon using sulphonated polystyrene as a wound dressing material. In a series of patents assigned to Aegis Biosciences, he describes the use of sulphonated styrene copolymer compositions in wound dressings, claiming their usefulness for inhibiting elastase and collagenase and for promoting angiogenesis in a wound (Vachon and Wnek, 2004; Vachon, 2006). In a subsequent publication he describes the applicability of his invention (Vachon and Yager, 2006). The material is based on a sulphonated triblock polymer and can be prepared with a range of sulphonation levels and thus hydrophilicities. It appears to be used most effectively as a fabric-supported dressing, which thus ensures good mechanical integrity of the hydrated materials. The dressings showed a significant (formulation-dependent) ability to sequester elastase and MMP-8, which was superior to a commercial dressing which claims to have protease-inhibiting properties. Given the description of the polymer and substrate, the logical conclusion is that the protease-inhibiting capability is related to the presence of the sulphonated polystyrene moieties and that the capability is related to the amount of sulphonated styrene available at the wound interface. There are interesting signs of related activity in which hydrogels based on aliphatic sulphonates, such as sulphopropyl acrylates or 2-(acrylamido)2-methyl propane sulphonates, are exploited in wound dressing materials. There is relatively little in the open literature apart from reports of materials primarily designed for use as burn dressings. Although this work is relevant, the published results do not directly illuminate the question of sulphonate-based efficacy in wound healing (Peplow, 2005; Nalampang et al., 2006; Nalampang et al., 2007). In contrast, the patent literature does exemplify the use in wound healing of sulphonate-based hydrogels – again based on sulphopropyl acrylates or 2-(acrylamido)-2-methyl propane sulphonates (Munro, 2010a; Munro, 2010b; Munro and Andrews, 2010a; Munro and Andrews, 2010b). It is important to consider the extent to which the use of sulphonate groups in polymers, particularly in gel structures that mimic the water levels in natural tissue, might legitimately be considered a biomimetic approach.

13.2

Polymers and biomimesis

13.2.1 Structural considerations The unique properties that polymers possess arise from the ability of carbon (C) atoms to link together to form stable bonds with four other atoms either of its own kind or alternatively atoms of, for example, hydrogen (H), oxygen (O), nitrogen (N), sulphur (S) or chlorine (Cl). Most of the polymers that we encounter are based on the extension of this principle to long chain

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structures in which the number of enchained backbone atoms, most commonly C, O and N, can readily exceed 10,000. These polymers may be purely natural (such as cellulose), modified natural polymers (such as cellulose acetate) or completely synthetic – such as poly(methyl methacrylate). The single characteristic that unites both natural and synthetic polymers is the fact that, as the name (poly-mer) suggests, they are composed of many units linked together in long chains. It is the gigantic length of polymers (sometimes called macromolecules) in relation to their cross-sectional diameter that gives them their unique properties, such as toughness and elasticity. The way in which individual polymer chains are arranged in space – sometimes called their tertiary structure – has a significant effect on their properties. These arrangements in space, or conformations, include random coils, helices, folded chains and extended chains. As a general rule, natural polymers are arranged much more precisely than synthetic polymers, both in respect of the arrangement of chemical groups along the chain (secondary structure) and the arrangement of chains in space (tertiary structure). These aspects of secondary and tertiary structure are important in underpinning the principles of biomimesis as they affect polymers.

13.2.2 Monomer structures The individual building blocks from which polymers are formed are termed ‘monomers’. To indicate that a polymer contains more than one type of repeating monomer unit, for example when two different monomers are polymerised together, the description ‘copolymer’ is used. ‘Copolymer’ is a general term and can be used to describe polymers obtained from mixtures of more than two monomers. The great majority of synthetic polymers used interfacially in wound healing (Chapter 11) are formed from monomers that are characterised by the presence of a carbon-to-carbon double bond that opens to form a linked chain. The process can be generalised as shown in Fig. 13.1 for the case of methyl methacrylate. It is the way in which the structural and functional groups attached to the carbon-carbon double bond interact with each other and with their surrounding environment that governs the resultant properties of the polymer. It is the unusual properties of sulphonates in this respect that underpins the basis of this chapter. Perhaps the best way of visualising the way in which polymer chains arrange themselves is by taking several pieces of string to represent individual molecules. The most usual arrangement will be a random one in which the pieces of string are loosely entangled rather than being extended. It is the interaction and entanglement of the individual molecules in this way that gives polymers their characteristic physical properties. By changing the chemical nature of the polymer chain and their arrangement together we can change the physical properties and thus obtain either hard glassy

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CH3

H C

C

H

CH3 C ]n

[ CH2 C

O

C

O

O

O CH3

CH3

methyl methacrylate

poly(methyl methacrylate)

13.1 Methyl methacrylate polymerisation.

behaviour or, at the other extreme, flexible, elastomeric behaviour. The best example of a hard glassy material is poly(methyl methacrylate) which is formed from methyl methacrylate monomer units (Fig. 13.1). There is an important way in which a hard glassy polymer can be converted into a flexible material and that is by the incorporation of a ‘plasticiser’. This is a mobile component, often an organic liquid having a high boiling point, that will act as an ‘internal lubricant’. Its presence separates the polymer chains and allows them to move more freely.

13.2.3 Functional groups, the importance of water and the unique position of hydrogels The principle that biomimetic polymers need to carry an array of functional groups to reflect the structures that they are designed to mimic is now commonplace but this was not always the case. To understand this we need to turn to the invention of poly (2-hydroxyethyl methacrylate), or polyHEMA, which was developed by Otto Wichterle and his co-workers in Czechoslovakia (Wichterle and Lim, 1960; Wichterle and Lim, 1961). PolyHEMA is in many ways typical of other hydrogels and is a logical place to start, as it was the first biomimetic biomaterial to be developed. Biomimesis is simply the principle of examining the natural structure and using it as a model or template to mimic the function of the component being replaced. Otto Wichterle enunciated the principle in connection with ophthalmic biomaterials. He criticised the idea of using metals in the eye, simply because they were inert. He proposed using slightly cross-linked hydrophilic threedimensional polymers, which would be water-swellable and match the mechanical properties of the eye, allow diffusion of metabolites and oxygen like natural tissue and have better compatibility than metal with the host structure. The concept of biomimesis can be taken to a much more detailed level but in order to do that the question of the structure and function of hydrophilic polymers must first be explored. The structure of poly (methyl methacrylate) shown in Fig. 13.1 can be made more hydrophilic by the incorporation of hydroxyl groups, because groups of this type have an affin-

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CH3 CH2

C C

O

O CH2 CH2 OH

13.2 2-Hydroxyethyl methacrylate (HEMA).

ity for water. The simplest methacrylate structure that can be made in this way is poly (2-hydroxyethyl methacrylate), or polyHEMA, which is obtained by polymerising 2-hydroxyethyl methacrylate (HEMA) monomer (Fig. 13.2). In the absence of water, polyHEMA is a hard glassy material which upon hydration is transformed into a soft hydrogel. It is simplest to regard hydrogels as ‘washing line’ polymers having a long (i.e. the ‘washing line’) backbone from which a variety of chemical groups may be suspended (the ‘washing’). The function of the chemical groups in hydrogels is primarily to attract and bind water within the structure. Greater physical stability is achieved by fastening the washing lines together at intervals by the use of cross-links. Cross-links are introduced by the use of cross-linking agents, which are simply monomers with two active carbon-carbon double bonds. Networks are never perfect and contain entanglements, chain loops and wasted chain ends. Two important aspects of hydrogel design are the control of network perfection by choice of cross-linking agent and polymerisation conditions, and the extension of the principle of hydroxyl group-based biomimicry to include other functional groups. Lightly cross-linked (ca 1%) polyHEMA hydrogel is a non-ionic hydrogel and only moderately hydrophilic. It will swell to an equilibrium point at which the swollen gel contains approximately 38% by weight of water. This is referred to as an equilibrium water content. The value of this equilibrium water content can be reduced by copolymerising with a hydrophobic monomer (such as methyl methacrylate) or increased by copolymerising with more hydrophilic monomers (such as N-vinyl pyrrolidone or methacrylic acid). N-vinyl pyrrolidone is a cyclic amide or lactam and is another neutral group. Methacrylic acid (Fig. 13.3) has one important new feature – it is an ionic monomer and will thus be more sensitive to pH. Another group of ionic monomers that is particularly important in this discussion is the

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CH2

Me | C | C O | OH

13.3 Methacrylic acid.

O SO3−K+

O

13.4 Acrylic acid bis-(3-sulphopropyl)-ester potassium salt.

+

Na−O

O S

O NH O

13.5 Sodium 2-acrylamido 2-methly propane sulphonic acid (NaAMPs).

sulphonate family. Two examples are particularly important, they are the potassium salt of sulphopropyl acrylate (Fig. 13.4) and the sodium salt of 2-(acrylamide)-2-methyl propane sulphonate (Fig. 13.5). At physiological pH carboxyl groups and sulphonate groups will be fully ionised and we can express the relative hydrophilicities of the hydrophilic groups considered so far as: sulphonate > > carboxylate > amide > hydroxyl. If we return to the ‘washing line’ analogy for polymers, these chemical groups will all act as the ‘washing’. In hydrogels the function of the pendant groups is primarily to attract and bind water within the structure. The extent of this water-binding ability controls the swelling characteristics and the water content, which is the single most important property of a hydrogel. Pendant functional groups (the washing) in addition to mimicking the water-binding behaviour of functional groups on natural polymers, can also in some circumstances mimic the function. This is a developing aspect of biomimetic behaviour and one that is extremely significant for sulphonate monomers which, as later discussion will show, can act as mimics of the sulphate motif on proteoglycan molecules.

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13.2.4 Shortcomings of conventional hydrogels Although conventional synthetic hydrogels have proved to be very effective biomimetic soft tissue biomaterials for a range of applications, they fail to match the behaviour of natural tissue in several respects. Two particularly relevant aspects in the present context are the water retention and wetting capabilities, which are closely related. Differences in the ability of natural and synthetic hydrogels to retain water when compromised are illustrated most readily by placing samples in an environment of reduced humidity and monitoring water loss. Whereas natural tissue shows a remarkable ability to retain its water content, conventional synthetic hydrogels lose water relatively rapidly. The remarkable exception to this generalisation is found in synthetic hydrogels that contain ionic sulphonate groups, which show an ability to both retain water and to regain it at relative humidities above approximately 55%. For most biomedical applications hydrogels need to show dimensional stability to changes in pH, osmolarity and temperature. The success of polyHEMA was in large part due to the fact that it behaves extremely well in these respects. Sulphonate-containing hydrogels (and to a much less extent, carboxyl-containing hydrogels) are responsive to environmental changes which renders them unsuitable – or at best difficult to manage – for most tissue replacement or tissue augmentation applications. Natural tissue contains both ionic carboxyl and ionic sulphate (a close relative of sulphonate) groups. Because all natural structures are composite materials it is not surprising that simple homogeneous synthetic hydrogels, which for the most part have isotropic properties, cannot produce equivalent properties by simply attaching an equivalent array of functional groups to a carbon backbone. It is necessary to examine how and where ionic groups are used in nature and to select applications where responsiveness, rather than inertness is required. Properties that are closely related to the structural aspects of water binding are wetting, surface hysteresis and interfacial tension that were considered in Chapter 11. They provide another key to the position of sulphate/sulphonate groups in biomimetic polymer design. An important aspect of that discussion is the fact that high surface energy differences between contacting surfaces are thermodynamically and biologically unstable (Chapter 11). The interfacial tension between water and hydrocarbon groups is around 50 mNm−1, which is a very significant energetic driver, given that the surface tension of water itself is not dramatically higher (72.8 mNm−1) and the interfacial tension between hydrocarbon groups and air is around 20 mNm−1. The consequence is that conventional hydrogels such as polyHEMA undergo chain rotation when exposed to air, thereby exposing the hydrocarbon groups and reducing interfacial tension.

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Effectively, the water binding properties of the pendant hydroxyalkyl group are inadequate to shield the hydrocarbon backbone with its pendant methyl group. Similarly, in contact with a biological fluid there is an energetic drive for molecules with hydrophobic domains to seek the hydrophobic domains in the polyHEMA chains. This is the same thermodynamic driving force that leads to marine fouling, spoilation of contact lens materials by tear components and the inability to successfully fabricate narrow diameter blood vessels. It also governs the interaction of the wound dressing at the wound interface. Although conventional hydrogels, typified by polyHEMA, show huge advantages over less hydrophilic polymers, they are unable to resist water loss, chain rotation and biological spoilation in the same way as do natural soft tissue surfaces. The reasons for this are explored in the next section.

13.3

Biomimetic models

13.3.1 Characteristics of some natural systems In order to further explore the model underpinning the concept of sulphonate-based technology it is necessary to examine the structural components and their arrangement in extracellular matrix. The extracellular matrix (ECM) is a complex structural entity surrounding and supporting cells that are found within mammalian tissues and it is often referred to as the connective tissue. It is composed of three major classes of biomolecules: • • •

structural proteins: collagen and elastin specialised proteins: e.g. fibrillin, fibronectin and laminin proteoglycans: which are composed of a protein with long chains of repeating disaccharide units termed glycosaminoglycans (GAGs).

In order to understand the way in which water binding takes place in the ECM it is useful to consider three key natural tissues in which hydration control is vitally important, but in different ways: cornea, cartilage and intervertebral disc.

13.3.2 Cornea Of all tissue surfaces it is the cornea that demonstrates the ability of natural surfaces to maintain hydration and wettability at the air interface and therefore under repeated dehydration stress. Although it is covered by the tear film, this breaks up at regular (ca 10–15 second) intervals. Despite this, the cornea does not show the tendency of conventional synthetic hydrogels to undergo surface hysteresis or dehydration. Consequently, unlike current synthetic hydrogel contact lens materials, the natural corneal surface does

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not promote the deposition of denatured proteins. The point has been made in Chapter 11 that the corneal surface has many similarities to, and provides a useful model for, the chronic wound. For all these reasons it is important to understand the structural make-up of the corneal surface and its intimate relationship with the tear layer. The cornea is an avascular disc-shaped ocular tissue located in front of the iris and the lens, which acts as a protective barrier. It is a natural superefficient hydrogel capable of retaining high hydration levels while possessing durability and excellent mechanical properties – especially considering that its bulk equilibrium water content is of the order of 80%. No current synthetic hydrogel can compete with tissues such as cornea, articular cartilage or intervertebral disc – all of which are sophisticated composite materials. It is with the surface characteristics of the cornea, however, that we are particularly concerned. Similar to the scleral epithelium (the adjacent tissue surface), the corneal epithelium has two main functions as a semi-permeable layer. It acts as a barrier protecting the integrity of the cornea itself and it also allows nutrients and oxygen to pass through to the cornea. It is 20–50 μm thick consisting of 5–6 layers of cells. The superficial cells at the top layer of the epithelium comprise microvilli at their interface with the mucus layer of the tear film. The mucus layer is itself associated the corneal glycocalyx. The whole arrangement, together with an indication of the structural chemistry of the surface is shown in Fig. 13.6. The outmost layer of the corneal and conjunctival epithelium is intimately interfaced with the tear film. It is composed of microvilli coated with a matrix of sulphated glycosaminoglycans and sugars, called the glycocalyx. This interface is considered to be fully part of the tear film; however, with its strategic location, the glycocalyx coat acts as an adhesive for the spreading of the tear film onto the epithelium (Gibson, 2004). The characteristics of the sulphated glycosaminoglycans and the glycocalyx are critical to the biomimetic interpretation of any suphonate-based philosophy and are developed in the next sections. It is also relevant to consider in passing the structure and role of the mucins, since these are not present exclusively in the ocular system but are widely distributed throughout the body. They are aggregates of proteins densely glycosylated mostly with sulphated glycoaminoglycans and mainly through non-covalent bonding. Those features, which again help to understand the biomimetic basis of the sulphonate-based philosophy, are responsible for the established water retention and water-binding properties of mucins in the body. They are the largest molecules present in the tear film with molecular weights in the order of 1000–2000 kDa. Mucus is found on most epithelial surfaces, where it has both physical and biochemical roles. It serves as a protective barrier against contact with

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aqueous layer

mucin layer corneal and conjunctival epithelium

glycolipid

glycoprotein glycocalyx

lipid bilayer

13.6 Schematic representation of the structure of the tear film with the magnification of the interface between the epithelium and the mucus layer.

toxic substances and acts as a lubricant to minimise shear stresses, for example. Additionally, given that most cellular responses to extracellular matrix require engagement of cell surface receptors, factors such as mucins that interfere with cell surface receptor interactions, also play key roles in regulating these events. Mucins also play complex roles since they not only limit certain cell-cell, cell-ECM interactions, but can also bind to other cell surface receptors, e.g. selectins, or in the case of transmembrane mucins, interact directly with intracellular signal transducers. As is the case for cell surface receptors, transmembrane mucins may be released by the action of cell surface proteases allowing for rapid modulation of mucin-dependent events. Like mucins, proteoglycans are spread throughout the body. They have a hugely important physicochemical role in the maintenance of hydration

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Chondroitin sulphate OH −

GlcUA

O

O−

H

H2C H O

CH O HO

GaINAc-6S

O3S-O

H

OH O H

HO H

OH

H

HN

H

H

H

H

C CH2

O Keratan sulphate OH −

GIcNA-6S

O-SO3

Gal

HO H2C H O

H H2C H O HO

H

OH O H

HO H

HN

H

H

H

H

H

C O

OH

CH2

13.7 Repeating sulphated glycosaminoglycan units of chondroitin sulphate and keratan sulphate (modified from Bayraktar et al., 2004). (Gal, galactose; GalNAc-6s, N-acetylgalactosamine 6-sulphate; GlcUA, D-glucuronic acid; GlcNAc-6s, N-acetylglucosamine 6-sulphate.)

levels. They are highly negatively charged due to the presence of sulphate and carboxyl groups on many of the sugar residues (Fig. 13.7). Proteoglycans are macromolecules composed of a core protein covalently linked to sulphated glycosaminoglycans GAGs (i.e., chondroitin sulphate, dermatan sulphate, heparan sulphate, heparin, keratan sulphate). These proteoglycan repeating units are linked into larger structures as illustrated in Fig. 13.8. The chemical structure of proteoglycans gives some clues about their hydrophilicity. The molecules are made of sugar chains with hydrophilic groups positioned at the periphery, showing clearly the significance of hydrophilic groups, and more importantly sulphate groups. Their brush-like structures enable the macromolecule to entrap and retain water more than other hydrophilic molecules found in the body. These characteristics occupied a centre place in the concept of the sulphonate-based platform.

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CH2OH O

CH2OSO3−

CH2OSO3− CH2OSO3−

O

O O

O

O O

O

CH2OH O

CH2OH O O

O

Fuc O CH3

CH2OH O O

Gal CH2OH O

HN-Ac

HN-Ac

GlcNAc6S

Gal6S GlcNAc6S

HN-Ac Gal

HN-Ac

HN-Ac

O

GlcNAc

CH2OSO3− CH2OSO3− CH2OSO3− CH2OH CH2OH O O O O O O O O O O

O CH2OH O

CH

Man

O

CH2OH O O

HN-Ac

O

Man

CH2 NH O H-C-CH -CH 2 H O C=O NH HN-Ac HN-Ac GlcNAc GlcNAc Asp O

core protein

linkage region

Dermatan sulphate proteoglycan SO3−CH2OH O O COO− O

O

SO3−CH2OH

O O − O COO O

HN-Ac IduA

GalNAc4S

O

− COO− SO3 CH2OH COO− CH2OH CH2OH O O O O O O O O O O O O

HN-Ac IduA

GalNAc4S

NH O

HN-Ac GluA

GalNAc4S

GluA

Gal

Gal

Xyl

O-CH-CH C=O NH Ser

core protein

linkage region

13.8 Representation of the molecular structure of keratan sulphate proteoglycan and dermatan sulphate proteoglycan (modified from Michelacci, 2003).

13.3.3 Articular cartilage and intervertebral disc The hydration of disc tissue and cartilage is compromised by their local environment, as is the hydration of cornea. In these cases it is compressive force rather than atmospheric exposure that is the causative factor. As a result the swelling pressure of the tissue, moderated by the retractive force of collagen fibrils, must be able to resist the compressive force of bodily weight. Exactly similar structural motifs are found in the hydrating structures arranged in structures of which aggrecan is typical. Figure 13.9 shows the arrangement of the three major molecular classes within extracellular matrix. The structural proteins, collagen and elastin are primarily concerned with structural stability whereas the specialised proteins such as fibronectin and laminin have a range of complex biological roles. The proteoglycans which, as we have seen, are composed of long chains of repeating disaccharide units attached to a protein core, have the primary role of hydration control. It is universally accepted that the primary drivers of hydration are the ionic carboxylate and sulphate groups – of which the sulphate group is a hugely more effective water-binding group. It is the recognition and exploitation of this fact that is the key to sulphonate-based technology.

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extracellular fluid proteoglycan collagen

adhesion molecule plasma membrane lipid bilayer protein cytoplasm microfilaments cholesterol

13.9 Schematic representation of the arrangement of the key components of extracellular matrix.

13.4

Sulphonated biomaterials in the context of biomimetic principles

The sulphonate-based platform is based on the use of a biomimetic gel that aims specifically to mimic the sulphate groups, which are essentially the hydration engine of proteoglycans. Proteoglycans, as we have seen, are the common feature employed throughout the body to maintain high levels of hydration. The biomimetic principle of using sulphonated proteoglycans as a model for hydrogel design is not unique to AMPS. Other examples will be described in a subsequent section. The sulphonate-based technology is unique in combining proteoglycan biomimetics with an effective and versatile manufacturing platform to produce an effectively designed product which has been applied with remarkable early success to chronic wound healing. All biomimetic approaches to proteoglycan chemistry to date have used the sulphonate group as a surrogate for the naturally occurring sulphate. AMPS products use two precursor monomers the sodium salt of 2-acrylamido 2-methylpropane sulphonic acid (NaAMPS) and the potassium salt of 2-sulphopropyl acrylate (KSPA) which are shown in Figs 13.4 and 13.5.

13.4.1 Structural, biological and toxicological considerations The questions that are usefully addressed at this point are these: is the sulphonate group a reasonable surrogate for the sulphate group, are there potential toxicity-related issues with the sulphonate group and what are the stereochemical implications of using a sulphonate group attached to a

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carbon-backbone chain in place of the sulphated sugar rings in proteoglycan species such as chondroitin sulphate? The molecular structures of alkyl sulphate and alkyl sulphonate are very alike – both have very much larger hydration shells than either hydroxyl or carboxyl groups, which means that they are able to more effectively ‘shield’ the hydrocarbon backbone. Indeed, the hydration shell of the sulphonate headgroup has been shown to extend over a larger portion of the hydrocarbon chain than that of the sulphate in simple alkyl derivatives. This point, together with other aspects of the similarities and differences of sulphates and sulphonates will be discussed in this section. Additionally, aspects of the stereochemistry of the proteoglycans in comparison with that of simple AMPS and SPA-based polymers will be highlighted. We are fortunate that various aspects of sulphate and sulphonate chemistry in compounds that come into bodily contact have been explored over many years. This is because of their use in the surfactant field. Indeed, the ability of these groups to completely solubilise hydrocarbons in water beyond C10 contrasts with the fact that the hydroxyl group cannot completely solubilise more than a C3 chain. In the context of the previous discussion of interfacial tension this is a very significant fact. The first surfactants to be developed in the late 1800s were the sulphates and sulphonates of vegetable oils, such as castor oil – made by reaction with sulphuric acid. Later, there was a move away from natural oils and fats to the sulphonation of petroleum products. The production of alkyl benzene sulphonates for example, was brought about by nucleophilic substitution in the benzene ring using oleum (H2S2O7) or sulphur trioxide. This has been widely used in contact with skin since the 1950s where the soap in the original formulation of Lever Brothers’ Persil (PERborate+SILicate+soap) was replaced by alkyl benzene sulphonates. The absence of major toxicity issues suggests that there is nothing to fear from the use of sulphonates per se in skin contact products. Because of the commercial importance of this area there have been many synthetic innovations in an attempt to establish superior performance or intellectual property positions. Much of this history is not directly relevant to this discussion but two or three brief statements will be useful. Innovation was not confined to the sulphonation of different oils and hydrocarbon feedstocks, but was soon accompanied by ethoxylation, in which a few or many ethylene oxide molecules react with a fatty alcohol – which may be synthetic or plant derived – to make the surfactant molecule. Thus, alcohol ethoxylates, alkyl phenol ethoxylates and more relevantly alcohol ether sulphates became available. There has therefore been a long history of the use of both the alcohol sulphates and the alkyl aryl sulphonates. The alkyl aryl sulphonates were used much more widely than alcohol sulphates for most purposes with two

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exceptions. The first was Teepol, a secondary alcohol sulphate which has historically been widely used and the second was the shampoo field where the alcohol sulphates have been important. Alkyl benzene sulphonate held almost undisputed sway as the major ingredient used in washing operations until the early 1960s when the problem of its poor biodegradability caused problems, whereas fatty acid sulphates were found to degrade very easily. This was found to relate to the difference in the hydrocarbon portion of the surfactant and nothing to do with sulphate-sulphonate differences. In summary, there is a long history of use of both sulphates and sulphonates without evidence of toxicity-related problems associated with either group. Since this is a commercially driven field, greater versatility of synthesis and ease of production has led to a dominance of sulphonate-based over sulphate-based products. Similar considerations underpin the availability of sulphonate-based monomer precursors used in the sulphonatebased formulations. On the basis of the long history of surfactant use and performance it seems clear that there are huge practical advantages and no perceived disadvantages in using sulphonates as water binding groups in hydrogel-based proteoglycan mimics. We now need to examine more specific evidence relating to molecular interactions and biological effects of the two groups. The properties of alkyl sulphate and alkyl sulphonate are very similar, although various workers have identified specific differences in their interaction with specific reagents. One area of difference is in their interaction with cationic surfactants such as the alkyl quaternary ammonium bromides. It was found that, in contrast to the single surfactants, the sulphonate-cation mixtures were much more soluble than the corresponding mixtures. The results were interpreted in terms of the distinction between alkyl sulphate and alkyl sulphonate in the molecular charge distribution. This is consistent with the observation, mentioned previously, that the sulphonate hydration shield extends further along the hydrocarbon chain. Similar, although more extensive, studies have been carried out on sodium decyl sulphonate and sodium decyl sulphate aqueous solutions in the presence and in the absence of poly(vinylpyrrolidone). The results suggest a preferential interaction of poly(vinylpyrrolidone) with sodium decyl sulphate in comparison to that with sodium decyl sulphonate. The aqueous poly(vinylpyrrolidone)-sodium decyl sulphate system has been extensively studied in order to obtain details of the micellar aggregates bound on the polymer, including an estimation of the number of micelle like clusters per polymer molecule and the number of surfactant molecules present in these micelles. The evidence for complex formation in the case of sodium decyl sulphonate is much weaker (Shimabayashi et al., 2003). The fact that alkyl sulphates and sulphonates are generally so similar is underlined by the fact that workers have sought to demonstrate wherein

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they differ and only in exceptional and relatively minor aspects, such as the ones outlined above have they been able to do so. In biological systems it is their interaction with proteins that are of much greater significance and both sulphates and sulphonates have been shown to have excellent binding and stability characteristics in protein chemistry. Here again the picture is one of overall similarity with observable differences in structural dependencies. An example of this is their use in conjunction with bovine serum albumin which has a total of ten binding sites to alkyl sulphates and sulphonates. The number of sites increases with hydrocarbon tail length, and while the interaction energy is not a function of chain length in the case of sulphates, it becomes chain length dependent in the case of sulphonates. Binding affinities are also dependent on the character of the polar portion of the ligand:sulphate and sulphonate groups. The overall picture is again consistent with the rather more extensive hydration shield associated with the sulphonate head group (Steinhart and Reynolds, 1969). Broader biological studies, including consideration of acute toxicity and genotoxicity have been carried out by various workers. The consensus picture emphasises the similarity of sulphonate and sulphate behaviour and removes any concern relating to toxicity and related problems associated with sulphonates and sulphates per se. It was found that interactions in biological systems had much in common with the in vitro studies of surfactant-protein complexes. The effects of sulphonate-type surfactants were attributed to the formation of complexes with the protein of (e.g.) skin and hair – an observation that extended to fish studies where interaction with gills was found to be the significant factor. In general adsorption of surfactant increased with time and was less biologically effective when protein was added. Studies with sulphonates and sulphates involving a wider range of aquatic organisms found no evidence of genotoxicity at a concentration 1000 mg l−1 and, interestingly, showed toxicity levels that were much lower than those of comparable nonionic surfactants (which gave LC50 values between 1 and 10 mg l−1 or in a few cases LC50 values below 1 mg l−1). Perhaps the most significant point was that toxicity increased with increasing molecular weight – which indicates that the toxicity effects were not specifically associated with the sulphonate and sulphate headgroups (Tomiyama, 1975; Miksch et al., 2005).

13.4.2 Stereochemical considerations To consider the question of stereochemistry we need to imagine the structures presented in Figs 13.3 and 13.4 in three dimensions, with appropriate representation of the bonds in space. We can achieve this using the chondroitin sulphate structure as an example. The local stereochemistry of

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O

O

OH SO3H O

HO

O

HO O

HO

O O NH O

n=15 to150

13.10 Stereochemical disposition of ring substituents in chondroitin sulphate repeat units (dotted lines indicate groups disposed below the plane of the page, wedges indicate groups disposed above the plane of the page).

chondroitin sulphate keratin sulphate

core protein

hyaluronan molecule

aggregan aggregate

1 μm

13.11 Stereochemical disposition of chondroitin sulphate and related repeat units around a protein core.

typical individual sulphate-containing linked ring structures found in proteoglycans, and exemplified by chondroitin sulphate is shown in Fig. 13.10. We then need to expand the larger proteoglycan structure with its central protein core (Fig. 13.8) into a three-dimensional representation as shown in Fig. 13.11. Finally, considering the chondroitin sulphate strands in Fig. 13.11, it is useful to acknowledge the steric accessibility of sulphate groups along an extended chondroitin sulphate chain. The sulphate groups are easily identified because the sulphur atoms are larger than any others in the structure. Figure 13.10 illustrates that because the sulphate group is O-linked to the

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sugar ring, in stereochemical terms it is effectively a sulphonate group with a ‘spacer’ oxygen atom. Additionally, the sulphate group is accessible to any molecule small enough to pass between the chondroitin sulphate strands. Although there is no way in which a single-strand carbon backbone polymer derived from the sulphonate monomers shown in Figs 13.4 and 13.5 can match the elegant stereochemistry of the carbohydrate chain, the local stereochemistry of the O-linked sulphate groups is well matched by that of the C-linked sulphonate. That is because the disposition of the three available oxygen atoms around the central sulphur is substantially identical. That leaves the question of chain conformation – essentially coiled versus extended. Although the carbohydrate chain is more extended than that of neutral hydrophilic carbon-backbone polymers (such as polyvinyl pyrrolidone or polyvinyl alcohol), the charge repulsion of the sulphonate groups in AMPS and SPA-based polymers causes these chains to exist in a very extended conformation. In consequence, the steric availability of sulphonate groups along a polyAMPS chain would not be expected to be significantly less than the availability of sulphate groups along a strand of chondroitin sulphate. There is one other point to consider here. The carbon backbone in polyAMPS will readily undergo chain rotation. In a lightly cross-linked polymer individual polymer chains will have considerable mobility. There is good reason to believe that they will, in that sense, be able to mimic soluble sulphonate-containing proteoglycan polymer fragment (i.e. not attached to a central protein core). Obvious examples of such structures are heparin, excreted by mast cells and heparin sulphates located at the epithelial cell surface, and which for their physiological functions need considerable mobility. In summary, stereochemical considerations suggest that AMPS-based structures would be able to mimic sulphonated proteoglycans in many respects. These considerations cannot imply or forbid any particular aspect of biochemical effectiveness. That aspect of behaviour is considered in a subsequent section.

13.4.3 Sulphonated biomaterials at other body sites Since the biomimetic logic of using sulphonate-containing polymers as surrogates for proteoglycans is clear, and since proteoglycans have important functions at many body sites, it would be anticipated that the idea of employing either proteoglycan fragments or synthetic sulphonate-based proteoglycan analogues would not be unique to AMPS. An examination of the available data will provide a useful basis to assess available evidence relating to the validity of the sulphonate-based concept. This will logically lead to a discussion of the possible physicochemical and biochemical mechanisms that contribute to the reports of clinical performance of sulphonatebased wound dressings.

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Work with sulphonate polymers based on AMPS and SPA salts involving cell-substrate interactions, haemoperfusion devices, intervertebral disc, intraocular lenses and both cosmetic and therapeutic contact lenses began in our laboratories in the early 1980s and is contained in PhD theses and publications (Minett, 1986; Fleming, 1999; Benning, 2000; Mahomed, 2005; Boote, 2007; Bahia, 2008; Tighe et al., 2008; Tighe et al., 2009). We observed early on that these polymers produced high levels of fibroblast cell adhesion and growth at very high water contents. Our interest in the use of the unusual properties of sulphonates became focused on the idea of proteoglycan analogues in the late 1990s and resulted in two major lines of work related to the eye and the intervertebral disc. The analogies between cornea and chronic wound previously discussed, coupled with the parallel work carried out on corneal wound healing and in particular post-laser surgery, give the results of this work particular relevance to the use of sulphonates in chronic wound care. The balance of functional groups, hydroxyl, amido, carboxyl and sulphonate has enabled effective mimicry of the natural disc behaviour and the way in which this changes with age. Again, the results of this study provide strong support for the concept of using sulphonate-based hydrogels to produce responsive surfaces that will produce a beneficial biomimetic environment for chronic wounds. Additionally, work on haemoperfusion devices, involving both extracorporeal circulation devices with rats and in vitro studies of blood clotting using the Lee-White method showed that whereas high levels of carboxyl groups incorporated via acrylic acid produced rapid clotting, high levels of sulphonate groups yielded extended clotting times. Limited use could be made of these observations because of the responsiveness of sulphonates to changes in osmolarity which results in inadequate dimensional stability for the applications in question. Interestingly, the potential of using carboxyl groups in tandem with sulphonate groups to achieve special effects has been recognised by various workers. These range from patents covering the preparation of carboxylatesulphonate polymers having cell proliferation-promoting properties and the application of this to production of a biomimetic prosthetic ligament to the concept of manipulating antibacterial properties of hydrogels (Hill et al., 2002; Brulez, 2004). This latter patent, based on the work of Jozefowic, describes the use of a molar ratio of carboxylate groups to sulphonate groups of greater than two as a method of reducing adverse events with contact lenses by preventing microbial growth (Johnson & Johnson Vision Care, 2004).

13.5

Sulphonated biomaterials and the chronic wound: possible modes of biomimetic behaviour

Having examined the wide range of physicochemical and biochemical factors that need to be taken together in assessing the biomimetic potential

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of the sulphonate-based approach to wound healing, we are in a position to examine, in turn, the possible modes of action of this family of materials in comparison to conventional neutral (non-ionic) hydrogels. The physicochemical aspects of behaviour are inevitably easier to speak about with certainty than are the biochemical mechanisms. Careful consideration of the relevant literature does provide clear indications of sulphate/sulphonate-related phenomena that provide the basis for the logical expectation of biomimetic effects.

13.5.1 Physicochemical factors: interfacial tension and hydrophobic shielding The first and most logical expectation arises from the discussion of the relative hydration capability of hydroxyl, carboxyl and sulphonate groups and the consequent difference in hydrophobic shielding. The experience with these surfaces in ophthalmic biomaterials bears out the prediction that sulphonates will maintain a lower interfacial tension than either hydroxyl or carboxyl-based hydrogels at the biological interface. Because proteins adsorb so rapidly at polymer surfaces, the first advantage that sulphonatemodified hydrogel surfaces bring is a reduction in irreversible non-specific protein adsorption. With a wound healing material the disadvantageous consequence of this is not spoilation of the dressing surface but rather the irreversible removal of key proteins from the tissue interface. This point is dealt with under biochemical factors relating to the specific proteins. In summary, there is a large amount of direct evidence to support the expectation that sulphonated surfaces will show tangible benefits.

13.5.2 Physicochemical factors: osmotic drive and charge repulsion These factors have been studied in detail at two body sites: the eye and the intervertebral disc. In both cases the effects are dramatic. By balancing the fixed charge density of the polymer (essentially the concentration of sulphonate groups along the backbone) the swelling response and hydrostatic pressure can be matched to that of the natural disc. The fixed charge density is of similar importance when sulphonates are used on the surfaces of contact lenses. The front surface is in contact with a constantly replenished tear film and is much less sensitive to this factor than the back surface. Here, the posterior surface is exposed to a relatively small tear volume which is relatively stagnant. If the level of sulphonation is set at too high a level, the appetite of the sulphonate for hydration takes water away from the posterior tear film producing a hypertonic tear film and consequent discomfort at the ocular surface.

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Volume swell (V/Vo)

300

200 High dilution: charge repulsion and Osmotic effects balanced by retractive force of cross-links

100

0 0.00001

0.0001

0.001

Increasing salt concentration Screens Neg charge repulsion Osmotic effects more significant

0.01

0.1

Salt screens charge repulsion Hydrophilicity of monomers predominates

1

10

Ionic strength NaCl

13.12 Volume swell vs. ionic strength for a cross-linked AMPS-based hydrogel.

Figure 13.12 shows the effect of osmolartity on the volume swell of a copolymer with a 75% AMPS content. The diagram explains two things. The first is the magnitude of the swelling (water uptake) of an AMPS-based gel – which explains why they are so good at fluid handling in wound healing applications. The second is that as the osmolarity increases the balance of factors driving that swell changes and diminishes. Despite that, in the region where the various bodily fluids lie (within the decade from 0.1 to 1.0 but mainly around 0.3) the curve is quite steep and the polymer still takes up around 25 times its own weight of fluid. The logical expectation is that sulphonate groups will confer excellent fluid handling properties and that in contact with an exuding wound this will confer enormous advantages over a neutral hydrogel. Additionally, the swell caused by fluid uptake would be expected to yield two advantages in a gel with a high AMPS content. The first is that it will dilute the effective concentration of sulphonate groups to a level that more closely matches that of the exposed tissue (which in biomimetic terms would be the target to aim for). The second is that the consequently greater chain separation will allow greater mobility to the long sulphonated chain segments between cross-links, which in turn will allow the segments greater mobility to act as heparin mimics – a point that will be dealt with later in a biochemical

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context. It is equally logical to expect that a high AMPS-containing gel will continue to exert an osmotic and charge repulsive drive to remove fluid from a non-exuding wound, thus causing potential disturbance to the wound in its healing phase. This points to the obvious future need to explore the use of synthetic sulphonate-containing gels that match the effective sulphonate content and fixed charge density of the exposed tissue. Such a development would be consistent with the biomimetic principle that underpins the claimed usefulness of high-AMPS gels with exuding wounds (Munro, 2010a; Munro, 2010b; Munro and Andrews, 2010a; Munro and Andrews, 2010b).

13.5.3 Physicochemical factors: ionic equilibria and selectivity The previous two sections have not considered the fact that wound fluid contains a range of ionic and neutral components differing in size and charge. We therefore have a situation where there is competition amongst these species whenever there is a net movement of fluid into the gel. Water is the smallest and most mobile species and, regardless of other factors such as charge repulsion and ion-binding that may occur in a membrane system, the drive to osmotic neutrality will always mean that movement of water molecules is an inevitable priority. Because the gel behaves as a semi-permeable membrane that contains a fixed (anion) charge the Donnan principle of membrane equilibrium will apply. Consistent with that there will be a gradual move to equilibrate the concentration of mobile anions and cations across the interface; this is consistent with the principle of electrical neutrality and ability to permeate through the membrane barrier. Since the AMPS-based gel contains a high concentration of sodium and (in some forms at least) a measurable potassium concentration, the relative potentials of these across the interface will be smaller than would be the case for a neutral gel. Since the gel is permeable to all mobile ions in the system the relative diminution of ions such as calcium would therefore be predicted to be greater than that of sodium. It is important to raise a word of caution about predictions of fluid and ion behaviour. Donnan equilibria are just what the name implies – equilibrium situations. In contrast, an exuding wound cannot be regarded as being in a state of equilibrium. Despite this it is possible to make general statements about the driving forces involved and the permeability of the highly hydrated sulphonate membranes that are produced by AMPS-based dressings in contact with an exuding wound. The AMPS-based platform has an infinitely greater degree of structural manipulation capability in relation to the selective movement of ions than does any neutral hydrogel. In conjunction with the osmotic drive this is a powerful tool. At present we do not know what specific

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benefits in wound management might be associated with the alteration in the balance of specific ions at the wound surface, although there are general logical principles. Here again, the dynamic behaviour of current AMPSbased products at the interface with exuding wounds gives demonstrable benefits that are consistent with the expectations of wound management principles.

13.5.4 Biochemical factors: matrix metalloprotease absorption and inhibition As we have seen already, there are several claims from different sources that sulphonates and related compounds inhibit the activity of matrix metalloproteases (MMPs) (Karim, 2005; Essler and Nisbet, 2007; Venkatesan and Baker, 2002). The fact that this occurred by either surface contact with sulphonated polymers or interaction with soluble sulphur-containing compounds is even more intriguing. This evidence suggests that there is validity in the expectation that the AMPS-based matrix, into which proteins of the size of the MMPs can diffuse, will also be capable of inhibiting MMP activity. The chelating group of the synthetic inhibitor (sulphonate, hydroxamate, etc.) binds the catalytic Zn2+ ion, blocking the active site (Jani et al., 2005). Chelation sequesters the MMPs, removing the entire active MMP entity together with the associated zinc. The catalytic domains of most MMPs have a high degree of homology and therefore exhibit a broad spectrum inhibition profile. The first and most extensively studied MMP inhibitor is batimasat, a so-called peptide-mimetic inhibitor, a low molecular weight broad spectrum inhibitor with an hydroxamate group as a zinc chelator (Botos et al., 1996; Wang et al., 1994). Recent evidence has shown that heparin can bind zinc through complexation involving both carboxylate and sulphate oxygens (Whitfield and Sarkar, 2004). This may offer some explanation for the success of oxygenated sulphur compounds and polymers previously referred to. In the case of AMPS-based gels, an additional or alternative factor is the osmotic drive and Donnan effect, which will lead to the rapid reduction of those ions that are not present in the gel as presented to the wound. Limiting MMP proteolysis at a stage in the healing process where there is the potential shift to a non-healing wound from less synthesis to more degradation would clearly be favourable. Protease reduction would reduce inflammation and local pain in the wound. Rapid and sustained pain reduction, which appears to be a feature of the clinical use of AMPS-based gels can be explained, at least in part by inhibition and absorbtion of matrix metalloproteases.

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13.5.5 Biochemical factors: cell-surface effects and neutrophil function It is widely recognised that cellular interaction with an artificial surface can affect the cell response. One study of interest investigated the interaction of neutrophils on polyurethanes with different ionic groups (Sundaram et al., 1996). Sulphonate groups (anionic) and quarternary amine groups (cationic) were compared to the base polyurethane (which is commonly used for a variety of blood-contacting biomaterials). The results demonstrated that not only were the numbers of adherent neutrophils on the sulphonated polyurethane higher compared to the base polyurethane, but these cells exhibited greater surface spreading – which would suggest a ‘happier’ cell. In parallel, the workers assessed integrin expression, and in particular, Mac-1 expression which is involved in many of the key cellular function including adhesion, spreading, chemotaxis and phagocytosis. The results demonstrated a significantly higher expression of Mac-1 by the adherent cells on the sulphonated polyurethane compared to the other surfaces. This is particularly significant in the context of studies that have shown that patients with leucocyte adhesion deficiency (which involves deficient expression of three related leukocyte adhesion glycoproteins including Mac-1) are more susceptible to bacterial infection (Kuijpers et al., 1997; Sergio et al., 2004). It is reasonable to assume a material that enhances or maintains integrin expression is extremely advantageous and will allow natural neutrophil function to be maintained against the constraints of bacterial phagocytosis and normal cellular migration.

13.5.6 Biochemical factors: vitronectin/plasmin effects Vitronectin has an important pro-active role in modulating the conversion of plasminogen to plasmin. The plasminogen-plasmin activation process is kept in equilibrium via the anatagnostic activities of the plasminogen activator system and plasminogen activator inhibitor (PAI) system. The most significant plasminogen activator inhibitor in cellular systems is PAI-1 which binds to vitronectin, thus localising PAI-1 to the pericellular compartment where active plasminogen activator is generated (Vaheri et al., 2002). If vitronectin is localised on a surface adjacent to the cellular site (such as a chronic wound), it removes plasminogen activator inhibitor from the reaction by fixing it, creating an imbalance in favour of the plasminogen activator and a production of active plasmin. This results in local upregulation of plasmin formation. The phenomenon of plasmin upregulation has been known in relation to corneal wound healing and in conventional cosmetic lens wear for several years. (Chapter 12).

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Because vitronectin is a sticky protein, once adsorbed on the biomaterial it remains there. The adsorption is very material-dependent and is promoted by high interfacial tension and the presence of hydrophobic domains within the material. Sulphonated biomimetic materials minimise this irreversible deposition. Increase in vitronectin associated with the material increases the potential for plasmin upregulation in the fluid held between material and tissue surface. The analogies between the cornea and the chronic wound have been outlined in Chapter 12. We have carried out extensive studies in this area, including the effect of sulphated biomimetic hydrogels in comparison with both neutral and carboxyl-containing hydrogels. The conclusion in relation to hydrogel wound dressings seems clear and is supported by experience in studies of contact lens-cornea interactions. The presence of hydrophobic domains within neutral and carboxylcontaining hydrogels leads to irreversible deposition of vitronectin and its consequent depletion in the tissue bed. Since this depletes active PAI-1 there is a consequent upregulation of plasmin – and ultimately collagenase. In the eye this effect is minimised by sulphonated biomimetic materials – it is logical to expect the same to apply to hydrogel wound dressings.

13.5.7 Biochemical factors: kinin effects, GAGs and pain inhibition potential The kinins are a family of potent bioactive peptides which directly mediate inflammation and which advance to produce the end product bradykinin, a nonapeptide with many pro-inflammatory functions. The consequences of kinin activation, and bradykinin generation, include an increase in vascular permeability, vasodilation, pain stimulation, and smooth muscle contraction. Initiation of the kinin cascade is brought about by a variety of natural and synthetic surfaces. The central species in the kinin cascade is high molecular weight kininogen (HMWK) which is a useful marker for kinin activity, and can be assayed following extraction from the biomaterial surface. The kinin system has many pro-inflammatory functions. High molecular weight kininogen, a key protein in the mediation of inflammation, has been implicated in a variety of disorders and allergic responses at other mucosal body sites. Additionally, kinin activation can indirectly stimulate the synthesis of other potent inflammatory agents such as, prostaglandins, leukotrines, histamine and platelet activating factors. Bradykinin, which is the end product of the kinin cascade, has the ability to induce pain, increase vascular permeability and mediate other signs of inflammation. Recently the attachment of high molecular weight kininogens (HMWK) to endothelial cell surfaces by means of GAGs, specifically heparan sulphate and chondroitin sulphate has been demonstrated (Renné et al., 2000; Renné and Müller-Esterl, 2001). GAGs were shown to ‘protect’ the HMWK

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from protelytic cleavage and prevent, or at least reduce, bradykinin release into the local environment. The GAG moieties hold on to the HMWK molecule and somehow interfere with the hydrolysis of the kininogens by the serine protease kallikrein which cleaves domain 4 of HMWK in two places to release the pro-inflammatory peptide, bradykinin. It has been postulated that the GAGs may induce a conformational change on the kallikrein molecule, which may serve to reduce or prevent its interaction with kininogen, or the GAGs may interact directly with the HMWK and neutralise its positive charges in domain 5 and consequently impede its hydrolysis (Gozzo et al., 2002). The incidence of pain may not always be a consequence of the natural or excessive immune response but may be initiated by specific non-host factors. Kinin activation, i.e. bradykinin release, has been implicated in the spread of infection by certain pathogens. The activation of bradykinin receptors through the release of bradykinin by bacterial derived cysteine proteinases has been determined. A specific example was demonstrated by one laboratory where the action of a specific proteinase, cruizipain, released from the pathogen Trypanosoma cruzi was observed (Lima et al., 2002). It was able liberate bradykinin resulting in the activation of bradykinin receptors, which in turn facilitated the cellular invasion by this protozoan parasite. Although it is clearly not possible to speak with certainty about the mechanism of action that governs the behaviour of sulphonated hydrogels in relation to kinin activity at the wound interface, there are several pointers. Significantly, heparan sulphate has been implicated as the site of initial adhesion for this pathogen to host cell surfaces and the fact that kinin release by T. cruzi increased 10-fold in the presence of heparan sulphate. Given that the provision of the docking site for the invading pathogen requires a high degree of specificity, there is a strong probability that synthetic GAG analogues will not provide ‘docking site’ characteristics requited by the invading pathogen but will provide protective HMWK binding preventing or reducing the release of pain-inducing bradykinin. Bradykinin (the pain-inducer) formation requires the local contact of both kininogen (HMWK) and the enzyme kallikrein. We know from ocular studies that surface adsorbtion is material dependent – which leads to lower levels of both retained kininogen and retained kallikrein. There is also the possibility that these materials can act as heparan sulphate analogues so providing protective binding for such kininogen as is adsorbed.

13.5.8 Biochemical factors: heparin/heparin mimicry Several of the observations of the activity of sulphated and sulphonated species reported from the literature show activity that is similar to that

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Sulphonated biomaterials as glycosaminoglycan mimics CH2SO3−

COO−

CH2SO3−

OH

OH

OSO3−

O

OH

CH2SO3− COO− OH O

O

O NHSO3−

NHSO3−

349

OH O

OSO3−

NH2SO3−

13.13 Representative structure of the heparin sulphate chain.

associated with heparin or heparan fragments or immobilised macromolecules. Additionally recent work has indicated that the functions of these two species may not be as distinct as was previously thought. There is an obvious contrast in chain rigidity between conventional proteoglycans, such as chondroitin sulphate, and the chains of AMPS anion segments in an expanded and lightly cross-linked state. Add to that the chain ends, loops and possible extra-network material in an AMPS-based gel and we have a degree of mobility that might be regarded as similar to that of soluble heparin. The point at issue therefore is whether there can be a reasonable presumption of heparin-like activity in structures such as that presented by AMPS-based materials? The first point to make is that the higher degree of sulphation of heparin does correspond to the fixed charge density of the AMPS anion repeat unit more closely than do either chondroitin sulphate or keratin sulphate. This can be seen by simply inspecting the AMPS repeating unit (Fig. 13.5), together with those of keratin and chondroitin sulphate (Fig. 13.7) and comparing them with the heparin sulphate chain structure illustrated in Fig. 13.13. The simple disaccharide repeating unit of heparin is decorated with sulphate substituents in a complex manner producing high degrees of sulphation. Additionally, compared to other GAGs, heparin, and heparan sulphate show considerable conformational flexibility due to the presence of L-iduronic acid (IdoA) residues, which change easily between chair and skew conformations. Perhaps the most convincing (though circumstantial) evidence to support the hypothesis that AMPS-based gels have enough structural similarity to act as partial heparin mimics comes from an unlikely source: the study of Alzheimer’s disease. In order to understand the structural implications more fully it is necessary to comment on the structure of heparan sulphate, which as has been mentioned is closely related in its function to heparin sulphate. Both heparan sulphate and its structural analogue heparin sulphate are composed of disaccharide units of either a-L-iduronate or b-D-glucuronate alternating with either N-acetyl or N-sulphonamido

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Direct interaction with BACE1 APP

HS chains

BACE1 HSPG

lumen

cytosol

α

β

membrane

γ

13.14 Schematic ‘interference’ of heparan sulphate (HS) with the regulating action of β-site amyloid precursor protein cleaving enzyme (BACE1) on amyloid precursor protein (APP) (modified from Patey et al., 2005).

a-D-glucosamine and contain complex patterns of additional sulphation and N-acetylation. Their source and size are different, however. Heparan sulphate is found predominantly on the cell surface and as a constituent of the extracellular matrix, attached to membrane-bound core proteins as heparin sulphate proteoglycans. Heparin, however, is a soluble molecule of varying molecular weight (ca 5–40 kDa) typically produced by mast cells. Heparan sulphate is known to play a pivotal role in the formation of plaques that characterise Alzheimer’s disease and other amyloid diseases. These plaques are deposits of proteinaceous material that have a core of fibrils formed from aggregates of amyloid β-peptide – the term amyloid is now widely used to describe plaque formation. Heparan sulphate can promote amyloid β-peptide aggregation and is associated with plaque deposition, essentially through sequestration of amyloid precursor protein and restricting the activity of β-site amyloid precursor protein cleaving enzyme (Patey et al., 2005). This interaction is shown diagrammatically in Fig. 13.14. Several approaches to prevent heparan sulphate-induced aggregation of amyloid β-peptide have been explored. Since heparin and heparan sulphate share the same disaccharide chain constituents, the target molecules need to be soluble and adequate structural mimics of these units. A range of compounds has been examined including derivatives or fractions of heparin and other proteoglycans and various sulphated compounds that act as heparin/heparan mimics. Heparin mimics usually consist of a carbon backbone with sulphate groups. Two groups are of particular interest. Low molecular weight (300–

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1000) anionic sulphonates and sulphates were examined in vivo in a model of serum amyloid amyloidosis (Kisilevsky et al., 1995). Both sulphates and sulphonates acted as mimics of the heparin/heparan chain sequence. Longer chain highly sulphated molecules of pentosan polysulphate and dextran sulphate have also been examined. Again, both groups of compounds were shown to be able to compete with heparan sulphate for amyloid β-peptide binding in vitro (Leveugle et al., 1994). It appears that neither the chain length, nor the nature of the oxidised sulphur function (i.e. sulphate vs. sulphonate) were particularly important. The characteristic of importance from these and other studies appears to be the presence of an adequate concentration of sulphate/sulphonate group substitution within the molecule. Heparin has the highest sulphate charge density of any known biomolecule. The polyAMPS repeat unit has a calculated sulphonate charge density of 4.18 equivalents/kg whereas the heparin sequence shown in Fig. 13.13 has a calculated sulphate charge density of 3.88 equivalents/kg. In biomimetic terms, in the light of the evidence discussed, it is reasonable to suppose that polyAMPS segments will show at least some heparin-like characteristics.

13.6

Conclusions

There appears to be considerable logic and supporting evidence for the use of a biomimetic approach to tissue contact biomaterials based on the important role of sulphate/sulphonate chemistry. There are several quite specific observations relating to the use of sulphonates, including: • • •

chondroitin sulphate hydrogels that ‘jump-started the healing process’ sulphated marine polysaccharide derivatives that inhibit secretion of proinflammatory cytokines and matrix metalloproteases oxidised sulphur-containing inhibitors of matrix metalloproteinases (MMPs) used for the treatment of abnormal wound healing.

The questions of suphonate-sulphate comparability, the stereochemistry of proteoglycan sulphated chains and the long history of use of sulphonates may be drawn together with the observations of interesting and relevant biochemical effects of a range of sulphonated compounds. All the available data, taken together, provide reassurance on two points. The first is that there can be no expectation of adverse biological consequences as a result of the use of biomimetic sulphonate-based polymers in chronic wound care. The second is that the clinical observations relating to reduction in pain and inflammation and increase in the rate of wound healing are consistent with observations on other sulphonated systems. There are three distinct strands to the materials aspect as related to wound healing. These are the incorporation of proteoglycan fragments in

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the form of glycosaminoglycans, the use of sulphonated aromatic rings, and the formation of hydrogels – essentially direct analogues of Wichterle’s model polyHEMA structure but incorporating sulphonated acrylic or acrylamido monomers.

13.6.1 Products derived from natural proteoglycan fragments There is clear evidence from the work of Prestwich and others (Prestwich, 2002; Gilbert et al., 2004; Kirker et al., 2004; Shu et al., 2004; Shu et al., 2006) that incorporation of chondroitin sulphate fragments within a suitable matrix (gelatin and hyaluronic acid have proved to be successful matrix components) produces a dressing that is capable of ‘kick-starting the healing process’. The gels were designed to integrate with the wound by a resorption-type process. The factors that limit their effectiveness as a competing technology include: • • •

the high cost of natural proteoglycans such as chondroitin sulphate and hyaluran the lack of adequate stability or fluid handling characteristics for routine use the gel-formation is not susceptible to high throughput–low cost processing.

13.6.2 Products based on sulphonated polystyrene The use of styrene sulphonate technology in wound healing applications is best exemplified by the work of Vachon (Vachon and Wnek, 2004; Vachon, 2006; Vachon and Yager, 2006). In practical terms, however, the Vachon technology falls significantly short of the biomimetic aspirations that underpin the hydrogel-based approach and was not driven by any biomimetic consideration. In essence this is because it is based on styrene sulphonate block copolymers with hydrophobic non-sulphonated hydrocarbon polymers – such as polyethylene. This means that the materials contain very sizeable hydrophobic blocks, which will produce high interfacial tension with the wound bed and lead to irreversible non-specific protein adsorption. The advantage of such block copolymer backbones is that they confer toughness and since the sulphonation of the styrene segments is carried out on commercially available block copolymers they are reasonably cheap to produce. Processing of these materials in film or foam form presents significant problems and thus the preferred method of use is to coat a fabric with a solution of the sulphonated copolymer in a carrier solvent.

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13.6.3 Hydrogels based on sulphonated monomers This technology is based on sulphonated aliphatic monomers – typically, the sodium salt of 2-acrylamido 2,2 methylpropane sulphonic acid (NaAMPS) and the potassium salt of 2-sulphopropyl acrylate (KSPA). Clinical assessment of AMPS-based wound dressings is claimed to show that they possess several advantageous and desireable characteristics, including pain relief, reduction of inflammation, angiogenesis and acceleration of the healing process. This chapter has examined the biological aspects of wound healing, together with current thinking relating to biomaterials and biomimetics and the ways in which biochemical processes at the wound interface are influenced by synthetic materials. The aim has been to assess the potential of this approach as a basis for the development of wound care therapies.

13.6.4 Synthetic and biologic dressings – bridging the gap The expectation that other biomimetic wound healing products will emerge is clear. There are many interacting factors and several possible explanations for the fact that materials in this and other fields have shown what Tirrell describes as ‘desirable biological interactions with their surroundings’ (Tirrell et al., 2002). There is not one single simple explanation for the interesting early clinical experience with sulphonate-based materials, nor is there a clear understanding of those molecular features that diminish rather than enhance effectiveness. The difficulties involved in establishing scientifically and statistically valid comparative clinical studies in order to probe such questions are a well-recognised feature of the wound healing field. For that reason progress in materials design is not really a matter of deduction from evidence that is clear cut arising from a small number of well-designed experiments; it depends rather on inspiration based on clues from as many sources as possible. The last decade has seen the developments of concepts and products that begin to bridge the gap between cellular tissue-engineered and wholly synthetic approaches to the facilitation of tissue repair and regeneration. Products based on naturally occurring materials such as collagen and alginate have been known for some time, as has the principle of incorporating scaffold-supported viable cells to a wound site. More recently, acellular materials have been developed that combine the structural properties of collagen with biochemical entities that are designed to promote biological activity. The approach is more that of a wound matrix rather than a wound dressing, but their apparent functional dependence on the role of glycosaminoglycans provides an obvious conceptual bridge to the biomimetic importance of the sulphate group in the promotion of wound healing. One such material is described as an extracellular matrix analogue. It is an

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acellular, collagen-glycosaminoglycan matrix in which the degree of crosslinking and glycosaminoglycan composition control the rate of matrix biodegradation (Orgill et al., 1999). Another is based on freeze-dried animal-derived extracellular matrix in which not only collagen and glycosaminoglycans, but also fibronectin and growth factors have been preserved (Mostow et al., 2005; Romanelli et al., 2010). The interesting differences and similarities between these materials, provides further ongoing information in understanding the molecular role of glycosaminoglycans in wound healing and the search for the ideal molecular paradigms for the design of synthetic wound healing materials.

13.7

References

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Science and Technology Conference, Thailand Science Park Convention Center, Bangkok, April (published online). Nalampang K, Molloy R, Witthayaprapakorn C and Suebsanit N (2007), ‘Design and preparation of AMPS-based hydrogels for biomedical use as wound dressings’, Chiang Mai Journal Sci, 34, 183–189. Orgill D P, Straus F H and Lee R C (1999), ‘The use of collagen-GAG membranes in reconstructive surgery’, Annals NY Acad Sci, 888, 233–248. Patey S J, Yates E A and Turnbull J E (2005), ‘Novel heparan sulphate analogues: inhibition of β-secretase cleavage of amyloid precursor protein’, Biochem Soc Trans, 33, 1116–1118. Peplow P V (2005), ‘Glycosaminoglycan: a candidate to stimulate the repair of chronic wounds’, Thrombosis Haemostasis, 94, 4–16. Prestwich D (2002), ‘Glycosaminoglycan hydrogel films as biointeractive dressings for wound healing’, Biomaterials, 23, 3661–3671. Renné T, Dedio J, David G and Müller-Esterl W (2000), ‘High molecular weight kininogen utilizes heparan sulphate proteoglycans for accumulation on endothelial cells’, J Biol Chem, 275, 33688–33696. Renné T and Müller-Esterl W (2001), ‘Cell surface-associated chondroitin sulphate proteoglycans bind contact phase factor H-kininogen’, FEBS Letters, 500, 36– 40. Romanelli M D, Valentina B and Maria S (2010), ‘Randomized comparison of Oasis wound matrix versus moist wound dressing in the treatment of difficult-to-heal wounds of mixed arterial/venous etiology’, Adv Skin Wound Care, 23, 34–38. Sergio D, Rosenzweig M D and Holland S M (2004), ‘Phagocyte immunodeficiencies and their infections’, J Allergy Clin Immunol, 113, 620–626. Shimabayashi S, Okuda M, Yagiu M and Nakagaki M (2003), ‘A comparison study between sodium decyl sulphonate and sodium decyl sulphate with respect to the interaction with poly(vinylpyrrolidone),’ Prog Colloid Polymer Sci, 122, 113–121. Shu X Z, Liu Y, Palumbo F, Luo Y, and Prestwich G D (2004), ‘In situ crosslinkable glycosaminoglycan hydrogels for tissue engineering’, Biomaterials, 25, 1339– 1348. Shu X Z, Ahmad S, Liu Y and Prestwich G D (2006), Synthesis and evaluation of injectable, in situ crosslinkable synthetic extracellular matrices (sECMs) for tissue engineering, J Biomed Mater Res, 79A, 902–912. Steinhart J and Reynolds J (1969), ‘Multiple equilibria in proteins’, Acac. Press, N.Y. Sundaram S, Lim F, Cooper S L and Colman R W (1996), ‘Role of leucocytes in coagulation induced by artificial surfaces: investigation of expression of MAC-1, granulocyte elastase and leucocyte adhesion on modified polyurethanes’, Biomaterials, 17, 1041–1047. Tighe B J, Nasso M, Benning B and Molock F J (2008), ‘Polymeric compositions comprising at least one volume excluding polymer,’ US 20080114123. Tighe B J, Franklin V J, Lydon F J, Roberts S, Urban J P G and Sarit S (2009), ‘Intervertebral Disc and Intraocular Lens’, WO/2009/127844. Tirrell M, Kokkoli E and Biesalski M (2002), ‘The role of surface science in bioengineered materials’, Surface Sci, 500, 61–83. Tomiyama S (1975), ‘Fundamental study of biochemical behavior of anionic sulphonate and sulphate-type surfactants’, J Am Oil Chemists’ Soc, 52, 135–138.

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Vaheri A, Bizik J, Salonen E M, Tapiovaara H, Siren V, Myohanen H and Stephens R W (2002), ‘Regulation of the pericellular activation of plasminogen and its role in tissue-destructive process’, Acta Ophthalmol, 70, 34–41. Vachon D J (2006), ‘Sulphonated styrene polymers for medical articles and barrier web constructs’, US2006292208, 2006-12-28. Vachon D J and Wnek G (2004), ‘Sulphonated styrene copolymers for medical uses’, to Aegis Biosciences, US2004142910, 2004-07-22. Vachon D J and Yager D R (2006), ‘Novel sulfonated hydrogel composite with the ability to inhibit proteases and bacterial growth’, J Biomed Mater Res A, 76, 35–43. Venkatesan A M and Baker J L (2002), ‘N-hydroxy-2-(alkyl, aryl, or heteroaryl sulphanyl, sulphinyl, or sulphonyl)-3-substituted alkyl, aryl or heteroarylamides as matrix metalloproteinase inhibitors’, to American Cyanamid Co, US2002188120, 2002-12-12. Wang T W, Sun J-S, Wu H-C, Huang Y-C and Lin F-H (2006), ‘Biomimetic bilayered gelatin-chondroitin 6 sulphate-hyaluronic acid biopolymer as a scaffold for skin equivalent tissue engineering’, Artificial Organs, 30, 141–149. Wang X, Fu X, Brown P D, Crimmin M J and Hoffman R M (1994), ‘Matrix metalloproteinase inhibitor BB-94 (Batimastat) inhibits human colon tumor growth and spread in a patient-like orthotopic model in nude mice’, Cancer Res, 54, 4726–4728. Whitfield D M and Sarkar B (2004), ‘Heavy metal binding to heparin disaccharides. II. First evidence for zinc chelation’, Biopolymers, 32, 597–619. Wichterle O and Lim D (1960), ‘Hydrophilic gels for biological use’, Nature, 185, 117–118. Wichterle O and Lim D (1961), ‘Method for producing shaped articles from three dimensional hydrophilic polymers’, US Patent 2 976 576.

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14 Drug delivery dressings K. H. M AT T H E W S, Robert Gordon University, UK

Abstract: This chapter concerns the targeted and sustained topical delivery of therapeutic agents to chronic wounds using natural and synthetic polymer carriers. It outlines the role of drug delivery dressings in wound management and focuses on the delivery of antimicrobials and growth factors to facilitate the healing process. Using the established dressing categories of hydrocolloids, hydrogels, collagen and alginates, it reviews recent research, limiting discussion to combinations of polymer and therapeutic agent. Traditional dressings composed of cotton wool, lint, natural or synthetic bandages and gauzes are only discussed in terms of secondary dressings for the primary dressings of interest. Key words: drug delivery dressings, topical delivery, natural and synthetic polymers in wound management, antimicrobials, growth factors.

14.1

Introduction

For the purposes of this chapter, the term ‘drug delivery dressing’ is defined as any substance applied directly to the surface of a chronic wound that contains an active ingredient intended to display therapeutic properties in terms of positively affecting or supporting the healing process. This could be the substance itself, e.g. manuka honey, or a therapeutic agent such as an antimicrobial compound or growth factor. For the former example, the honey is both vehicle and therapeutic agent, and likewise for many other primary dressings made from natural therapeutic polymers such as alginate, chitosan, collagen or hyaluronic acid; the therapeutic agent is normally contained within or attached to the vehicle or carrier in direct contact with the wound bed. The physical form of the primary dressing can be solid, semisolid or liquid (related to the rheological nature of the dressing) and include general classification types such as powder, gauze, lint, film, sponge, fibre, wafer, gel, paste, ointment, cream, suspension, emulsion or solution. Traditional dressings, composed of cotton wool, lint, natural or synthetic bandages and gauzes, have varying degrees of absorbency and can be used as primary or secondary dressings. The generally non-occlusive nature of these wound dressings (in the absence of an occlusive outer film) encourages evaporation of moisture, resulting in a dry wound bed, in direct contrast to the universally preferred conditions for optimum healing, i.e. moist wound bed. These traditional dressings, sterilised or not, account for the 361 © Woodhead Publishing Limited, 2011

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vast majority of wound dressings and do not generally contain recognised therapeutic agents. They are primarily designed to maintain the optimum conditions for a given type of wound in terms of fluid absorption (moisture balance), environmental protection (from exogenous bacterial infection) and occlusive/non-occlusive properties. Within the scope of this chapter, these traditional dressings will be of little interest other than as secondary dressings for primary drug delivery dressings as defined. It should be stressed from the outset that the term ‘dressing’, as used in this chapter, should not be confused with the bulk of traditional or modern dressings readily available on national drug tariffs and familiar to health workers and patients alike. The vast majority of drug delivery dressings discussed are not commercially available wound management products and there are few commercialised examples beyond those containing antimicrobial reagents or growth factors. Indeed, only one commercial preparation of a growth factor is generally available (see Section 14.3.2).

14.2

The role of drug delivery dressings in wound management

Within the wound management/wound healing area, it is generally accepted that modern dressings fall into various categories that include, foams, films, hydrocolloids, alginates, hydrogels and bioactive dressings. All these dressing types are potentially suitable as vehicles or matrices for the delivery of therapeutic agents. There are many text books on wound management that deal with the daily reality of caring for acute and chronic wounds and a plethora of clinical studies documented in the medical literature that consider all manner of wound aetiologies and the efficacy, with respect to improved or complete healing, of one particular treatment or another. Many of these trials avoid the use of therapeutic agents and may focus, for example, on the merits of one type of dressing or bandage compared with another. In most cases at large, wound healing is termed acute and progresses in non-compromised, healthy human beings by the recognised stages of haemostasis, inflammation, proliferation and maturation (or ‘remodelling’) (Myers, 2008). Within these phases, the complex biochemistry of vascular and cellular response and the role of proteins, cytokines, growth factors, chemotactic agents, proteases and a variety of cell types all have a defined role (Stadelmann et al., 1998a) the intricacies of which are constantly evolving in the scientific and medical literature. When there is a prolonged healing time, the orderly series of events from wounding to healing have been disrupted for a number of reasons ranging from underlying disease states like diabetes or anaemia, which can cause compromised circulation

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and hypoxia; poor nutritional status and old age, causing a deficiency in essential elements; foreign bodies causing a perpetual inflammatory response, and infection resulting from a compromised immune system and abnormal colonisation by pathogenic bacteria (Stadelmann et al., 1998b; Boateng et al., 2007; Myers, 2008). It is within the vast medical field of chronic wounds that the use of therapeutic agents is most commonly encountered. Effective wound management relies on some basic operations that include the removal of necrotic tissue (debridement), control of exudates, protection from foreign bodies and maintenance of the ideal healing conditions of moisture balance, temperature, pH and circulation. Topically applied therapeutic agents can generally aid with debridement and/or control infection and these are by far the most commonly encountered chemical treatments applied as dressings. Less common but arguably of greater current interest on the frontiers of wound chemotherapy are the use of growth factors and proteinase inhibitors. The ideal dressing will not only deliver precise therapeutic quantities of these carefully chosen ‘drugs’ at the right time in the right place, but will be conducive with the ideal healing conditions discussed and be practicable with respect to handling, application and removal. This ideal drives current research and development of drug delivery dressings in the quest to better manage and, where possible, heal chronic wounds. The following sections discuss in more detail some of the therapeutic agents and drug delivery dressings of current interest.

14.3

Topically delivered therapeutic compounds

14.3.1 Antimicrobials Antimicrobial compounds are by far the most commonly used therapeutic agents for the topical treatment of wounds. The term ‘antimicrobial’ refers to a substance that is able to destroy unicellular microorganisms and they may be delivered topically or systemically (Myers, 2008). Systemic antimicrobials are commonly referred to as ‘antibiotics’ and are normally reserved for patients showing signs of sepsis or advancing infections stemming from the wounded area. Of course, there is much concern about the dangers of systemic antibiotic use with regards to adverse reactions and bacterial resistance despite the convenience to the patient, especially if given perorally. There can be little doubt, though, that in patients with decreased tissue perfusion and local ischemia, adequate levels of the active agent in infected wound tissue are unlikely and the increased doses required, normally by infusion, pose a severe threat to liver and kidney function. Likewise, the topical use of antibiotics is fraught with concerns about bacterial resistance and skin sensitivity (Thomas, 1990) and it is generally advised that they

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should not be applied topically (Morgan, 1999; BNF 2009). Despite these warnings, many authors continue to develop dressings incorporating antibiotic substances. Simple solutions of cefazolin applied as an irrigation have been shown to produce higher and more protective concentrations of the antibiotic in wound drainage fluid compared to intravenous delivery (White et al., 2008). Collagen sponges (Sripriya et al., 2007), alginate/chitosan sponges (Ozturk et al., 2006) and a synthetic poly(2-hydroxyethyl methacrylate) hydrogel (Tsou et al., 2005) have been used for the sustained release of ciprofloxacin; doxycycline has been incorporated in crosslinked gelatine microspheres as an inhibitor of matrix metalloproteinases (MMPs) and tested in National Institute of Health (NIH) 3T3 mouse embryonic fibroblasts (Adhirajan et al., 2007); ampicillin incorporated in chitosan-graft-poly(methyl methacrylate) microspheres (Changerath et al., 2009); clindamycin in a poly(vinyl alcohol)/sodium alginate hydrogel (Kim et al., 2008b) and minocycline in poly[hydroxyethyl methacrylate-co-poly(ethylene glycol)-methacrylate] films (Bayramoglu et al., 2009). Erythromycin has been delivered to deep dermal, Staphylococcus aureus (S. aureus) infected wounds in mice in an ethosomal carrier (modified lipid vesicles) with an efficacy comparable to systemically delivered antibiotic (Godin et al., 2005) and vancomycin has been incorporated in lyophilised calcium alginate (Lin et al., 1999) and plasticised glycerol/gelatine sponges (Drognitz et al., 2006). A review article by Zilberman and Elsner (2008) covers the incorporation of antibiotics in a wide range of medical devices including wound dressings but it is possibly in intracorporeal implants and/ or where there is clear indication of complicated infection in ischemic and necrotic tissue that antibiotic eluting polymers have a brighter future. In contrast, there appears to be less preoccupation with the topical application of broad spectrum antimicrobial compounds to infected wounds. Many types of antimicrobial-impregnated wound dressings are commercially available. Many are based on the use of iodine, or more correctly an iodophor (complex) of poly(vinyl pyrrolidone) and iodine (PVP-I); or crosslinked starch and iodine (cadexomer iodine, CI). The former of these common antimicrobial preparations is widely available as a solution or ‘scrub’, paint, powder-spray, ointment or hydrophilic poly(ethylene glycol) fabric dressing (BNF, 2009). The latter as a powder of micronised beads, a poly(ethylene glycol)-based ointment, or a paste of hydrophilic beads sandwiched in a protective gauze (Morgan, 2004). For PVP-I, triiodide (I3−), is released from the polymer complex depending on the absorption of wound exudates whereas diatomic iodine is released from CI (Noda et al., 2009). It appears that much of the inconclusiveness of clinical studies relating to the use of iodine-releasing dressings may be related to the relative uptake of water from the different dressings, presumably resulting in variations in

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the concentrations of antimicrobial agent released. It also appears that the amount of available iodine, free iodine, iodide and triiodide varies significantly at different dilutions in the wound environment for PVP-I and is related to a complex equilibrium between the different iodine species (Atemnkeng et al., 2006). A recent review on the efficacy of antimicrobials on the healing of venous ulcers identified five trials using PVP-I and ten using CI and concluded, after much statistical consideration, that there was some evidence to support the use of CI but emphasised the limiting of the use of topical antimicrobials to clear cases of clinical infection (O’Meara et al., 2010). The argument generally supports the use of iodine delivery systems in wound management. The other most popular topical antimicrobial is without doubt that of silver and its salts, mainly silver sulfadiazine. From a wound management point of view, as for iodine dressings, silver dressings are generally contraindicated for uninfected wounds and should be used with caution where new epithelial or granulating tissue is present as silver is cytotoxic to fibroblasts and has been shown to retard epithlialisation both in vitro and in vivo (Ovington, 2004). Cynicism in many clinicians is apparent from the literature (Brett, 2006; Myers, 2008) but there can be no doubting the fact that as a broad spectrum antimicrobial, silver cations (Ag+) liberated from its salts, elemental or colloidal preparations is effective against aerobic, anaerobic, gram-positive and gram-negative bacteria, yeast, fungi and viruses (Sibbald et al., 2003). The continuing popularity of antimicrobial silver is evident from the scientific literature and a recent review examines all the available evidence about effects of silver on wound infection control and wound healing (Atiyeh et al., 2007). A balance between infection control and cytotoxicity appears to be the ultimate goal. Some examples of the continued incorporation of silver as a drug delivery dressing, beyond a common topical cream preparation containing silver sulfadiazine and an activated charcoal dressing containing silver (Morgan, 2004) include a collagen scaffold impregnated with alginate microspheres containing silver sulfadiazine (Shanmugasundaram et al., 2006). The authors claim an initial in vitro burst release of 47.5% followed by a controlled release for 72 hours with an equilibrium concentration of 68.8%, obtaining minimum bactericidal concentrations against Klebsiella pneumoniae (K. pneumoniae), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and S. aureus. Incorporation of silver (I) in itaconic acid-based poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels demonstrated that the silver ions were embedded throughout the polymer network and leached out in a controlled manner in an aqueous medium (Micic et al., 2009). Atomic force microscopy (AFM), metal sorption analysis, antibacterial activity assays and mass spectroscopy (MS) were used to identify the silver co-ordination sites within the gel network and confirm the in vitro

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stability. A semi- interpenetrating network (sIPN) of poly(ethylene glycol) and gelatine was loaded with both silver sulfadiazine and bupivacaine for ‘multiple hit’ management of dermal wounds whereby the pain-killing effect of bupivacaine would work in tandem with the antimicrobial silver (Kleinbeck et al., 2009). These authors reported that the absorbent properties of the IPN would absorb exudates, minimise infection, manage pain and facilitate epidermal growth. Like most of the antimicrobial-loaded hydrogels reported in the scientific literature, this multi-purpose wound dressing only possesses the ‘potential’ to improve wound management and remains untested in a clinical situation. Chitosan films plasticised with glycerol or sorbitol have been produced as carriers of silver sulfadiazine (Azevedo et al., 2006) and although naturally sourced films have been used as wound dressing materials since the documented use of isinglass or oiled silk in the 18th century and collodion in the 19th century, medicated films (containing iodine and chlorhexidine) were not widely used until the late 20th century and primarily as catheter dressings (Thomas, 1990). High conformability, transparency and the semiocclusiveness of films are dressing traits best suited to non-infected, dry to lightly exuding wounds, firstly as a barrier to exogenous bacteria, secondly as retaining moist healing conditions and preserving endogenous enzymes that naturally deslough necrotic tissue (Myers, 2008). It would appear a natural progression to incorporate antimicrobial agents in either the film adhesive or the film itself. Incorporation of silver and its compounds in semiocclusive films and dressings such as cloths, fabrics, foams, gauzes, hydrocolloids, alginates and hydrofibres have all been demonstrated and many are commercially available as selective tools for the management of infected wounds (Ovington, 2007; Myers, 2008). A natural hurdle to the incorporation of high loadings of therapeutic agents, relative to hydrogels say, may still hinder the development of drug delivery dressings based on films alone. Even the development of ‘nanocrystalline’ silver technologies may have its own problems according to the findings of Taylor et al. (2005) who concluded that nanocrystalline silver dressings lost their antimicrobial properties following heat treatment (for sterilisation) for 24 hours at temperatures above 75°C. Synthesis of hydrogel-silver nanocomposites based on poly(acrylamide-co-acrylic acid) with potential applications in the field of biomaterials and ‘fair’ antibacterial activity against E. coli was recently demonstrated (Thomas et al., 2007). Other more recent research involving silver dressings have highlighted the negative effect of silver on human keratinocytes (Wiegand et al., 2009). Earlier work by Lee et al. (2005) used EGF in a silver sulfadiazine-loaded collagen sponge dressing to reverse such cellular damage, the latter authors advocating the combination of growth factor and silver sulfadiazine in clinical dressings. An attempt to improve the clarity of evidence for the use of

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silver in the treatment of contaminated and infected acute and chronic wounds failed to do so, concluding, after consideration of three large and relevant clinical trials comprising a total of 847 participants, that there was insufficient evidence to recommend the use of silver-containing dressings or topical agents for the treatment of infected or contaminated wounds (Vermeulen et al., 2007). Such advice does not seem to deter many scientists who continue to pursue the incorporation of silver in drug delivery dressings. One quite different delivery system concerning the development of a ‘mesoporous silica sphere’ as a biodegradable haemostat for traumatic wounds, recently demonstrated the antimicrobial efficacy of silver ions (Dai et al., 2009). No doubt the debate over silver drug delivery dressings will continue. As a final, brief addition to this section on antimicrobials, the case for the continued use of chlorhexidine salts, particularly the acetate and gluconate, will be summarised due to its abundant use in general hospital antisepsis. As a broad spectrum antimicrobial it is generally thought to be as safe as iodine or silver (Morgan, 1999) and a 0.05% solution is recommended for antiseptic treatment of wounds (Morgan, 2004) although many clinicians still believe that its use, like all established antiseptics, should be restricted to inanimate objects or intact skin (Myers, 2008). It is true that its main use is with vascular and epidural catheters, where it is recognised as being effective in reducing bloodstream or central nervous system (CNS) infections from developing (Ho and Litton, 2006); however, it has been used as a prophylactic burn dressing in the form of a paraffin impregnated gauze containing 0.5%w/w chlorhexidine acetate (Morgan, 2004). It is known to bind strongly to cellulose materials and that its antibacterial activity is reduced in the presence of blood or pus (Thomas, 1990). More recent in vitro data has demonstrated that chlorhexidine digluconate has considerably decreased antimicrobial activity against methicillin-resistant S. aureus (MRSA) in the presence of 3% bovine serum albumin (BSA) (Labovitiadi, 2011). A few severe reactions and side-effects have been reported over the years and acquired resistance by P. aeruginosa documented (Hammond et al., 1987). The consensus still seems to be that there is insufficient data to assess its safety and efficacy (Drosou et al., 2003; O’Meara et al., 2010) although recent research in this author’s laboratory has shown it to be as effective as PVP-I, silver sulfadiazine and neomycin sulphate against planktonic colonies of MRSA and methicillin-susceptible S. aureus (MSSA) when released in vitro from lyophilised wafers (Labovitiadi et al., 2009).

14.3.2 Growth factors The role of growth factors in the wound healing process is an ongoing quest. It is well-established that in acute wounds there is a finely balanced environ-

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ment in which proteolytic activity and matrix synthesis occur under tight biochemical regulation. Chronic wounds, on the other hand, have lost this fine balance (Stadelmann et al., 1998a) and for more than fifteen years it has been known that chronic wounds have reduced levels of plateletderived growth factor (PDGF), β-transforming growth factor (β-TGF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for starters (Cooper et al., 1994). EGF was the first growth factor to be isolated and used successfully to treat acute wounds but its degradation in the chronic wound environment has limited its use in clinical practice. At that time it was also appreciated that before growth factor deficiencies could be addressed, impediments to wound healing such as hypoxia, infection, wound debris, necrotic tissue, nutritional deficiencies, environmental factors and metabolic disorders were of arguably greater significance (Stadelmann et al., 1998b). Accepting such facts and acknowledging that there is still a requirement for increasing the level of key growth factors in particular wound management situations, it is necessary to deliver these expensive compounds efficiently and accurately to the target area where they are most effective without premature degradation. Indeed, it may be possible to design delivery systems that can protect growth factors such as EGF long enough for it to be effective (Hardwicke et al., 2008b). One such approach considers conjugating the growth factor with a suitable polymer that is degraded in vivo by naturally occurring enzymes present in wound fluid. The ability of a succinoylated dextrin to protect recombinant human epidermal growth factor (rh-EGF) towards proteolytic degradation by trypsin and neutrophil elastase in vitro has recently been demonstrated (Hardwicke et al., 2009). Addition of α-amylase degraded the dextrin component leading to sustained release of 53% of rh-EGF over a period of 168 hours thus the principle of bioresponsive nanomedicines for use in tissue repair was simulated. Another approach to improving the stability of growth factors in wound fluid involves targeted therapy whereby the growth factor is attached to a suitable protein binding agent to produce a recombinant protein-growth factor that specifically binds to the target wound protein. Zhao et al. (2008) attached bFGF to a fibrin-binding peptide called ‘Kringle 1 (K1)’ specific for fibrin clots and when applied to wound sites K1bFGF induced ‘robust neovascularisation and improved wound healing’. For wounds where no fibrin clot is evident, the authors attached bFGF to a prefabricated fibrin scaffold to serve as the vehicle for the peptide-bound growth factor. As fibrin functions as the natural haemostat for wounds and a matrix for fibroblast migration, leading to the production of glycosaminoglycans and collagen formation, fibrin itself may be classified as a natural vehicle for delivering therapeutic compounds. A haemostatic dressing that forms a

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fibrin clot immediately on contact with wound fluids (blood) is available to the military in the form of a lyophilised bilayer of fibrinogen and thrombin (Larson et al., 1995). The use of naturally occurring proteins as delivery systems is well-established for collagen and an assortment of examples are given in this chapter (see Section 14.6). Collagen-derived gelatine has also been incorporated in hydrocolloid dressings for almost thirty years and despite being a thermally degraded collagen-derived product, is often referred to wrongly as ‘collagen’ (Ruszczak and Freiss, 2003). Huang et al. (2005) used the correct term when describing the incorporation of bFGF in gelatine microspheres prepared by a modified coacervation technique whereby a simple gelatine in paraffin oil emulsion was cooled and filtered, the micro-sized disperse gelatine phase collected, dried, crosslinked with gluteraldehyde, washed, centrifuged and freeze-dried. Microspheres were suspended in a further solution of gelatine that was subsequently crosslinked, poured to suitable moulds, flash-frozen with liquid nitrogen and freeze-dried to form a shaped sponge. The growth factor was incorporated in the microspheres by addition to the gelatine solution prior to initial emulsion formation or directly into the gelatine sponge without microsphere formation for comparative purposes. Release of bFGF for both types of dressing was measured in vitro using ELISA and in vivo using a full-thickness wound, porcine model. The dressing containing the bFGF-containing microspheres showed the best results in terms of epidermis formation and wound closure, followed by bFGF-incorporated gelatine sponge, control samples of both designs (minus bFGF) and a further petroleum jelly control. Presumably the slower, more sustained release of growth factor from microspheres exercised greatest effect as expected for controlled release systems. Some other select examples of incorporating growth factors in crosslinked polymers include TGF-β1, representative of the TGF-β family, in a synthetic thermosensitive or thermoresponsive hydrogel (see Section 14.5) composed of a triblock poly(ethylene glycol) – poly(lactic-co-glycolic acid) – poly(ethylene glycol) polymer (PEG-PLGA-PEG) (Lee et al., 2003). On this occasion, the authors used a plasmid TGF-β1 attached to DNA as a ‘release carrier’ for the growth factor based on earlier evidence that exogenous TGF-β1 injected intradermally accelerated wound healing in diabetic mice (Chesnoy et al., 2003). They described how the triblock hydrogel formed an adhesive (and occlusive) film at the wound site on evaporation of the solvent (water) at skin temperature, exhibiting a ‘wound dressing effect’ i.e. preventing wound desiccation and providing a barrier to exogenous bacterial infection. They concluded that this delivery dressing was superior to a control hydrogel dressing composed of chondroitin sulphate, a naturally occurring glycosaminoglycan used increasingly in cartilage tissue engineering as a biodegradable scaffold with an ability to bind to

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growth-active molecules and play an active role in the healing process (Malafaya et al., 2007). The recognised advantages of hydrogels as drug delivery dressings are further illustrated with recent work to deliver constant amounts of insulinlike growth factor-1 (IGF-1) to wounds where the healing is suppressed by steroid treatment (Beckert et al., 2007). This study compared the topical delivery of IGF-1 using a methylcellulose gel (0.2% of an unspecified grade) and a 10% poly(vinyl alcohol) film placed on a commercial polyurethane hydrogel to steroid-suppressed, full-thickness wounds on Sprague Dawley rats. Unadulterated hydrogel dressing was used as a control. There was no doubt that the healing action of the growth factor was enhanced when incorporated in the hydrogel dressing, leading the authors to conclude that the mechanism of IGF-1 action was dependent on its application mode. This supported the much earlier conclusions of Puolakkainen et al. (1995) that the vehicle for topical growth factor delivery significantly affects its benefit in wound healing. Judith et al. (2010) have studied the effects of collagen-chitosan-PDGF in a rat model and monitored the healing process using a variety of biochemical, histological and transmission electron microscopic methods. They found that the combination of chitosan and collagen with PDGF halved the time to wound closure (10 days) compared with the control (20 days). A chitosan-PGDF gel without collagen caused complete epithelialisation at 14 days. The presence of exogenous collagen in the topical gel was concluded to supplant endogenous collagen allowing fibroblasts to immediately attach, migrate and proliferate at the wound site, producing their own collagen without waiting on the natural collagen-forming process to take place. Chitosan in combination with collagen better mimics the extracellular matrix possibly due to the amine groups of the solubilised chitosan promoting cellular binding. Presumably the growth factor in these studies had improved distribution, residence and hence activity on the target surface by way of its association with these two biocompatible polymers. The attractions of chitosan as a wound contacting biomaterial and matrix for the topical delivery of therapeutic agents to wounds are described in a recent review by Bhattarai et al. (2009). Growth factors and their improved delivery to chronic wounds continue to be of considerable interest in wound healing and the recognised problems associated with maintaining them at the correct site in appropriate concentration, where they can exercise a positive effect on the healing process, will sustain efforts to improve the delivery system. At present, there is only one commercial formulation of rh-PGDF, becaplermin, available on the drug tariff (Morgan, 2004; BNF, 2009) as a sodium carboxymethylcellulose gel formulation, but like all therapeutic compounds that are topically applied as hydrated gels, dilution of the vehicle in wound fluid and depletion

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of gel consistency (viscosity) will continue to restrict its efficacy as a topical treatment. Braund et al. (2007) recognised this and prepared dried films of hydroxypropylmethylcellulose (hypromellose) containing bFGF onto a commercial, perforated polyester film that normally functions as the wound contact layer of an absorbent polyester (viscose)/cotton pad, with the intention of creating a controlled release platform for the growth factor. The rates of hydration and inherent gel viscosities of the three grades of hypromellose tested (E4M, K4M and E10M) were expected to produce different release profiles according to the different degrees of methoxyl and hydroxypropyl substitution (E and K grades); and different barriers to the diffusion of bFGF due to different molecular weights, and hence gel viscosities, at equivalent concentration (E4M and E10M). This was indeed the case although no clear, overall fit to established release models was realised. The integrity of the gel layers formed appeared to have desirable properties for the sustained delivery of growth factors directly to exuding wound surfaces. This principle of manipulating the rheological properties of hydrophilic polymers to control the topical release of wound healing agents had also been demonstrated by Matthews et al. (2005) using combinations of sodium alginate and methylcellulose on a model wound surface with sodium fluorescein as a visible solute. One other selected approach to incorporate a growth factor involves the use of a plasma discharge to the surface of a polymer scaffold to produce a functionalised surface on the substrate material without damaging the bulk physicochemical properties of the selected polymer. Shen et al. (2008) used a carbon dioxide plasma to introduce carboxylic acid functional groups on the surface of poly(lactide-co-glycolide) (PLGA) films. They immersed the activated film in a phosphate buffer solution containing bFGF and found that the binding efficiency of growth factor was significantly enhanced. Furthermore, the release of bFGF from the film was continuous in both static and dynamic dissolution experiments. The increased binding efficiency of plasma-treated PLGA to bFGF was most noticeable in the dynamic experiment conducted in a parallel plate flow chamber, where sustained release of the growth factor was measured for seven days. This was in contrast to the untreated films where no bFGF was detected under shear flow conditions. Although these particular studies were concerned with cell adhesion to plasma-treated PLGA surfaces, with the aim of improved substrates for tissue culture as opposed to release of growth factor to a chronic wound, the advantages of plasma-treated surfaces with respect to improved binding and subsequent sustained release of bioactive bFGF were clearly demonstrated. The universal interest in growth factor delivery dressings might be expected to continue into the future providing the costs of production decrease and clinical efficacy continues to be established.

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14.3.3 Less common therapeutic agents Although it is clear that antimicrobial compounds and growth factors are by far the most studied therapeutic agents for drug delivery dressings, there is in fact no real limit to the type of compound that can be incorporated in a polymeric carrier. Provided there is a sound rationale for the topical application of a substance to a wounded area of skin, it is possible to select an appropriate carrier for that substance. There may be as much science in the optimum design of a suitable polymeric carrier that satisfies all the desirable attributes of the ideal dressing, of which there is in reality probably no such thing, than in the active agent and its function in the immensely complex and variable physiology of a chronic wound. A few more unusual therapeutic agents are occasionally encountered and some examples include the vitamins A, C and E, as well as the essential elements at the centre of many matrix metalloproteinase (MMP) enzymes i.e. zinc and copper (Agren, 1990; Lansdown, 1996; Lansdown, 2002; Cangul et al., 2006). Although the vitamins are normally administered orally as dietary supplements in chronic wound patients, there are one or two examples that include the incorporation of vitamin E in silicon gel sheets (Palmieri et al., 1995) and vitamins A and C applied with collagen sheets (Lazovic et al., 2005). Of course, the topical application of zinc oxide is well-established in wound management and classically applied as a solid dispersion in an oil base ‘zinc ointment’. One recent study (Kietzmann and Braun, 2006) concluded that the combination of zinc oxide and cod liver oil was superior to controls containing one or the other in dexamethasone-retarded rat wounds. Clearly, zinc paste bandages are still useful as a control when assessing the performance of other drug delivery dressings (Thomas, 1990). Furthermore, it is still accepted that topical application of zinc appears to be superior to oral therapy and therefore controlled topical application of this essential element to wounds may still be desirable; however, as the role of zinc in wound healing is diverse and outside the scope of this chapter, the reader is directed to a comprehensive review article by Lansdown et al. (2007). Other unusual combinations of dressings and therapeutic agents include a proprietary hydroxamate inhibitor of stromelysin-1 (MMP-3), known to be up-regulated in chronic wounds, as a solid dispersion within a lyophilised xanthan wafer (Fray et al., 2003; Matthews et al., 2008b); a cotton gauze saturated with adenosine triphosphate (ATP) vesicles (Chiang et al., 2007); and a poly(ethylene oxide -co-propylene oxide) hydrogel (Pluronic F127) containing the nitrogen oxide (NO) donor, S-nitrosoglutathione (Amadeu et al., 2008). These three examples of novel therapeutic agents contained in both traditional (cotton) and non-traditional (lyophilised wafer and hydrogel) carriers offer some examples of therapeutic agents, other than growth

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factors or antimicrobials, that may have a role to play in the future treatment of chronic wounds.

14.4

Hydrocolloids

The term ‘hydrocolloid’ describes wound dressings that are traditionally fabricated from a blend of hydrophilic colloidal polymers of a natural or semi-synthetic origin. They are invariably presented in the form of a flexible and self-adhesive foam or film consisting of a bespoke combination of synthetic elastomers and pressure-sensitive adhesives. Foams and films are commonly fabricated from polyurethanes, polyesters, poly(vinyl chloride) and poly(ethylene); and pressure-sensitive adhesives from plasticised poly(butylene), poly(isobutylene), styrene-isoprene copolymers and poly(cyclopentadiene-dioctyladipate). The hydrocolloid can be contained within the foam matrix or adhesive layer, or as a separate laminate on a permeable, semi-permeable or impermeable film. Originally composed of gelatine, pectin and carboxymethylcellulose (CMC), they can contain other polysaccharides such as alginate, guar, karaya and xanthan. In addition to the standard sheets and films, hydrocolloids are also available in pastes and powders, or granules, to fill the cavities of heavily exuding wounds and increase the absorbency of the dressing (Myers, 2008). For the most common manifestation of these popular and effective products, a piece of release paper normally covers the dressing (Thomas, 1990). Hydrocolloids are often referred to as ‘interactive’ dressings because gel formation on the slow absorption of wound exudates creates the recognised conditions of the ideal wound dressing, i.e. moist conditions, thermal insulation, non-adherence, permeability to oxygen, carbon dioxide and water (Morgan 2004) and a barrier to exogenous bacteria (Lawrence and Lilly, 1987; Bowler et al., 2001; Thomas, 2008). On their own as contacting wound dressings, a plethora of clinical studies have debated the relative efficacy of hydrocolloids over the years, and many conflicting views have been expressed. A recent systematic review of ninety-nine studies considered the literature on the efficacy of modern dressings for the healing of acute and chronic wounds and concluded that for chronic wounds, hydrocolloids resulted in a ‘statistically significant improvement in the complete healing rate of leg ulcers and pressure sores’ when compared to traditional paraffin and wet-to-dry gauze dressings (Chaby et al., 2007). This research was published shortly after a preceding statistical analysis of 42 randomised control studies on the efficacy of dressings for venous leg ulcers (23 hydrocolloids, 6 foams, 4 alginates, 6 hydrogels and ‘3 miscellaneous’ dressings) had concluded that there was no evidence to ‘suggest that any one dressing type was better than others in terms of number of ulcers healed’ (Palfreyman et al., 2006).

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Despite this ongoing debate, it is clear that the formation of a physical gel in contact with the affected wound area has all the credentials to provide a useful matrix for the topical delivery of therapeutic agents. Unfortunately, there is also confusion in some quarters over the use of the term ‘hydrocolloid’ as applied to wound dressings and this is highlighted by Thomas (2008) in a recent review of the merits of traditional hydrocolloid dressings in clinical wound management. In the field, ‘hydrocolloid dressing’, rightly or wrongly, refers specifically to a hydrophilic, gel-forming mass applied in a semisolid form to a flexible semi-permeable carrier (Thomas, 2008). Some authors use the term ‘hydrocolloid’ when they really mean ‘hydrogel’ (see Section 14.5) and so, if we accept this thesis, traditional hydrocolloids are not designed as drug delivery dressings as they do not contain added therapeutic agents, irrespective of the perceived healing properties of certain natural polymers. Despite this dichotomy, it is clear that traditional hydrocolloids could provide a suitable matrix for such compounds if desired, although no examples are to be found in the literature.

14.5

Hydrogels

The term ‘hydrogel’ is loosely used throughout medical science to describe any polymeric material that has a gel-like form on absorption of water or aqueous fluids such as wound exudates. In the last 20 years, it has been generally accepted in wound management that two basic types of gel dressing exist, those with a fixed three-dimensional macro structure that do not change physical form as they absorb fluid, and ‘amorphous hydrogels’ that do not have limiting absorption characteristics. The latter class of hydrogels continue to absorb fluid until they form dilute solutions or dispersions of the polymer in water while the former class of hydrogels swell until they become fully saturated or a state of equilibrium is reached (Thomas, 1990). Since then, the field of wound management has not, by and large, kept apace of polymer science where the definition of a hydrogel is more specific. To date, it is universally recognised that hydrogels can essentially be of a ‘physical’ or ‘chemical’ nature. Physical hydrogels consist of polymer chains that form a weak gel structure on absorption of water due to polymer binding via non-covalent physical associations such as hydrogen bonding of contained moieties; ionic attractions between oppositely charged groups or counter-ions; hydrophobic interactions; physical entanglement and/or weak dispersive forces such as Van der Waals attraction (Hoffman, 2002). The particular gel structure can also be pH and temperature dependent. This is in contrast to ‘chemical’ or ‘neutral’ hydrogels where strong covalent bonding between adjacent polymer chains prevents simple solution.

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Provided no part of these chemically crosslinked, polymer networks is hydrolysable or biodegradable, they possess a shape memory for indefinite periods of time and have maximum swelling properties. Both chemical and physical hydrogels can be produced from a limitless variety of natural, synthetic and semi-synthetic polymers. Natural polymers are of interest as biomaterials due to inherent biocompatibility based on the presence of biologically recognisable moieties that support cellular activities and/or biodegradable qualities initiated by in vivo enzymatic processes (Bhattarai et al., 2009); however, they may also contain unwanted pathogens or even invoke immune or inflammatory responses (Lin and Metters, 2006). Synthetic hydrogels can be carefully fabricated and tailored to produce precise degradation characteristics and biological functionality but can produce toxic by-products also resulting in unwanted inflammatory responses. As primary wound management dressings, pre-swollen hydrogels can rehydrate dry wounds, softening eschar and facilitating autolytic debridement; however, they are contraindicated for heavily exuding and infected wounds (Myers, 2008). In cases where surgical debridement is not the desired option, improved debridement may be achieved by the inclusion of enzymatic or chemical agents. Substrate-specific, exogenous enzymes that include proteolytic, fibrinolytic and collagenase enzymes, e.g. papain-urea, chlorophyllin copper, trypsin, streptokinase/streptodornase (proteolytic); fibrinolysin, deoxyribonuclease (fibrinolytic) and collagenase can be included in hydrogels as the relatively mild network fabrication and encapsulation conditions are suitable for thermosensitive proteins and peptides (Peppas et al., 2000). These enzymes will work in combination with the idealised properties of hydrogels with respect to the desirable characteristics of the ideal dressing (Morgan, 1999). Hydrogen peroxide, on the other hand, is a small molecular weight compound traditionally used for desloughing that, despite its known cytotoxicity (Brown and Zitelli, 1993; Mertz and Ovington, 1993) has recently been incorporated in wholly synthetic hydrogels of poly(vinyl alcohol) and poly(acrylic acid) (Smith et al., 2009). These authors claim that differing functionality created by variations in hydrogen peroxide concentration produce thermal properties in the hydrogels that cause a weakening of the hydrogel at bodily temperatures and may make them useful as (controlled) delivery systems. This is an example of how release of active agents can be drastically affected by the specific design of the hydrogel network. The hydrodynamic size of the biomolecule to be delivered and the physical properties of the hydrogel that affect its swelling characteristics, such as hydrophilicity (‘wettability’), molecular weight, crosslink density, pore or mesh size and hence ‘tortuosity’ (Peppas et al., 2006, Bhattarai et al., 2009), are all critical for control of its release properties.

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Some relatively recent examples of the use of hydrogels as drug delivery dressings for the topical application of therapeutic agents in wound management include well-established examples from the armoury of synthetic polymers produced by the free radical reaction of substituted acrylic esters and acrylic amides. Jones et al. (2008) report the loading of poly(2-hydroxyethyl methacrylate) (pHEMA), poly(4-hydroxybutyl methacrylate (pHBMA), poly(6-hydroxyethyl methacrylate) (pHHMA) and copolymers composed of N-isopropylacrylamide (NIPAA), methacrylic acid (MA) and the above monomers with the broad spectrum antimicrobial, chlorhexidine diacetate, using the thermoresponsive nature of polymers containing, in particular, HEMA, MA and NIPAA. At a temperature below a ‘lower critical solution temperature’ (LCST) the polymers will readily absorb water and any water-soluble agent it may contain, e.g. chlorhexidine diacetate, hence effective loading of the hydrogel can be achieved. When the temperature increases to above the LCST, hydrophobic-hydrophobic interactions between polymer chains cause a deswelling and a ‘pulsed’ release (of the antimicrobial). Although such hydrogels are more routinely used as injectable delivery systems, in principle they could be used topically as a primary dressing, especially where a wound cavity is present. This inherent property has been known for many years (Yoshida et al., 1991) and thermosensitive or ‘smart’ hydrogels have been thoroughly reviewed (Jeong et al., 2002; Ruel-Gariepy and Leroux, 2004). Poly(hydroxyethyl methacrylate) as an ‘active’ wound dressing has been demonstrated by Cabodi et al. (2007) who formed a bilayer of porous pHEMA and silicone (as vapour barrier and support) and monitored convective mass transfer using mathematical modelling methods. They also demonstrated a uniform mass transfer, both extraction and delivery, of low molecular weight solutes and a model protein on a simulated wound bed made from the ionic hydrogel, calcium alginate. The synthetic, neutral hydrogel formed by crosslinking poly(vinyl alcohol) (PVA) with sodium borate has recently been shown to be an effective reservoir system for soluble agents that could be applied topically to lacerated wounds (Loughlin et al., 2008). Release of the topical anaesthetic, lidocaine hydrochloride, was facilitated by the incorporation of mannitol to circumvent network constriction attributed to ionic and pH effects that initially prevented release of the soluble salt. PVA has also been crosslinked with the natural polymer, sterculia gum (karaya) and loaded with the antimicrobial agent, tetracycline hydrochloride (Singh and Pal, 2008) as a novel wound dressing. The use of natural gums as lyophilised topical hydrogels for the containment and sustained release of antimicrobials such as povidone-iodine, chlorhexidine digluconate, silver sulfadiazine and neomycin sulfate has also been investigated (Matthews et al., 2008a).

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One other recent claim for the utility of PVA as a neutral hydrogel for advanced wound dressings, formed by the long established method of radiation crosslinking, has been made (Gwon et al., 2009) but no therapeutic agent was included and its use as a traditional absorbent hydrogel may be limited to specific wound types free from infection, as is the case for nonmedicated hydrogels in general. Indeed, the lack of biodegradable properties and local inflammatory issues with the synthetic hydrogels discussed in this section, e.g. NIPAM, HEMA and PVA, probably restricts their use as drug delivery dressings. Unless they are modified with biocompatible polymers, they may be more suited to uses in the biomaterial field other than topical wound management. Examples of hydrogels more suited to the controlled and targeted release of wound healing agents, e.g. collagen/gelatine sponges, hydrocolloids, alginates, semisolids, wafers, foams, films, fibres and microspheres, are discussed throughout this chapter.

14.6

Collagen

Collagen is recognised as the principal building block of connective tissue and as such has been popular as a matrix, carrier or scaffold for therapeutic agents. For more than a decade, drug delivery dressings fabricated from type-1 collagen of animal origin have centred on the ubiquitous collagen sponge (Ruszczak and Friess, 2003). Produced by air, vacuum or freezedrying (Schwarzer, 1999; Radu et al., 2002; Huang et al., 2005; Matthews, 2008) these highly porous scaffolds are able to contain the active agent(s) until they contact wound exudate, absorbing fluid and releasing their contents at a rate determined by the physical properties of the particular sponge – typically within 1–2 hours (Zilberman and Elsner, 2008). The mechanism of release involves a combination of diffusion and enzymatic breakdown of the collagen structure within the biological environment. In addition to simple release of contained compounds, these pure collagen products; also available in the form of gels, pastes, powders, granules and sheets; are known to be active in the healing process, stimulating fibroblast and macrophage production (Mian et al., 1992). It is well established that fibroblasts produce collagen in vivo (Dielgelmann et al., 1981) during the proliferative phase of the healing process until homeostasis is achieved (Stadelmann et al., 1998a) therefore it is also believed that exogenous collagen present in the dressing has a similar effect in the wound environment to endogenous collagen i.e. its ability to recruit and stimulate the proliferation of cells, facilitating the healing process (Ovington, 2007). The term ‘bioactive dressing’ would appear appropriate even in the absence of contained therapeutic agents. Recent attempts to further improve the bioactive nature of collagen dressings have centred on modification of its chemical and physical

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properties. The three main polypeptide building blocks of type-1 collagen, composed of the amino acids glycine, proline, hydroxyproline or hydroxylysine (Robinson and Friedman, 1995) are prone to degradation by collagenase, gelatinase and several other non-specific proteinases (Freiss, 1998). Changes to the rate at which the collagen matrix degrades and releases its therapeutic contents can be controlled by chemically crosslinking with a suitable aldehyde such as glutaraldehyde (GTA). One recent study prepared a range of collagen sponges containing recombinant human epidermal growth factor (rhEGF) with three different types and concentrations of crosslinking agents that included GTA, genipin (a natural aglycone derived from the fruit of Gardenia jasminoides) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Yang 2008). Controlled release profiles of the crosslinked sponges compared with non-crosslinked ones indicated that GTA had the most potent controlling effect on the release of growth factor. Increased concentrations of GTA resulted in a more rigid matrix that demonstrated a decreased release rate presumably due to an increased crosslink density, as might have been expected. Collagen as a delivery system for growth factors was studied by Marks et al. (1991) who considered the effect of fibroblasts and basic fibroblast growth factor (b-FGF) on dermal wounds, and by Royce et al. (1995) who used a collagen implant containing platelet-derived growth factor (PDGF) in rats, however, Grzybowski et al. (1999) recognised that no standardised formulation for the topical delivery of growth factors to wounds had been established or accepted for clinical use. Consequently, they described the preparation of collagen dressings containing human recombinant granulocyte colony stimulating factor (rhGCSF), granulyte-macrophage colony stimulating factor (rhGM-CSF) and recombitant human epidermal growth factor (rhEGF) by saturating small pieces of bovine collagen sponge with prepared solutions of the respective cytokines at a concentration of 500 ng/cm2. Subsequent storage at 4°C in a moist chamber followed by lyophilisation (freeze-drying) produced impregnated sponges for release testing. This was conducted by contact with polyurethane sponges impregnated with the dilution medium and used as collectors of the cytokines under controlled conditions at 24-hour periods for three days. Unfortunately, the release of rhG-CSF and rhEGF from the lyophilised sponges was extremely low (3%) and the reasons largely inconclusive but a combination of the extraction methods employed, possible denaturing of the cytokines over the course of the experiment and degradation of the collagen sponge at the low pH necessarily used (pH = 4) were discussed. Despite these initial drawbacks, the application of collagen sponges for the topical administration of growth factors to wounds continued.

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Other preliminary studies using collagen focussed on beta-transforming growth factor (β-TGF) (Pandit et al., 1999) and human growth hormone (hGH) (Maeda et al., 2001); however, the major application of collagen has been without doubt in tissue engineering, where its application as a matrix or scaffold for cell and gene delivery with respect to the reconstruction of intervertebral discs, bone, cartilage, skin, teeth, adipose, cardiovascular, genitor-urinary tract and renal glomerular tissue have all been investigated (Malafaya et al., 2007). Where the application of collagen to topical drug delivery has been relatively limited for growth factors, much more success has been in its application as a vehicle for topical antimicrobial compounds, in particular gentomycin. A review by Ruszczak and Friess (2003) discussed in considerable detail the popular choice of collagen as a natural polymer for drug delivery and described the rationale for local antibiotic delivery in ophthalmic, periodontal, bone and soft tissue infections, including wound healing. The goal of obtaining local efficacy without systemic effects could be achieved by the physical and/or chemical incorporation of antimicrobial compounds within a collagen matrix. It was claimed that the combined results of randomised, controlled clinical studies in humans (n = 661) treated with gentamycin-containing collagen sponges indicated that significantly more patients, 95.6%, as opposed to 72.5%, treated for potential soft tissue infections following surgery or trauma, healed by either primary intention or without evidence of post-operative infection (Ruszczak and Friess, 2003). These authors predicted in 2003 that both drug combination and different release profiles would lead to better infection control. Technical developments in collagen processing as well as a combination of collagen with other materials held the key. Other contemporary studies had already combined a collagen membrane with hyaluronan microparticles containing silver sulfadiazine (SS) (Lee et al., 2002) and collagen with hyaluronic acid (HA) containing tobramycin and ciprofloxacin respectively (Park et al., 2004) with limited clinical success compared to the controls; the latter study revealing that 0.4 g/mL ciprofloxacin was cytotoxic to fetal human dermal fibroblasts. In contrast, incorporation of b-FGF or PDGF in the collagen-HA-tobramycin matrix, in the same study, was shown to significantly enhance wound healing hence the combination of growth factors and antimicrobials was suggested. Further studies concerning the effect of EGF to reverse the apparent delay in wound healing caused by SS have also been undertaken with some success (Lee et al., 2005). Another promising approach has been to succinylate collagen so that ionic bonds can interact with the cationic antibiotic ciprofloxacin and restrict its diffusion (Sripriya et al., 2004). Controlled in vitro release of the antibiotic over five-days was demonstrated and subsequently tested in vivo with success in rats (Sripriya et al., 2007).

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More examples of the combination of collagen with other polymeric materials include those with alginate microspheres containing SS (Shanmugasundaram et al., 2006) and poly(caprolactone) (Prabu et al., 2006). The former authors advanced the application of SS-impregnated collagen (without alginate) in infected, deep partial thickness burn wounds with considerable success according to the levels of inoculated bacteria (Pseudomonas aeruginosa), pro-inflammatory cytokines (interleukin-6, 1-β and tumour necrosis factor-a) and matrixmetalloproteinases-2 and 9 (MMP-2 and MMP-9) detected. The inherent nature of the collagen dressing to cause complete healing at day 27 (control ≥37 days) and maintain therapeutic levels of SS for 72 hours after application was reported and concluded to ‘accelerate the magnitude and sequence of reparative events’ (Shanmugasundaram et al., 2009). The ability of collagen to attract MMPs has been exploited in commercially available collagen matrix dressings which act as sacrificial substrates, diverting the action of these proteinase enzymes to degradation of the exogenous collagen of the dressing in preference to viable collagen in the wound bed. Conversion of type 1 collagen to gelatine is desirable in the healing process as gelatine has many chemotactic sites that attract fibroblasts and endothelial cells responsible for creating granulation tissue. An overabundance of MMPs-1, -2 and -9, however, contribute to the chronicity of wounds, so it is desirable to inhibit and control their action (Brett, 2008). This can be done in a variety of ways that include incorporation of zinc-binding compounds such as ethylendiaminetetraacetic acid (EDTA) that inactivate the MMPs attracted to the applied collagen by complexing with zinc ions at the core of the enzymes (BiostepTM, Smith & Nephew). Another method employed involves the use of oxidised regenerated cellulose (ORC) to create an acidic environment that hydrolyses and deactivates the proteinases (Promogran PrismaTM, Systagenix) although the low pH of ~2.5 can also hydrolyse growth factors essential to the healing process. Incorporating other biocompatible polymers such as sodium alginate and carboxymethylcellulose in collagen dressings to absorb exudate and control the moisture balance is also employed. One further improvement to the efficacy of such dressings involves the addition of low levels of silver in an appropriate form (see Section 14.3.1) that includes a silver-ORC combination. It is clear that due to its inherent bioactivity and suitability as a carrier matrix for therapeutic agents, collagen will continue to be used as a primary dressing in wound management.

14.7

Alginates

Alginates constitute one of the larger classes of wound dressing and their use as haemostats and dressings in clinical practice dates back to

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the late 1940s and early 1950s. Essentially a linear polysaccharide composed of guluronate and mannuronate units, adjacent chains can be ionically crosslinked by a variety of di- and trivalent metal ions of which calcium is by and large the most common. A major attraction of alginates was the apparent lack of toxicity of these materials when absorbed by living tissue (Blaine, 1947; Thomas, 1990); however, by the 1970s, the small proportion of all alginate fibres (10%) used in medical applications became economically unviable as the bulk-use of alginate fibres, i.e. the textile industry, opted for newer, more economic and betterperforming synthetic yarns. It is only in the last twenty years that interest in the use of alginates in wound management products has been rekindled mainly due to improved production methods in the textile industry and an increased understanding of the mechanisms of wound healing (Thomas, 1990). Alginate wound dressings are composed of the sodium and calcium salts of alginic acid that occurs naturally in brown seaweeds such as kelp. Dressings and haemostats are normally constructed from woven or non-woven fibres and fleeces that absorb wound fluid and form non-occlusive gels which are reputed to limit infection by entrapping endogenous and excluding exogenous bacteria in much the same way as attributed to hydrocolloids (Thomas, 1985; Lawrence and Lilly, 1987; Thomas and Loveless, 1992; Heenan, 1998). It would appear that the use of alginates as matrices for therapeutic agents is as viable as that of hydrogels and hydrocolloids as previously described in this chapter. A recent example where therapeutic agents have been incorporated with alginates as drug delivery dressings concerns an in vitro study on the comparative properties of alginate and silver-containing alginate fibres with respect to the cytotoxicity, antimicrobial activity and binding of pathophysiological factors in chronic wounds (Wiegand et al., 2009). Pro-inflammatory cytokines MMP-2, tumour necrosis factor-alpha (TNF-α) and interleukin-8 were reduced in the wound with alginate alone and the effect was increased with the presence of ionic and nanocrystalline silver. Calcium, present in alginate fibres as the crosslinking agent, is thought to have a pharmacological function that includes increased fibroblast production (Schmidt and Turner, 1986; Doyle et al., 1996) and increased haemostasis (Lansdown, 2002); however, reduction of TNF-α as concluded by Wiegand et al. (2009) appears to contradict the earlier work of Thomas et al. (2000) that reported the activation of macrophages by some alginate dressings to produce increased amounts of TNF-α. Jude et al. (2007) completed a randomised control study concerning the treatment of diabetic foot ulcers with both a non-woven calcium alginate (CA) dressing and similarly non-woven carboxymethylcellulose (CMC) dressing containing silver ions. All ulcer healing outcomes improved in the

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out-patient groups studied but CMC dressings containing ionic silver were observed to reduce the wound depth ‘twice as much as CA-treated ulcers’. In the same year, the purported lack of clarity concerning the effectiveness of silver and silver dressings in contaminated or infected wounds was investigated by Vermeulin et al. (2007) who identified a total of three randomised control trials, including that of Jude et al. (2007), involving a total of 847 participants. Two of the three trials compared commercially available silver impregnated polyurethane foams, the other comparing the results from the ionic silver containing CMC/CA study discussed. The authors concluded that there was insufficient evidence to recommend the use of silver-containing dressings or topical agents for treatment of infected or contaminated chronic wounds (Vermeulin et al., 2007). This argument is compounded by the earlier findings of Parsons et al. (2005) who tested seven proprietary silver-containing dressings, two of which contained alginate, with respect to silver content, rate of release and antibacterial activity against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). They found no direct correlation between silver content, release or antibacterial activity and concluded that dressing choice, with respect to fluid absorption and retention under compression, wound type and condition were as important. It appears that there is still some disagreement as to the efficacy of silver and its compounds in wound healing, although there is plenty of evidence to the contrary as evidenced by the plethora of silver-containing wound management products that are commercially available. The role of calcium, released naturally from CA dressings in contact with wounds, is also still to be completely resolved. Other di- and trivalent metal ions capable of ion-exchange with sodium to produce crosslinked alginate hydrogels have been investigated for antimicrobial properties as has their compatibility with commonly used topical antimicrobials (Goh et al., 2008). A microbiological broth dilution method was used to evaluate the alternative crosslinking agents against S. aureus and P. aeruginosa. Minimum inhibitory concentrations (MICs) revealed that the antibiotics (ciprofloxacin, gentamycin, tetracyline, erythromycin and sodium sulfadiazine) were the most active, followed by the antiseptics (chlorhexidine, chloroxylenol, cetrimide and proflavine) and crosslinking agents (Al3+, Ca2+, Cu2+ and Zn2+). Calcium ions were observed to exercise very weak antimicrobial activity and a higher fractional inhibitory concentration than the other crosslinking agents, although aluminium, zinc and copper were more potent and did not appear to interact with the antimicrobials. The authors concluded that calcium alginate dressings could be potentially incompatible with certain antimicrobials and even antagonistic towards bacteria as evidenced by the calcium/gentamycin combination towards S. aureus. The alternative use of aluminium, copper and zinc ions

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would serve to crosslink sodium alginate without compromising antimicrobial efficacy. Alginates have also recently been used as polyelectrolyte complexes in conjunction with chitosan as a biocompatible vehicle for the delivery of silver sulfadiazine (SS) (Yu et al., 2005), ciprofloxacin (Ozturk et al., 2006) and smad3 antisense oligonucleotides (ASOs) (Hong et al., 2008). In the former study, super-absorbent chitosan-alginate sponges were prepared and their stability in phosphate buffered saline (PBS) and ability to release SS against P. aeruginosa and S. aureus investigated to good effect. Control of a ‘ladder-like’ structure between both polymers by the use of crosslinking enabled a controlled release of antimicrobial. The second study prepared sponges by gelation of SA followed by lyophilisation, crosslinking with calcium ions and subsequent coating with chitosan. Antimicrobial activity was concluded to be proportional to the release rate of ciprofloxacin resulting from increased water uptake. The latter study concluded that the healing process, in an in vivo murine model, was faster than that in control groups due to increased collagen and decreased TGF-β production. The more general debate about the precise function of growth factors can be facilitated by incorporating them in relatively benign alginate dressings and a possible method may be by forming shaped ‘wafers’ or ‘fleeces’ from freeze-dried sodium alginate solutions (Matthews et al., 2005). Another method may involve the production of microspheres which can be incorporated in an appropriate sponge-forming material such as collagen (Shanmugasundaram et al., 2006). Two final examples of alginate as a component of drug delivery dressings (Kim et al., 2008a,b) concern the incorporation of nitrofurazone and clindomycin in crosslinked hydrogels composed of repeatedly ‘freeze-thawed’ poly(vinyl alcohol) (PVA) and sodium alginate (SA). In both studies, published separately, it was concluded that an increased concentration of SA had an insignificant effect on the release of both antibiotic compounds but stated that increased absorption of blood proteins was evident on those hydrogels containing increased amounts of SA. Wounds induced in rats were observed to heal faster than the control. Clearly, the use of alginates as drug delivery dressings will continue to be attractive in wound management.

14.8

Honey

Honey qualifies for a place in this chapter, not because of its ancient and universal use for the treatment of wounds (Thomas, 1990), but because it fits the term ‘drug delivery dressing’ as defined in the introduction, i.e. any substance applied directly to the surface of a chronic wound that contains an active ingredient/s intended to display therapeutic properties in terms

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of positively affecting or supporting the healing process. There have been an increasing number of reviews and published papers from many parts of the world in the last decade, particularly in the last five years, that acknowledge that many different honeys appear to convey improvements in the healing rate of all manner of wound types and even the nature of resulting scar tissue. A recent systematic review (Jull et al., 2008) attempted to determine whether honey increased the rate of healing in most types of acute and chronic wounds. Nineteen trials (n = 2554) were identified that met the inclusion criteria of randomised and quasi-randomised studies with a view to determining wound healing efficacy. The authors concluded that ‘honey may improve healing times in mild to moderate superficial and partial thickness burns compared with some conventional dressings’ but that there was ‘insufficient evidence to guide clinical practice in other areas’. A commentary by Gethin (2008) on a systematic review that identified 33 random controlled trials (RCTs) on the use of honey in wound healing and its potential value in oncology care (Bardy et al., 2008), included an extra nine RCTs not identified by Bardy et al. (2008) and acknowledged the rapidity with which new studies were being published. This author considered the relevance of meta-analysis of clinical studies and its drawbacks with respect to provision of a definitive answer as to the efficacy of honey in general wound management. They concluded that of the 42 RCTs to the date of publication, there was ‘an overall trend that demonstrates efficacy (of honey) in management of burns, venous ulcers, pressure ulcers and more unusual wound aetiologies’. So what is it about the properties of honey that are ensuring its continuing use as a wound management product? Briefly, it is a concentrated solution of sugars (up to 80% of predominantly sucrose, maltose, fructose and glucose) and contains enzymes such as invertase, diastase, catalase and glucose oxidase; eighteen free amino acids and a complex mixture of phytochemicals, many with antioxidant properties (Gheldof et al., 2002; Molan, 2006). The high concentration of sugars in honey render it hygroscopic and so it can dehydrate environmental bacteria; however, sugar content alone does not explain its established antimicrobial properties (Cooper et al., 2002; Simon et al., 2008). Dilution of honey with water activates glucose oxidase which produces gluconic acid and traces of hydrogen peroxide. It has been assumed that catalase present in both honey and wound fluid destroys the activity of hydrogen peroxide and that the production of this antiseptic compound varies from one source of honey to the next (Henriques et al., 2006). Moreover, heat treatment of natural honeys destroys both glucose oxidase and hydrogen peroxide but certain nonperoxide honeys, typified by those originating from Leptospernum scoparium or ‘manuka’, appear to maintain their antimicrobial properties despite the application of heat, light, gamma-irradiation or the addition of catalase

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(Adcock, 1963; Molan, and Allen; 1996; Molan, 1992; Henriques et al., 2006). Medical grade honey is available as both an antibacterial honey tulle (gauze) impregnated with medical grade manuka honey or an impregnated gel sheet of sodium alginate (BNF, 2009). Although there are innumerable sources of honey around the world, there seems to be a general consensus that manuka, originating from Australia and New Zealand where at least 79 species of Leptospernum have been described (Thompson, 1988), is the most noteworthy. Scientists from countries as diverse as Nigeria (Efem, 2009), Malaysia (Tan et al., 2009), South Africa (Basson and Grobler, 2008) and Iran (Jalali et al., 2007) have all recently commented on the anti-inflammatory, anti-allergenic, antibacterial, debriding and cost-effectiveness of particular types. These disparate honeys do not appear to have the remarkable antibacterial effects of manuka, with other honeys varying by as much as 100-fold in antibacterial potency (Simon et al., 2008). Cooper et al. (2008) published an article that tested 139 local Welsh honeys against manuka and concluded from studying the mean TNF-α response from monocytic cells (Monomac 6) that the local honeys produced a range of values from 7.6 to 1437 pg/mL TNF-α with a mean value of 547 pg/mL compared to manuka at 320 pg/mL. Despite these notable differences in the honeys’ wound healing properties; the function of TNF-α being to stimulate fibroblasts, inflammatory mediators, angiogenesis and the activation of neutrophiles (Myers, 2008); it was acknowledged that the local honeys had weak antiseptic potential compared with manuka. Moreover, it was concluded that TNF-α response and antimicrobial activity were not associated. Another recent study accredited the immunomodulatory properties of honeys to naturally present endotoxins (Timm et al., 2008). In summation, there is no doubt that the debate will continue for the foreseeable future. The very fact that the BNF (2009) lists a total of nine commercial products based on ‘medical grade honey’, i.e. a standard mixture of different Leptosporidium honeys with a known antibacterial activity; gamma-irradiated to inactivate Clostridium botulinum spores as a safety precaution; bears some testament to the growing popularity of this very ancient, yet modern, wound treatment.

14.9

Future trends

Drug delivery dressings have been defined within the context of this book chapter and restricted to delivery systems, normally hydrogels of a neutral, ionic or physical nature, containing a therapeutic agent conducive with

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improved wound healing. Clearly, there is a plethora of literature on the abilities of one type of drug delivery dressing or another to hasten the time to closure of acute wounds or improve epithelial processes in chronic wounds. These studies often use more conventional dressing types as controls for the efficacy of the technology of interest and inevitably highlight the advantages of the proposed systems over existing or competing technologies. The reader must be appreciative of the difficulty in organising a clear control group, especially where such studies are in man, due to multivariable wound aetiologies, patient condition, co-morbidities and healing stage. Despite these largely insurmountable difficulties, there can be no doubt that the drive to shorten treatment times and target wound beds with the correct quantities of efficacious agents will continue to provide a challenge to clinical scientists. As our understanding of the complexities of the healing cycle improves with time, as it surely must, there will be a continuous demand for primary drug delivery dressings that work. There is no reason to suspect that existing materials will necessarily be replaced with ever newer synthetic polymers, especially where treatment costs are a concern, and so the existing classes of dressing discussed in this chapter should be around for the foreseeable future. With respect to the therapeutic agents to be delivered topically, it would appear that the use of growth factors remains attractive but hampered by a few key issues relating to their high cost, effective delivery, stability in vivo and an incomplete understanding of their precise roles within the complete biochemical cascades associated with the different stages of healing. In an ideal situation, correct diagnosis of wound aetiology and the health of the patient, coupled with astute wound management practice, may nullify the requirement to deliver therapeutic agents at all. Conversely, where there are clear metabolic deficiencies or signs of significant infection, precise quantities of known active agents will require to be delivered topically and accurately to wound beds irrespective of the degree or rate of exudation. On the latter point, no single dressing type will be suitable for all wounds and one might envisage a series of graded dressing types containing varying quantities of the desired agent(s) targeted at specific wound types and the particular healing stage. Despite all of these ideals, clinical practice might be slow to change and the inevitably increased costs of pioneering drug delivery dressings may prohibit the drive for future development and confine this interesting and challenging area of science and medicine to the laboratory. Optimistically, increased availability of active agents such as key growth factors or safe and effective antimicrobials, and indisputable evidence of their efficacy in chronic wound treatment, will ensure that major interest in drug delivery dressings continues.

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14.10 References Adcock D (1963), ‘The effect of catalase on the inhibine and the peroxide values of various honeys’. J Apic Res, 1, 38–40. Adhirajan N, Shanmugasunderam N and Babu M (2007), ‘Gelatin microspheres crosslinked with EDC as a drug delivery system for doxycycline’, J Microencapsulation, 24(7), 659–671. Agren MS (1990), ‘Studies ion zinc in wound healing’, Acta Derma Venereologica Suppl, 154, 1–36. Amadeu TP, Seabra AB, de Oliveira MG and Monte-Alto-Costa A (2008), ‘Nitric oxide donor improves healing if applied on inflammatory and proliferative phase’, J Surg Res, 149, 84–93. Atemnkeng MA, Plaizier-Vercammen J and Schuermans A (2006), ‘Comparison of free and bound iodine and iodide species as a function of the dilution of three commercial povidine-iodine formulations and their microbicidal activity’, Int J Pharm, 317(2), 161–166. Atiyeh BS, Costagliola M, Hayek SN and Dibo SA (2007), ‘Effect of silver on burn wound infection control and healing: review of the literature’, Burns, 33(20), 139–148. Azevedo EP, Saldanha TDP, Navaro MVM, Medeiros AC, Ginani MF and Raffin FM (2006), ‘Mechanical properties and release properties of chitosan films impregnated with silver sulfadiazine’, J App Pol Sci, 102(4), 3462–3470. Bardy J, Slevin NJ, Mais KL and Molassiotis A (2008), ‘A systematic review of honey uses and its potential value within oncology care. J Clinical Nursing, 17, 2604–2623. Basson NJ and Grobler SR (2008), ‘Antimicrobial activity of two South African honeys produced from indigenous Lucospermum cordifolium and Erica species on selected micro-organisms’, BMC Complimentary and Alternative Medicine, 8, 41, Article No.14. Bayramoglu G, Batislam E and Arica MY (2009), ‘Preparation and drug release behavior of minocyclin-loaded poly[hydroxyethyl methacrylate-co-poly(ethylene glycol)-methacrylate] films’, J App Pol Sci, 112(2), 1012–1020. Beckert SB, Haak S, Hierlemann H, Farrahi F, Mayer P, Königsrainer A and Coerper S (2007), ‘Stimulation of steroid-suppressed cutaneous healing by repeated topical application of IGF-1: Different mechanisms of action based upon the mode of IGF-1 delivery’, J Surg Res, 139, 217–221. Bhattarai N, Gunn J and Zhang M (2009), ‘Chitosan-based hydrogels for controlled, localized drug delivery’, Adv Drug Del Rev, 62(1), 83–99. Blaine, G (1947), ‘Experimental observations on absorbable alginate products in surgery’, Ann Surg, 125, 102–114. Boateng JS, Matthews KH, Stevens HNE and Eccleston GM (2007), ‘Wound healing dressings and drug delivery systems: a review’, J Pharm Sci, 97(8), 2892–2923. Bowler PG, Duerden BI and Armstrong DG (2001), ‘Wound microbiology and associated approaches to wound management’, Clinical Microbiol Rev, 14(2). 244–269. Braund R, Tucker IG and Medlicott NJ (2007), ‘Hypromellose films for the delivery of growth factors for wound healing’, J Pharm Pharma, 59, 367–372. Brett D (2008), ‘A review of collagen and collagen-based wound dressings’, WoundsA Compendium of Clinical Research and Practice, 20(12), 347–356.

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Brett DW (2006), ‘A discussion of silver as an antimicrobial agent: alleviating the confusion’, Ostomy Wound Management, 52(1), available online at http://www.owm.com/article/5125 British National Formulary (BNF), 58 (Sept 2009), Appendix 8.3.1, p.920. Brown CD and Zitelli JA (1993), ‘A review of topical agents for wounds and methods of wounding:guideline for wound management’, J Dermatol Surg Oncol, 19, 732–737. Cabodi M, Cross VL, Qu Z, Havenstrite KL, Swartz S and Strook AD (2007), ‘An active wound dressing for controlled convective mass transfer with the wound bed’, J Biomed Maters Res Part B – App Biomaters, 82B(1), 210–222. Cangul IT, Gul NY, Topal A and Yilmaz R (2006), ‘Evaluation of the effects of topical tripeptide-copper complex and zinc oxide on open-wound healing in rabbits’, Vet Dermat, 17(6), 417–423. Chaby G, Senet P, Vaneau M, Martel P, Guillaume JC, Meaume S, Teot L, Debure C, Dompmartin A, Bachelet H, Carsin H, Matz V, Richard JL, Rochet JM, SalesAussias N, Zagnoli A, Denis C, Guillot B and Chosidow O (2007), ‘Dressings for acute and chronic wounds – a systematic review’, Arch Dermatol, 143(10), 1297–1304. Changerath R, Nair PD, Mathew S and Nair CPR (2009), ‘Poly(methyl methacrylate)grafted chitosan microsperes for controlled release of ampicillin’, J Biomed Mat Res-Part B:App Biomat, 89B(1), 65–76. Chesnoy S, Lee PY and Huang L (2003), ‘Intradermal infection of transforming growth factor-β1 gene enhances wound healing in genetically diabetic mice’, Pharm Res, 20, 345–350. Chiang B, Essick E, Ehringer W, Purphree S, Hauck MA, Li, M and Chien S (2007), ‘Enhancing skin wound healing by direct delivery of intracellular adenosine triphosphate’, Am J Surg, 193, 213–218. Cooper DM, Yu E, Hennessey P, Ko F and Robson MC (1994), ‘Determination of endogenous cytokines in chronic wounds’, Annals Surgery, 219(6), 688–692. Cooper RA, Molan PC and Harding KG (2002), ‘The sensitivity to honey of Grampositive cocci of clinical significance isolated from wounds. J Appl Microbiol, 93, 857–863. Cooper RA, Wheat EJ and Burton NF (2008), ‘An investigation into the wound healing potential of Welsh honeys’. J Apic Res, 47(4), 251–255. Dai CL, Yuan Y, Liu CS, Wei J, Hong H, Li XS and Pan XH (2009), ‘Degradable, antibacterial silver exchanged mesoporous silica spheres for hemorrhage control’, Biomaterials, 30(290), 5364–5375. Dielgelmann RF, Cohen IK and Kaplan AM (1981), ‘The role of macrophages in wound repair: a review’, Plast Reconstr Sur, 68, 107–113. Doyle JW, Roth T and Smith M (1996), ‘Effects of calcium alginate on cellular wound healing processes modelled in vitro’, J Biomed Mater Res, 32, 561– 568. Drognitz O, Thorn D, Kruger T, Gaterman SG, Iven H, Bruch HP and Muhl E (2006), ‘Release of vancomycin and teicoplanin from a plasticised and resorbable gelatin sponge: in vitro investigation of a new antibiotic delivery system with glycopeptides’, Infection, 34(1), 29–34. Drosou A, Falabella A and Kirsner RS (2003), ‘Antiseptics on wounds – an area of controversy’, Wounds, 15(5), 149–166.

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Efem S (2009), ‘Clinical observations on the use of honcrivine (honey + acriflavine) in the chemical debridement of wounds’, Nigerian J Clinical Practice, 12(4), 412–415. Fray MJ, Dickinson RP, Huggins JP and Occleston L (2003), ‘A potent, selective inhibitor of matrix metalloproteinase-3 for the topical treatment of chronic dermal ulcers’, J Med Chem, 46, 3514–3525. Friess W (1998), ‘Collagen: biometarial for drug delivery’, Eur J Pharm Biopharm, 45, 113–136. Gethin, G (2008), ‘Commentary on Bardy J, Slevin, NJ, Mais KL & Molassiotis A (2008): A systematic review of honey uses and its potential value within oncology care. J Clinical Nursing, 17, 2661–2664. Gheldof N, Wang XH and Engeseth NJ (2002), ‘Identification of antioxidant components of honeys from various floral sources. J Agric Food Chem, 50, 5870–5877. Godin B, Touitou E, Rubinstein E, Athamna A and Athamna M (2005), ‘A new approach for treatment of deep skin infections by an ethosomal antibiotic preparation – an in vivo study’, J Antimicrobial Chemotherapy, 55(6), 989–994. Goh CH, Heng PWS, Huang EPE, Li BKH and Chang LW (2008), ‘Interactions of antimicrobial compounds with cross-linking agents of alginate dressings’, J Antimicrob Chemotherapy, 62(1), 105–108. Grzybowski J, Oldak E, Antos-Bielska M, Janiak MK and Pojda Z (1999), ‘New cytokine dressings. I. Kinetics of the in vitro rhG-CSF, rhGM-CSF and rhEGF release from the dressings’, Int J Pharm, 184, 173–178. Gwon HJ, Lim YM, An SJ, Youn MH, Han SH, Chang HN and Nho YC (2009), ‘Characterisation of PVA/glycerine hydrogels using gamma-irradiation for advanced wound dressings’, Kor J Chem Eng, 26(6), 1686–1688. Hammond SA, Morgan JR and Russell AD (1987), ‘Comparative susceptibility of hospital isolates of gram-negative bacteria to antiseptics and disinfectants’, J Hospital Infection, 7(3), 213–225. Hardwicke J, Ferguson EL, Moseley R, Stephens P, Thomas DW and Duncan R (2008a), ‘Dextrin-rhEGF conjugates as bioresponsive nanomedicines for wound repair’, J Controlled Release, 130(3), 275–283. Hardwicke J, Schmaljohann D, Boyce D and Thomas D (2008b), ‘Epidermal growth factor therapy and wound healing – past, present and future’, Surgeon-J Royal Colleges of Surgeons of Edinburgh and Ireland, 6(3), 172–177. Heenan A (1998), ‘Alginates: frequently asked questions’, World Wide Wounds, 1, 1–7. Henriques A, Jackson S, Cooper R and Burton N (2006), ‘Free radical production and quenching in honeys with wound healing potential’. J Antimicrob Chemother, 58, 773–777. Ho KM and Litton E (2006), ‘Use of chlorhexidine impregnated dressing to prevent vascular and epidural catheter colonisation and infection : a meta analysis’, J Antimicrob Chemother, 58(2), 281–287. Hoffman AS (2002), ‘Hydrogels for biomedical applications’, Adv Drug Delivery Reviews, 51(1), 3–12. Hong HJ, Jin SE, Park JS, Ahn WS and Kim CK (2008), ‘Accelerated wound healing by smad3 antisense oligonucleotides-impregnated chitosan/alginate polyelectrolyte complex’, Biomaterials, 29(36), 4831–4837.

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Huang S, Deng T, Wu H, Chen F and Jin Y (2005), ‘Wound dressings containing bFGF-impregnated microspheres’, J Microencapsulation, 23(3), 277–290. Jalali FSS, Saifzadeh S, Tajik H and Farshid AA (2007), ‘Experimental evaluation of repair process of burn-wounds treated with natural honey’, J Animal Vet Adv, 6(2), 179–184. Jeong B, Kim SW and Bae, YH (2002), ‘Thermosensitive sol-gel reversible hydrogels’, Adv Drug Del Rev, 54, 37–51. Jones DS, Lorimer CP, McCoy CP and Gorman SP (2008), ‘Characterisation of the physicochemical, antimicrobial, and drug release properties of thermoresponsive hydrogel copolymers designed for medical device applications’, J Biomed Maters Res Part B – App Biomaters, 85B(2), 417–426. Jude EB, Apelqvist J, Spraul M and Martini J (2007), ‘Prospective randomised controlled study of Hydrofiber® dressing containing ionic silver or calcium alginate dressings in non-ischaemic diabetic foot ulcers’, Diabetic Med, 24(3), 280– 288. Judith R, Nithya M, Rose C and Mandal AB (2010), ‘Application of a PDGF-containing novel gel for cutaneous wound healing’, Life Sciences, 87(1–2), 1–8. Jull AB, Rogers A and Walker N (2008), ‘Honey as a topical treatment for wounds’, Cochrane Database of Systematic Reviews, Issue 4, Article No.CD005083. Kietzmann M and Braun M (2006), ‘Effects of zinc oxide and cod liver oil containing ointment Zincojecol® in an animal model of wound healing’. Deutsche Tierarztliche Wochenschrift, 113(9), 331–334 (English abstract). Kim JO, Choi JY, Park JK, Kim JH, Jin SG, Yong CS, Li DX, Choi YJ, Woo JS, Yoo BK, Lyoo WS, Kim JA and Choi, HG (2008a), ‘Development of polyvinyl alcoholsodium alginate gel-matrix-based wound dressing system containing nitrofurazone’, Int J Pharm, 359(1–2), 79–86. Kim JO, Choi JY, Park JK, Kim JH, Jin SG, Chang SW, LI DX, Hwang MR, Woo JS, Kim JA, Lyoo WS, Yong CS and Choi HG (2008b), ‘Development of clindamycin-loaded wound dressing with polyvinyl alcohol and sodium alginate’, Biol Pharm Bull, 31(12), 2277–2282. Kleinbeck KR, Bader RA and Kao WJ (2009), ‘Concurrent in vitro release of silver sulfadiazine and bupivacaine from semi-interpenetrating networks for wound management’, J Burn Care Res, 30(1), 98–104. Labovitiadi O (2011), PhD thesis, Robert Gordon University, Aberdeen UK. Labovitiadi O, Matthews KH and Lamb A (2009), ‘Modified Franz diffusion for the in vitro determination of the efficacy of antimicrobial wafer against methicillinresistant Staphylococcus aureus’, J Pharm Pharma, 61(Supp.), A-19, 27. Lansdown ABG (1996), ‘Zinc in the healing wound’, Lancet, 347, 706–707. Lansdown AB (2002), ‘Calcium: a potential central regulator in wound healing in the skin’, Wound Repair Regen, 10, 271–285. Lansdown ABG, Mirastschijski U, Stubbs N, Scanlon E and Agren MS (2007), ‘Zinc in wound healing: theoretical, experimental and clinical aspects’, Wound Repair and Regeneration, 15(1), 2–16. Larson MJ, Bowersox JC, Lim RC and Hess JR (1995), ‘Efficacy of a fibrin hemostatic bandage in controlling hemorrage from experimental arterial injuries. Arch Surg, 130, 420–422. Lawrence JC and Lilly HA (1987), ‘Are hydrocolloid dressings bacteriaproof?’, Pharm J, 239, 184.

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Molan PC (1992), ‘The antibacterial activity of honey. 1. The nature of the antibacterial activity’. Bee World, 73, 5–28. Molan PC (2006), ‘The evidence supporting the use of honey as a wound dressing’. Int J Low Extrem Wounds, 5, 40–54. Molan PC and Allen KL (1996), ‘The effect of gamma-irradiation on the antibacterial activity of honey’. J Pharm Pharmacol, 48, 1206–1209. Morgan DA (1999), ‘Wound management products in the drug tariff’, Pharm J, 263, 820–825. Morgan DA (2004), Formulary of wound management products – a guide for healthcare staff, Haslemere, Surrey, Euromed Communications Limited. Myers BA (2008), Wound management – principles and practice, New Jersey, Pearson Prentice Hall. Noda Y, Kiori F and Satoshi F (2009), ‘Critical evaluation of cadexomer-iodine ointment and povidone-iodine sugar ointment’, Int J Pharm, 372, 85–90. O’Meara S, Al-Kurdi D, Ologun Y and Ovington, LG (2010), ‘Antibiotics and antiseptics for venous leg ulcers’, Cochrane Database Syst Rev, (1):CD003557. Ovington LG (2004), ‘The truth about silver’. Ostomy Wound Management, 50(9A), 1S–10S. Ovington LG (2007), ‘Advances in wound dressings’, Clinics in Dermatology, 25, 33–38. Ozturk E, Agalar C, Kececi K and Denkbas EB (2006), ‘Preparation and characterisation of ciprofloxacin-loaded alginate/chitosan sponge as a wound dressing material’, J App Pol Sci, 101(3), 1602–1609. Palfreyman SJ, Nelson EA, Lochiel R and Michaels JA (2006), ‘Dressings for healing venous leg ulcers’, Cochrane Database of Systematic Reviews, (3), Art.No. CD001103. Palmieri B, Gozzi G and Palmieri G (1995), ‘Vitamin E added silicone gel sheets for the treatment of hypertrophic scars and keloids’, Int J Dermatol, 34, 506–509. Pandit A, Asher R and Feldman D (1999), ‘The effect of TGF-beta delivered through a collagen scaffold on wound healing’, J Invest Surg, 12(2), 89–100. Park SN, Kim JK and Suh H (2004), ‘Evaluation of antibiotic-loaded collagenhyaluronic acid matrix as a skin substitute’, Biomaterials, 25(17), 3689–3698. Parsons D, Bowler PG, Myles V and Jones S (2005), ‘Silver antimicrobial dressings in wound management: A comparison of antibacterial, physical and chemical characteristics’, Wounds- Compend Clinical Res Pract, 17(8), 222–232. Peppas NA, Bures P, Leobandung W and Ichikawa H (2000), ‘Hydrogels in pharmaceutical formulations’, Eur J Pharm Biopharm, 50, 27–46. Peppas NA, Hilt ZJ, Khademhosseini A and Langer R (2006), ‘Hydrogels in biology and medicine: from molecular principles to bionanotechnology’, Adv Mater, 18, 1345–1360. Prabu P, Dharmaraj N, Aryal S, Lee BM, Ramesh V and Kim HY (2006), ‘Preparation and drug release activity of scaffolds containing collagen and poly(caprolactone), J Biomed Mater Res A, 79(1), 153–158. Puolakkainen PA, Twardzik DR, Ranchalis JE, Pankey SC, Reed MJ and Gombotz WR (1995), ‘The enhancement in wound healing by transforming growth factorβ1 (TGF-β1) depends on the topical delivery system’, J Surg Res, 58, 321–329. Radu FA, Bause M, Knabner P, Lee GW and Friess WC (2002), ‘Modeling of drug release from collagen matrices’, J Pharm Sci, 91(4), 964–972.

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15 Molecular and gene therapies for wound repair E. K I WA N U K A, F. H AC K L, D. N O W I N S K I and E. E R I K S S O N, Harvard Medical School, USA

Abstract: The basic concept of gene therapy is to introduce into a cell a therapeutic gene whose expression can offer a transient advantage for tissue growth and regeneration or lead to the cure of a disease. In this chapter, we look at current gene therapy for cutaneous wound healing, focusing on the most commonly used genes for wound healing enhancement. We have included a short account of the important ethical issues related to gene therapy and conclude with a discussion on future prospects of gene therapy. Key words: skin gene therapy, tissue repair, wound healing, gene delivery, gene therapy.

15.1

Introduction

The term gene therapy describes a research field aimed at the development of methods for genetic modification of cells for therapeutic purposes. The basis for gene therapy is the possibility to transfect cells with genetic material, to bring about permanent or transient cellular transformation. Transient transformation finds its potential application in the enhancement of temporally limited biological processes such as wound repair and regeneration. The cellular events driving these processes are tightly regulated by highly complex molecular mechanisms. Manipulation of these mechanisms at the genetic level offers several advantages over exogenous application of substances. This chapter describes the current state and future prospects of gene therapy for cutaneous wound healing. The first section is a brief historic account of the early advances in gene therapy. Next follows a review of the various growth factors that regulate the cellular behavior in wound healing. An understanding of these factors is the basis for target gene selection in the various gene therapy approaches. A description of the characteristics of the skin as a target organ for gene therapy is followed by a review of the various methodologies for gene delivery. The most commonly targeted genes for wound healing enhancement are described, followed by a description of the porcine wound healing model established at the Laboratory of Tissue Repair 395 © Woodhead Publishing Limited, 2011

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and Gene Therapy. We have included a short account of the important ethical issues related to gene therapy. The chapter concludes with a discussion on future prospects followed by a list of sources for further information.

15.1.1 History of gene therapy The concept of gene therapy arose during the late 1960s and early 1970s. Friedman and Roblin published their paper in Science in 1972, and proposed guidelines for the future development of gene therapy in humans (Friedmann and Roblin, 1972). Initially the main focus was the treatment of monogenetic, inherited disease. The concept to replace deficient genes with transgenes encoding normal functional proteins, seemed to be a promising approach for permanent cure of several severe and untreatable conditions. Early interest was also placed on gene therapeutic approaches for the treatment of advanced malignancies with poor prognoses. The approach was to use viral vectors for in vitro transformation of target cells obtained from the patient, followed by reimplantation of the genetically modified cells. The first approved phase I gene therapy trial in the US was launched in 1990 (Blaese et al., 1995). Peripheral blood lymphocytes from patients with ADA deficiency were retrovirally transduced with cDNA encoding for the missing enzyme. The same year gene therapy for metastatic melanoma was reported. Tumor infiltrating lymphocytes were retrovirally transduced with interleukin-2 before infusion to the patient (Rosenberg et al., 1990). Long-term follow-up from the first ADA deficiency trial demonstrated that the chosen approach was safe and that a relatively large proportion of the patients’ lymphocytes still expressed the transgene (Muul et al., 2003). The successful treatment of two patients with Severe Combined Immuno Deficiency (SCID), caused by ADA deficiency (SCID-ADA), using hematopoietic stem cells, was published in 2002 (Aiuti et al., 2002). The first effective anti-cancer treatment was published in 2006 (Morgan et al., 2006). Regression of metastatic melanoma was observed with an approach where a retroviral vector encoding a T-cell receptor was used to transduce T-lymphocytes thereby enhancing tumor recognition. Gene therapy suffered a major setback in 1999 with the death of a patient participating in a clinical trial for ornithine transcarboxylase deficiency (OTCD) (Raper et al., 2003). The patient died from multiorgan failure four days after infusion of the adenoviral vector into the hepatic artery. However, the development of new gene therapy protocols for treatment of a wide array of monogenetic as well as multifactorial diseases has continued. In part this progress has depended on the establishment of methods for celland tissue-specific transduction. Examples are technologies to deliver transgenes to brain tissue across the blood-brain barrier or to retinal cells. Today, there are about 1500 approved clinical trial protocols worldwide; 64.5% of

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all ongoing trials concern cancer disease, 8.7% are for cardiovascular disease and 7.9% concern monogenetic disease (Wiley Gene Therapy Clinical Trial Database, www.wiley.co.uk). These trials deploy various approaches for gene delivery. About two-thirds of the trials are based on viral vectors, while 5–10% deploy lipofection as a mode of gene delivery. Gene therapy for wound treatment has thus far mainly been represented by experiments in various animal wound models. The commonly deployed strategy has been to enhance the reparative process by overexpressing genes coding for various growth factors. Recently the results of the first clinical trial for gene therapy in wound healing were reported. The safety and feasibility of peri-ulcer injection of adenoviral vectors containing a transgene coding for platelet derived growth factor-beta was demonstrated (Margolis et al., 2009).

15.1.2 Growth factors in wound healing The peptide growth factors play a pivotal role in the temporal and spatial regulation of cellular behavior during wound healing. Cells are activated by growth factors to migrate, proliferate, differentiate and synthesize components of the extracellular matrix in a highly coordinated manner. Growth factors are biologically active peptides that alter growth, differentiation and metabolisms of the targeted cell. The source of these growth factors is the cells that participate in the reparative process and secreted factors act by autocrine, paracrine, juxtacrine or endocrine signaling. Upon binding to the cell receptors, the growth factors trigger a cascade of intracellular events, which lead to the binding of transcription factors to various gene promoters activating gene expression. Of particular importance are the epidermal growth factor (EGF) family, fibroblast growth factor (FGF) family, transforming growth factor beta (TGF-β) family, platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). The characteristics of these factors are summarized in Table 15.1. Since the development of recombinant growth factors many animal studies on the effect of topically applied factors on wound healing have been performed (Pierce and Mustoe, 1995). Among others, PDGF, TGF-β, EGF, bFGF, insulin growth factor-1 (IGF-1) have been tested. The effects of recombinant growth factors were initially promising. However, direct application of peptide factors to the wound environment carries several limitations. Half-time is generally short necessitating repeated administration. The factors are susceptible to degradation by the abundant proteolytic enzymes in the wound environment. Furthermore, sequestration of growth factors by the wound matrix may hinder binding to receptors at the surfaces of cells. In order for recombinant growth factors to become an attractive option for wound therapy, improved delivery systems, or carriers, will have to be developed (Robson et al., 1998).

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TGF-β family TGF-β1 – β2

KGF

FGF family FGF

EGF family EGF

Growth factor

Platelets Keratinocytes Macrophages Lymphocytes Fibroblasts

Keratinocytes Mast cells Fibroblasts Endothelial cells Smooth muscle cells Fibroblasts

Platelets Macrophages Fibroblasts

Source

Table 15.1 Growth factors in wound healing

Keratinocytes Fibroblasts

Epidermal

Fibroblasts

Epidermal Mesenchymal

Target cell

Reepithelialization and inflammation. Granulation tissue formation, fibrosis and tensile strength. Increased levels in the acute wound, decreased levels in the chronic wound.

Epidermal motility and proliferation.

Cell motility and proliferation, granulation tissue formation and remodeling. Increased levels in the acute wound, decreased levels in the chronic wound.

Cell motility and proliferation. Increased levels in the acute wound, decreased levels in the chronic wound.

Effect during wound haling

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IL-1α and β

VEGF

IGF-1

Other PDGF

Platelets Keratinocytes Macrophages Endothelial cells Fibroblasts Plasma Platelets Platelets Neutrophiles Macrophages Endothelial cells Smooth muscle cells Fibroblasts Neutrophiles Monocytes Macrophages Keratinocytes Macrophages Epidermal cells Fibroblasts

Endothelial Fibroblasts Endothelial Fibroblasts

Fibroblasts Macrophages

Inflammation and reepithelialization. Increased levels in the acute and chronic wound.

Angiogenesis and granulation tissue formation. Increased levels in the acute wound, decreased levels in the chronic wound.

Mitogen.

Chemotactic and inflammation. Granulation tissue formation and matrix remodeling. Increased levels in the acute wound, decreased levels in the chronic wound.

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15.1.3 Skin as a target for gene therapy As the body’s largest and most accessible organ, the skin is an attractive target for gene therapy. The skin is easy to access, allowing direct DNA transfer by many techniques, including injection, microseeding and topical application. Thus, systemic delivery can be avoided. The molecular and cellular biology of the skin has been well characterized. The predominant cells of the dermis, the fibroblasts, and the epidermal keratinocytes can be easily harvested and cultured, which allows for gene transfer in vitro. The superficial location of the skin enables repeated monitoring of the effects of the therapy. The high turnover time of the epidermis limits long-time gene expression, which reduces the risks for long-term side effects (Bevan et al., 1999).

15.2

Methods of gene delivery

Genes can be delivered to the target tissue by either in vitro or in vivo approaches (Tepper and Mehrara, 2002). The in vitro approach utilizes wellestablished cell culture techniques. The cells are isolated, cultured and genetically modified in vitro and subsequently implanted back into the tissue. While cumbersome and more laborious, the in vitro approach enables selective gene transfer to the specific cell type targeted. The efficiency of the transfection can be quantified and by avoiding administration of the vector directly to the patient, the risk of systemic contamination is decreased. Furthermore, the risks of detrimental effects related to systemic vector administration are avoided. The in vivo approach involves direct delivery of the gene to the targeted cell in a comparatively technically straightforward way, facilitating clinical application. Vectors may be administered directly to the site of tissue repair by topical application, injection or carried by biomaterial scaffolds. The low transfection efficiency compared to the in vitro approaches and the lack of complete specificity are limitations of this method. In both in vitro and in vivo approaches, the selection of an appropriate delivery system is paramount for successful gene therapy. The common goal for all gene delivery systems is to incorporate the therapeutic gene and deliver it to the cell by a reproducible method (Braddock et al., 1999). The cells must be able to take up the transgene and express the product in a required amount during a desirable time. Moreover, any gene delivery technology should be non-toxic, atraumatic and should not evoke immune responses. The various delivery systems can be categorized into biological, physical and chemical techniques. Biological methods can be further divided into viral and non-viral methods. Viral methods use viruses as vectors for

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delivery of the genetic material to the recipient cell. Non-viral biological vectors may be bacteria, bacteriophages, virus-like particles or biological liposomes. The non-biological methods for gene delivery may be divided into physical and chemical approaches. The transgenes to be delivered are incorporated into closed circular DNA molecules called plasmids. Physical approaches such as needle injection, electroporation, ballistic, ultrasound or hydrodynamic delivery, employ a physical force to deliver the transgene into the cell through the plasma membrane. Chemical approaches use synthetic or naturally occurring chemical compounds as transgene carriers. The common techniques for gene delivery are summarized in Table 15.2.

15.2.1 Biological methods (viral vectors) By far the most common biological vectors are different types of viruses. Commonly used viruses are retroviruses, adenoviruses, herpes simplex viruses and adeno-associated viruses. Viral gene delivery is based on the natural ability of viruses to infect host cells with genetic material. In order to be used as vectors, viruses first have to be genetically modified. The genes required for viral replication are deleted and replaced by the gene of interest. To compensate for this inability to replicate, the viral vectors are propagated in packaging cell-lines that provide the genes coding for the structural proteins for the viral particle. In viral gene delivery the vector must first bind to cell-surface receptors. The cell-membrane associated molecules that bind viral particles may be divided into attachment factors, primary receptors and co-receptors. Attachment factors are molecules such as lectins and Intercellular Adhesion Molecules (ICAM) that bind with relatively low specificity to aggregate viral particles at the cell surfaces. Primary and co-receptors bind with higher specificity and participate in the internalization of the viral particles. The virus can enter the cell through the cell membrane using different routes. The most common and best-characterized mechanism is clathrin coated endocytosis. Viruses may also enter cells through so-called caveolar endocytosis, through clathrin and caveolar independent endocytosis or through direct fusion with the plasma membrane. After nuclear delivery the transgene may be integrated into the host genome or stay in the nucleosome as an episome, an independently replicating extrachromosomal particle. Retrovirus Members of the retroviruses include murine leukemia virus (MuLV) and lentiviruses. The retroviral single stranded RNA genome is reversetranscribed into double stranded DNA and the transgene is integrated into

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Large amounts of DNA can be delivered technically simple.

Gene gun

Herpes simplex virus 1

Adeno-associated virus

Adenovirus

Retrovirus

Cationic liposomes

Electroporation

Technically simple with local delivery and low immunogenicity. All cell types can be transfected. Long-term gene expression with high efficiency. Transduces many different cell types. Transfects all cell types, dividing and nondividing. Large transgene size and no integration into host genome. Good transfection efficiency in vitro and in vivo. Long-term gene expression with low immunogenicity. Transduces dividing and non-dividing cells and integrates to specific site at chromosome 19. Neurotropism with low immunogenicity. Large transgene size, transduces wide variety of cell types. Long-term expression feasible with low immunogenicity.

Large amount of DNA of different types can be delivered. Large transgenes can be delivered technically simple. Non-toxic.

Local delivery of the transgene and unlimited gene size. Non-toxic and non-immunogenic.

Direct injection of naked DNA/plasmid DNA

Microseeding

Advantages

Gene delivery technique

Table 15.2 Common techniques for gene delivery

Very low transfection efficiency and transient gene expression. Target tissue has to be accessible for direct injection. Non-specific method causing physical damage to cell. Low transfection efficiency and potential foreign body reaction. Low transfection efficiency causing cellular damage. Non-specific method requiring complex equipment. Cell membrane perforation for DNA uptake. Low transfection efficiency with transient transfection. No targeting and transient expression. Risk of insertional mutagenesis. Limited transgene size. Inefficient transduction in vivo, difficult to propagate in culture. Non-permanent expression. Immune response and preexisting antibodies are common. Potential wild-type breakthrough infection. Risk for insertional mutagenesis and limited transgene size. Complex and expensive to manufacture, difficult to grow to high titers. Difficult to manipulate due to complex life cycle. Risk of wild-type breakthrough infection. Pre-existing antibodies.

Disadvantages

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the genomic DNA of host cells. Retroviruses offer the possibility of stable transfections. The first gene therapy protocols for ADA deficiency combined gene and cell therapy. The first generation retroviral vectors were transfected into autologous T-cells, which were then infused into the patients. Viable and transgene expressing transfused T-cells were demonstrated in patients treated for ADA deficiency after more than 10 years. Retroviral vectors have been associated with a risk for insertional mutagenesis. Other potential drawbacks are relatively small maximum transgene size (<8 kb), lower yield of virus particles compared to adenoviruses and the inability to infect non-replicating cells. Nevertheless retroviral vectors have so far been the most frequently used viral vectors in clinical trials. Retroviral gene therapy has mainly been attempted in cancer treatment and to treat several types of inherited single gene disorders.

Adenovirus Adenoviruses are non-enveloped double-stranded DNA viruses that may be used to transfect dividing as well as non-dividing cells with high efficiency. A broad range of cells, in different tissues such as muscle, brain, lung, skin and vessels, are susceptible to adenoviral transfection. Furthermore, high virus titers are obtained with these systems. Thus, the adenoviral technology is a highly interesting option for therapeutic gene delivery. However, the adenoviral genome does not integrate into the host genome, but stays in the nucleoplasm as an independently replicating particle (episome). Furthermore, adenoviral gene delivery may evoke immunologic reactions that hinders repeated applications and contributes to the short-lived effects. The modifications to render the adenoviral vectors replication-defective, and to increase space for transgene insertion, commonly entail deletion of the genes called early gene 1 (E1) and E3. This renders a packaging capacity of 7–8 kb. Further modified adenoviruses have been established primarily with the purpose of reducing immunogenicity, thereby prolonging the life span of transgene expression. The so-called “gutless” or high-capacity HSV, with totally deleted virus-encoded sequences, have produced long-term transgene expression for more than one year in mice. Moreover, these later generation adenoviral vectors may accommodate larger transgenes of up to 36 kb. The high trophism to epithelial cells has made adenoviral vectors the primary choice for delivery of the CFTR gene in clinical trials for treatment of cystic fibrosis. Adenovirus has also been used for gene therapy in vascular disease with the aim to stimulate angiogenesis through the transfection of vasculogenic factors such as vascular endothelial growth factor (VEGF). The capacity to achieve high transgene expression during a relatively short period of time may prove to be of great value in cancer treatment.

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Adeno-associated virus Adeno-associated viruses (AAV) are single stranded DNA viruses that have not been found to cause pathology in humans. The AAV is dependent on helper viruses for replication and for productive infection. AAV contains two genes, rep and cap, that are required for its replication. With adenoviruses as helpers, the adenoviral E1 and E4 gene products stimulate AAV replication. Furthermore, E4 is required for synthesis of the second DNA strand to produce a double-stranded and functioning transgene molecule. The difficulty with AAV as vectors is to completely remove the helper viruses. Co-transfection of packaging cells with AAV and a plasmid containing E1 and E4 may circumvent this problem. AAV vectors may be used for small transgene molecules of 4.1–4.9 kb. Immunogenicity is low compared to adenoviruses and a wide range of cell types are susceptible to infection. AAV protocols have mainly been used in clinical trials for the treatment of various monogenic inherited disorders, such as Sickle-cell anemia, Fabconis anemia, β-thalassemia and cystic fibrosis. Herpes simplex virus Herpes simplex viruses (HSV) replicate in epithelial cells and have the ability to cause lifelong, latent infections in the neurons of sensory ganglia. HSV vectors can, however, be used to transfect into a wide range of other cell types and into dividing as well as non-dividing cells. The relatively large genome of HSV enables transfection of large (30–50 kb) transgenes that are not integrated into the host cell genome. The natural trophism for neuronal cells renders HSV interesting as a vector for cancer gene therapy of neurogenic tumors. In addition to latent infections, HSV may cause cytolytic infections through the secretion of viral proteins called infected cell proteins (ICP). The development of gene therapy for neuronal tumors has mainly involved the use of oncolytic HSV vectors with maintained replicative capacity. Strategies to increase specificity for tumor cells and reduce neurotoxicity include development of attenuated vectors and genetic modifications to loose the replicative ability in non-dividing cells.

15.2.2 Biological methods (non-viral vectors) The limitations of viral vectors have lead to a search for alternative biological vectors. Unconventional biological vectors that have the capability to infect cells with high specificity and maintained efficiency, without evoking immune responses, are being developed. In bactofection, bacteria that are invasive and non-pathogenic deliver the transgene. Bacteria have naturally occurring plasmids of 2–200 kb and may

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accommodate large transgene constructs or multiple genes for gene therapy. Furthermore, bacteria do not require the propagation in a packaging cell line, which simplifies the gene delivery protocol. Another characteristic of bacteria is their ability to act as a so-called expression cassette, expressing the transgene themselves within the host cell. In this way, the foreign transgene DNA evades immune recognition during the processes of endosomal escape and nuclear delivery. The natural trophism of many bacteria for certain cells or tissues enables gene delivery with high specificity. In the absence of natural trophism, specificity may be increased by modification of bacterial surface proteins through conjugation to a cell specific ligand. However, just like viruses, bacterial vectors may provoke toxic reactions and immunological responses. Attenuation of bacteria is one way to reduce toxicity and immunogenicity. Interestingly, native bacterial flora, such as bacteria of the gut or respiratory tract, has an inherent ability for cellspecific transfection without evoking immune response. The use of native bacterial flora to deliver transgenes to the epithelia of the respiratory tract, gastrointestinal tract or vagina, appears to be the most promising future application for bacterial vectors. Clinical trials, using protocols with bacterial vectors, have been launched for the treatment of different cancers and for ulcerative colitis. Another option for non-viral biological gene delivery is to use bacteriophages, natural viruses that exclusively infect bacteria. Bacteriophages are resistant to non-enzymatic degradation, and survive in a wide range of pH, which could make them suitable for oral delivery. However, bacteriophages are usually rapidly eliminated by the reticuloendothelial system, and modified bacteriophages that specifically target cells of interest and escape degradation in spleen, liver and lungs are being developed. Intravenous administration of bacteriophages has been performed in humans without toxic reactions. However, phages are potent antigens that generate antibodies, which reduces efficiency at second administration. Applications based on single administration may become a future niche for bacteriophages as vectors in gene therapy. Transfecting a packaging cell line with a plasmid that contains only the structural proteins of the viral capsid generates virus-like particles (VLP). The capsid proteins may be purified and combined with plasmids containing the transgene to form a vector for gene delivery. This technology is frequently used for vaccination and delivery of genetic adjuvant. VLP may be generated from many different types of viruses with different trophisms. For example, liver may be targeted by hepatitis B VLPs, spleen with papilloma or polyoma VLPs, antigen presenting cells with certain papilloma VLPs and JC virus VLPs may be used for glial cells. Thus, without further modifications, VLP vectors may be utilized to target specific cells and tissues. There are, however, several drawbacks with this technology. The yield of

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functional VLP is low, due to a complex and inefficient manufacturing process and, just as viral vectors, VLPs stimulate the immune response. VLPs are particularly suitable for the transfer of small oligonucleotides such as small inhibitory RNAs, and are therefore potentially interesting vectors for gene therapy based on RNA interference. Biological liposomes are spherical lipid particles produced by certain cell types. These particles could be derived from primary cells obtained from the patient, packed with transgene, and re-delivered to the patient without stimulating the immune response. This is a technology still in its infancy and further development is needed before it can be considered in gene therapy.

15.2.3 Physical methods Direct injection The simplest way of transgene delivery is through direct injection of naked DNA plasmid molecules locally into the tissues. This approach has the advantage of being non-toxic and non-immunogenic. However, the large and hydrophobic DNA molecules are kept from entering cells by several barriers and naked DNA is susceptible to degradation by nucleases. As a consequence the transfection efficiency is very low. Modifications to increase the efficiency of naked DNA delivery include the addition of water-immiscible solvents, non-ionic solutions, surfactants, hypotonic solutions or nuclease inhibitors. Injection of naked DNA is used for vaccinations and has been attempted for gene therapy against certain cancers. Microseeding Microseeding is an in vivo gene transfer method where recombinant viral vectors can be delivered directly to the skin (Eriksson et al., 1998). Using oscillating solid microneedles driven by a modified tattooing the DNA solution can be injected into the cell. The device has the capability to introduce plasmid DNA into the skin or wound bed at a depth of 2 mm and at a rate of 7500 injections per 2.25 cm2 surface area (Yao et al., 1998). The advantage of microseeding is that the technique requires limited preparation and no foreign material is introduced to the tissues (Yao and Eriksson, 2000). Ballistic gene delivery Gold particles may be used as carriers in gene delivery. Plasmids containing the transgene are coated with gold particles and the gold-plasmid complexes are accelerated into the tissue using a gene gun. The generated physical force renders the cellular plasma membrane permeable to the

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plasmids, which are released from the gold particles at the transfection site. This technology is mainly suitable for gene delivery to cells of the skin, mucous membranes or surgically exposed tissue. Side effects such as microscopic hematoma, infection or cellular damage, have been observed only in a minority of transfection sites (Dileo et al., 2003). Electroporation Plasmid based gene transfer may be facilitated by the application of electrical pulses. An electrical field generated between two electrodes opens up the cell membranes to plasmid delivery through mechanisms that are incompletely understood. Electroporation has been studied in a wide range of tissues. A 100–1000 times higher transgene expression compared to injection of naked DNA has been achieved. DNA as large as 100 kb has been effectively delivered into muscle cells (Magin-Lachmann et al., 2004). Drawbacks with electroporation are limited range of the electric field, need for surgical procedure to implant electrode in deep tissues and risk for local thermal injury from high voltage. Ongoing development of this technology aims at optimizing the design of electrodes, the field strength, duration and frequency of electric pulses.

15.2.4 Chemical methods Cationic liposomes By far the most common non-viral gene delivery technology is the use of cationic liposomes. The positively charged cationic lipids are usually combined with co-lipids, which may be non-charged phospholipids or cholesterol, to form liposomes. These liposomes are mixed with transgene containing plasmids to form particles called lipoplexes. The DNA bound in these particles is resistant against degradation by nucleases. Cellular uptake is presumed to occur through endocytosis and the DNA exits the intracellular vesicles before degradation in lysosomal compartments. Although this technology is widely used for in vitro gene transfer, it is limited by its low specificity and the low rate of stable integration into the targeted cell.

15.3

Gene therapy for wound healing

The increased understanding of the complex molecular mechanisms that regulate cells participating in tissue repair, has laid a foundation for therapeutic interventions with the purpose of enhancing wound healing. Such therapies could be applied to increase the rate of wound healing and enhance the quality of newly formed tissue in wounds complicated by

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delayed, insufficient healing. Furthermore, molecular mechanisms could be manipulated to prevent excessive scarring in fibrotic conditions such as hypertrophic scars and keloids. In this context gene therapy offers several advantages over direct administration of peptide factors. Peptides delivered to the wound environment are highly susceptible to proteolytic degradation, lowering the effective dose. Furthermore, sequestration by the wound matrix may prevent the binding to receptors at the surfaces of cells. Gene therapy allows sustained and regulated secretion of factors in their proper spatial and temporal context. This aspect is particularly important as growth factors may have different effects depending on cell-type, concentration and on other simultaneous signals from soluble factors, adhesion molecules and matrix components. Normally, the skin is a barrier against mechanical and chemical insults and the cutaneous tissue provides substantial obstacles to effective insertion of foreign genes. However, upon cutaneous injury, the epidermal barrier is disrupted and the integrity of the skin is reduced which facilitates gene transfer to the wound bed (Khavari et al., 2002). A wide range of gene therapy approaches have been utilized for wounds in the experimental setting.

15.3.1 Genes with wound healing effects Growth factor genes have been widely used in experimental gene therapy to enhance wound healing. The most commonly used approach has been to overexpress genes that stimulate reepithelialization, angiogenesis or fibroplasia. Various different methods for gene delivery have been deployed. Furthermore, transgene containing vectors have been directly applied to wounds for in situ transformation, or used in vitro in approaches based on cell therapy. An evolving field is the use of biomaterial scaffolds as carriers of in vitro transformed cells to the sight of tissue repair. Platelet derived growth factor-B (PDGF-B) was delivered by adenoviral vectors to wounds in the ischemic rat ear model (Liechty et al., 1999). PDGF-B increased the rate of re-epithelialization compared to control. However, the adenoviral particles themselves impaired wound healing, presumably due to a local inflammatory response. Adenovirally delivered PDGF-B was used in the first clinical trial of gene therapy in wound healing, which demonstrated that peri-ulcer injection with replication incompetent vector was well tolerated and improved wound healing (Margolis et al., 2000). It appeared that PDGF-B overexpression enhanced vasculogenesis and the authors concluded that the protocol should be considered for further studies in humans. Several authors have used gene therapy with VEGF to enhance healing in diabetic as well as non-diabetic rodent models (Brem et al., 2009) (Saaristo et al., 2006) (Ailawadi et al., 2002, Romano Di Peppe et al., 2002). Other adenovirally delivered growth factors in animal

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wound models are TGF-β, Placenta growth Factor (PGF) and Keratinocyte Growth Factor (KGF) (Lee et al., 2004) (Cianfarani et al., 2006) (Escamez et al., 2008). Wound healing was improved by delivery of Insulin Growth Factor 1 (IGF1) by local injection of cationic liposome vectors in thermally injured rats (Jeschke et al., 1999). A similar protocol was then used with Keratinocyte Growth Factor (KGF) (Jeschke et al., 2002). Recently, the same group demonstrated that liposomal transfection of IGF1 and KGF in combination, increased neovascularization and wound expression of VEGF more than the transfection of either factor alone (Jeschke and Herndon, 2007). Biological scaffolds have been used to deliver transduced cells to the site of cutaneous wound healing. Accelerated wound healing was achieved by a polyglycolic acid scaffold carrying mouse dermal fibroblasts, which had been retrovirally transfected in vitro with PDGF-B (Breitbart et al., 2003). Increased formation of granulation tissue was observed with collagen matrices, loaded with adenoviral vectors carrying PDGF-B, and applied to the wound surface in a rodent model (Gu et al., 2004). Transgene constructs may be deployed to achieve selective expression in specific cell-types. Adenoviral vectors carrying a growth factor inducible element (FIRE) were used for targeted transgene delivery to wound edge keratinocytes in a murine wound healing model (Jaakkola et al., 2000). VEGF associated with protein ORP150 (inducible endoplasmic reticulum chaperone oxygen-regulated protein 150) was delivered by adenoviral vectors and enabled targeted expression in wound macrophages in diabetic mice (Ozawa et al., 2001). Increased secretion, neovascularization and improved healing were observed. A fibronectin promoter delivered by a retroviral vector was used to target quiescent fibroblasts in vitro (Wei et al., 1999). Grafting of the transformed fibroblasts resulted in sustained transgene expression in a murine wound model. Genes other than growth factors may be used in gene therapy to improve healing. Inducible nitric oxide synthase (iNOS) was locally delivered and reversed impaired wound healing in iNOS deficient mice (Yamasaki et al., 1998). Another group managed to increase NO levels, rate of re-epithelialization and inflammation, by delivering adenoviral vectors carrying e (endothelial) NO in fibrin scaffolds (Breen et al., 2008). Overexpression of human Telomerase Reverse Transcriptase (hTERT) improved healing in an ischemic rat wound model (Mogford et al., 2006). The cyclin-dependent kinase inhibitor p21WAF-1/Cip-1 decreased formation of PDGF-B induced granulation tissue (Gu et al., 2005). The signaling of growth factors may also be inhibited by gene therapy. The truncated TGF-β receptor II, delivered to a rodent wound model by adenoviral vectors, inhibited TGF-β signaling, inflammation and scar formation (Liu et al., 2005).

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15.3.2 Gene transfer of growth factors to full thickness wound in a porcine experimental model A porcine wound model was established at the Laboratory of Tissue Repair and Gene Therapy. Full thickness wounds are created on the back of Yorkshire pigs. A variant with induction of a diabetic state with Streptozotocin has been used to investigate the process of delayed diabetic wound healing. A polyurethane wound chamber is used as an incubator for wound healing in a moist environment. This wound chamber system facilitates analysis of wound fluid as well as topical administration of various agents. The porcine model has been exploited for the development of gene therapy technologies, and for investigations based on transgene growth factor delivery. In an initial approach, EGF was delivered by gold particle bombardment to partial thickness wounds using a gene gun (Andree et al., 1994). The concentration of EGF in the wound fluid increased 190 times and wounds healed 20% faster compared to control. Transgene expression was maintained for 5–6 days. Subsequently, we developed a device for microseeding, which was shown to produce 2–3 times higher concentrations of EGF in partial thickness wound fluid compared to particle bombardment (Eriksson et al., 1998). More recently, we recovered keratinocytes for in vitro transfection with EGF by liposome delivery, before transplantation to full thickness wounds (Vrannckx et al., 2007). Wounds grafted with transfected keratinocytes healed faster compared to control and EGF was sustained in the wound fluid up to 5 days. Using a similar approach with combined gene and cell therapy, IGF-1 overexpression was demonstrated to increase the rate of healing in full thickness wounds in the diabetic porcine model (Hisrch et al., 2008). We used adenoviral technology to overexpress VEGF in full thickness wounds without any effects on neovascularization or rate of wound healing (Vranckx et al., 2005). This finding was contradictory to several reports from other groups demonstrating enhanced healing from VEGF gene therapy. Most probably VEGF overexpression in our model was unable to further improve healing in the wet environment created by the chambers. This illustrates that a gene therapy strategy aimed at growth factor overexpression usually presupposes a state of impaired healing and relative deficiency of the gene of interest.

15.4

Ethical issues

The possibility of therapeutic gene modification raises several ethical issues. The prospect of finding permanent cures for severe or life threatening diseases by gene therapy could come to great benefit for many patients. Moreover, such progress would be highly rewarding for individual scientists, institutions and companies. However, clinical gene therapy trials are associ-

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ated with risks for severe, even life-threatening side effects from vector administration or from the genetic modification itself. Thus, the basic principles of medical ethics such as the principles of autonomy, nonmalefience and beneficence, may be challenged. Particularly complex moral and ethical issues are raised by the prospect of germ line gene therapy and cloning. On one hand, fetal therapy could prevent life threatening disease or dangerous postnatal surgery. On the other hand, the same technologies could be used for eugenic purposes, i.e. for genetic enhancement with the goal to maximize well-being or promote certain traits for individuals or societies. In an attempt to address these concerns many regulatory bodies have been established to overlook the process involved and enforce some basic principles. The Recombinant DNA Advisory Committee was one such initiative drawn up by the NIH, and the Food and Drug Administration (FDA) focuses on safety and efficacy of genetically altered products. Regulations were established whereby research proposals went through a review process consisting of a preliminary approval by the home institution’s Institutional Biosafety Committee and Institutional Review Board before final approval from the Recombinant DNA Advisory Committee. The American Society of Gene and Cell Therapy was established in 1996 and has provided a policy for ethical standards involving gene therapy clinical studies. The guiding principle is that the clinical investigators must be able to design and carry out clinical research studies in an objective and unbiased manner, free from conflicts caused by significant financial involvement with the commercial sponsors of the study. In March 2000, as part of ongoing efforts to ensure patient protection in gene therapy trials, the FDA and NIH announced two new initiatives to strengthen further the safeguards for individuals enrolled in clinical studies for gene therapy: Gene Therapy Clinical Trial Monitoring Plan and the Gene Transfer Safety Symposia. The ultimate aim of all societies concerned with ethical issues is to ensure that clinical trials are conducted maintaining the principles in the constitution of the United Nations Educational, Scientific, and Cultural Organization of the “dignity, equality and mutual respect of men”.

15.5

Future trends

Gene therapy has shown great promise in the experimental setting as a way to enhance wound healing. Clinical trials are under way to assess the safety of various gene therapy protocols in human subjects. Further development will, however, be needed to bring gene therapy from the experimental setting to established clinical practice. Several issues have to be addressed. Technologies for gene delivery will have to be further improved to achieve celltype specific and efficient transformation without eliciting immune response or provoking toxic reactions. Gene therapy in wound healing should not be

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limited to overexpression of single growth factors with the aim to accelerated tissue repair. The identification of new target genes will be an important step to increase the possibilities for quantitatively as well as qualitatively enhanced wound healing. In particular the targeting of genes coding for intracellular factors may become a way to increase the therapeutic specificity. Furthermore, simultaneous delivery of several transgenes, and at different time-points, will increase the possibilities to stimulate several events, acting in concert to enhance healing. Still another approach is the use of transgene constructs, designed for cell type specific transgene expression. Another development is the use of cell and gene therapy in combination with biomaterial scaffolds. These matrices may be utilized to deliver transduced cells or gene therapy vectors to the site of tissue repair for sustained slow-release of gene products. The three-dimensional scaffold-cell/vector composite occupies the wound space and facilitates locally confined delivery of cells or vectors. Moreover, scaffolds stimulate the migration of cells and promote the formation of new extracellular matrix. However, further development of biomaterials will be required to circumvent the problems of inflammatory reactions and induction of scar formation caused by the scaffolds themselves. Lastly, in order for gene therapy to reach the bedside of patients with complicated wounds, the developed technologies and protocols have to be adapted for practical use in the clinical setting.

15.6

References

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Robson, M. C., Mustoe, T. A. & Hunt, T. K. 1998. The future of recombinant growth factors in wound healing. Am J Surg, 176, 80S–82S. Romano Di Peppe, S., Mangoni, A., Zambruno, G., Spinetti, G., Melillo, G., Napolitano, M. & Capogrossi, M. C. 2002. Adenovirus-mediated VEGF(165) gene transfer enhances wound healing by promoting angiogenesis in CD1 diabetic mice. Gene Ther, 9, 1271–7. Rosenberg, S. A., Aebersold, P., Cornetta, K., Kasid, A., Morgan, R. A., Moen, R., Karson, E. M., Lotze, M. T., Yang, J. C., Topalian, S. L. & et al. 1990. Gene transfer into humans – immunotherapy of patients with advanced melanoma, using tumorinfiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med, 323, 570–8. Saaristo, A., Tammela, T., Farkkila, A., Karkkainen, M., Suominen, E., Yla-Herttuala, S. & Alitalo, K. 2006. Vascular endothelial growth factor-C accelerates diabetic wound healing. Am J Pathol, 169, 1080–7. Tepper, O. M. & Mehrara, B. J. 2002. Gene therapy in plastic surgery. Plast Reconstr Surg, 109, 716–34. Vranckx, J. J., Hoeller, D., Velander, P. E., Theopold, C. F., Petrie, N., Takedo, A., Eriksson, E. & Yao, F. 2007. Cell suspension cultures of allogenic keratinocytes are efficient carriers for ex vivo gene transfer and accelerate the healing of fullthickness skin wounds by overexpression of human epidermal growth factor. Wound Repair Regen, 15, 657–64. Vranckx, J. J., Yao, F., Petrie, N., Augustinova, H., Hoeller, D., Visovatti, S., Slama, J. & Eriksson, E. 2005. In vivo gene delivery of Ad-VEGF121 to full-thickness wounds in aged pigs results in high levels of VEGF expression but not in accelerated healing. Wound Repair Regen, 13, 51–60. Wei, M. Q., Lejnieks, D. V., Ramesh, N., Lau, S., Seppen, J. & Osborne, W. R. 1999. Sustained gene expression in transplanted skin fibroblasts in rats. Gene Ther, 6, 840–4. Wiley Gene Therapy Clinical Trial Database. Yamasaki, K., Edington, H. D., McClosky, C., Tzeng, E., Lizonova, A., Kovesdi, I., Steed, D. L. & Billiar, T. R. 1998. Reversal of impaired wound repair in iNOSdeficient mice by topical adenoviral-mediated iNOS gene transfer. J Clin Invest, 101, 967–71. Yao, F. & Eriksson, E. 2000. Gene therapy in wound repair and regeneration. Wound Repair Regen, 8, 443–51. Yao, F., Svensjo, T., Winkler, T., Lu, M., Eriksson, C. & Eriksson, E. 1998. Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. Hum Gene Ther, 9, 1939–50.

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16 Antimicrobial dressings M. I P, Chinese University of Hong Kong, Hong Kong

Abstract: A wide range of antimicrobial dressings is commercially available and has been heavily marketed for treatment of various wounds. The choice of dressings depends on the type of dressing material, antimicrobial agent incorporated, mode of delivery to wound, and characteristics of wound, e.g. amount of exudates, bacterial bioburden or infection. With advances in molecular therapy and technology, dressings of specific characteristics will be made available, and a ‘mix and match’ approach of ‘antimicrobials’ and ‘dressing materials’ specifically targeted to a particular wound may be the solution to the ideal management of wound care. Well designed prospective studies based on direct head-on-head comparisons of these dressings in a well-defined wound type would be warranted to elucidate the applicability of these dressings. Key words: antimicrobial dressings, silver, antiseptic(s), burn(s), ulcer(s), chronic wound.

16.1

Introduction

16.1.1 Historical perspective of antimicrobial dressings Dressings with or without topical applications of drugs, e.g. antiseptics, constitute a major component in the local management of acute and chronic wounds. Dressings act as barriers against exogenous bacteria (Sharp, 2001) and topical agents may be used with or coated on dressings to treat or prevent wound infections, so that these microorganisms are destroyed or their growth limited. Silver is one of most popular topical agents added or incorporated into dressings. The use of metallic silver in the treatment for diseases dates back to 1000 BC, and its use as an antimicrobial agent has long been recognized (Klasen, 2000a,b; Lansdown, 2002). Back in the early 19th century it had been shown that a diluted solution of silver nitrate could effectively inhibit the growth of Staphylococcus aureus (Klasen, 2000a,b). Silver-wire sutures and silver-foil dressings have been used to help prevent postoperative wound infections, and one of the first documented uses in wound care was silver nitrate soaks to prevent infection in burns (Moyer, 1965; Moyer et al., 1965). This was subsequently replaced with silver sulphadiazine cream (Fox, 416 © Woodhead Publishing Limited, 2011

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1968). Silver sulphadiazine cream is relatively insoluble and releases silver ions slowly. Later in the early 1990s, silver-coated dressings were introduced and with technological advances in the application of topical silver, e.g. nanocrystalline silver, a wider range of wound care dressings have been made commercially available and the composition of the dressing has become much more sophisticated.

16.1.2 Rationale of effects of applications of antimicrobials to dressings Antibiotic resistance is a global problem and is an alarming concern in clinical practice. Increasingly, agents with ‘antimicrobial’ effect are being coated on materials and medical devices (Darouiche, 1999) as a prophylaxis for prevention of bacteria from growing or for therapeutic use. The presence of bacteria in a wound may result in contamination, colonization whereby bacteria multiply and become a ‘bioburden’ to the wound, or infection when there is tissue damage, inflammatory response from the host and disrupted healing. Bacteria release a variety of enzymes and toxins, and can seriously impede wound healing. A bacterial population size of 105 colony forming units (cfu)/g or cm2 indicates an infected wound and 104 cfu/g in complex wounds (White, 2002). The bacterial load can be reduced by debridement and the use antimicrobial dressing can produce a sustained release of the agent, e.g. silver, to suppress growth. In chronic wounds, prolonged inflammatory response and elevated levels of matrix metalloproteinase and inflammatory cytokines lead to tissue destruction. The infection may also spread locally and systemically, resulting in fever and severe sepsis. Other host factors, e.g. diabetes, may also impact on and further delay wound healing. Bacteria in wounds usually exist as complex communities within biofilms, consisting of several types of bacteria embedded in a polysaccharide matrix excreted by the microbes (Woodward, 2005). These sessile bacteria develop a slower metabolic and growth pattern, and are protected by the matrix, which inhibits both penetration and susceptibility of systemic antibiotics. Antimicrobial dressings claim to manage the bioburden of the wound locally: they are thought to reduce the risk of invasive infection by minimizing the bacterial colonization of wounds. Specialist products containing antimicrobials include the Acticoat range (delivering nano-crystalline silver) and the Iodosorb range (cadexomer iodine). Several types of product have now been produced with added silver, including; Contreet (Hydrocolloid with silver), Avance (Foam with silver) and Aquacel Ag (Fibre dressing with silver, ConvaTec). The advantage of coating or impregnation of antimicrobial agents onto the dressing is that it enables a continual exposure of the agent directly at

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the wound surface, and provides adequate contact time for action. Topical agents that are used to irrigate or cleanse wounds may have a very limited brief contact exposure for prolonged inhibitory effects. At the same time, toxicity and adverse effects from systemic absorption of the agent may be minimized. One benefit of silver was that it did not appear to promote the development of bacterial resistance (White, 2001a). Because systemic antibiotics are ineffective in reducing bacterial counts in granulating wounds, the use of a suitable topical antibacterial agent may substantially decrease wound sepsis and benefit overall management (Manafin, 2008). Thus, the role of these dressings may be twofold: in prophylaxis of contaminated wounds and in chronically infected wounds. Antimicrobial dressings may serve as a protection for contamination on cultured skin substitutes, e.g. grafting, used for wound closure (Fong, 2006). They may reduce the bioburden to those at risk of infection, e.g. in burns and wounds from major trauma, and facilitate healing, whilst in the latter category, also as treatment to those with signs of infections.

16.2

Types of currently available dressings/ formulations

A large array of dressings is available in the market, and a number of factors need to be considered when evaluating the efficacies of these ‘unformulated’ dressings in a particular clinical application. In general, the purpose of a dressing is to be placed over a wound to prevent soiling of clothing, and to aid healing and prevent contamination. An ‘ideal’ dressing has been described (Goodman, 1992) as having these properties as outlined: • • • • • • • • • • •

Allow excess exudates to be removed from the wound surface Provide a moist micro-environment Be sterile/contaminant free No dressing material should be left in the wound Reduce wound pain Be easy to change Not cause an allergic reaction Act as a semi-permeable membrane Cause no trauma when removed Be impermeable to micro-organisms Provide thermal insulation.

Besides the above considerations, a particular dressing may be of limited value in healing if the underlying pathology, e.g. vascular supply, poor control of diabetes mellitus, necrotic tissue debridement , etc., has not been adequately addressed.

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The types of dressings available for wound care include gauzebased dressings, e.g. dressing pads, tulle, paraffin gauze, or more modern dressings, e.g. hydrocolloid, hydrogels, alginate dressings, foam, and bead dressings. In general, from a previous review by Wiechula (2003), it was concluded that moist wound healing has a distinct clinical advantage over non-moist products with respect to wound healing, pain, comfort, and infection rates. Semi-permeable film dressings, which are applied to catheter care, e.g. chlorhexidine dressings, will not be discussed in this context. The types of ‘antimicrobial formulation’ incorporated in dressings currently are as follows: • • • • •

silver-based, as silver coat, impregnation, nano-crystalline form chlorhexidine iodine-based polyhexamethyl biguanide (PHMB) triclosan.

Examples of common antimicrobial dressings available commercially are listed in Table 16.1.

16.2.1 Evidence on clinical efficacy of antimicrobial dressings Besides the intrinsic variables associated with the hosts’ response to wound healing, and extrinsic risks to infections which are not readily adjustable in evaluations of clinical trials, the efficacy of any ‘antimicrobial’ dressings depends largely on the combined effects of the materials of the dressing itself, the choice of the ‘antimicrobial’, and how it was formulated onto the dressing. There are a large number of studies that examined various types of dressings against their comparators, and in different types of wounds. However, there are many methodological shortcomings for comparisons in many of these studies, with limitations due to small sample size, inadequate randomization, unclear definition of wound types and characteristics (e.g. depth), and variable outcome measures for wound healing, ranging from different timepoints, re-epithelization endpoints, complete healing, and systemic infection rates. However, some trends may be drawn based on the metaanalyses of these studies with different dressings applied in various wound types. Table 16.2 lists some of studies that provide clinical evidence on silver and other ‘antimicrobials’ dressings. The evidence will be discussed under categories of wound types for which these dressings have commonly been applied.

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© Woodhead Publishing Limited, 2011

silver-impregnated activated charcoal cloth silver coated nylon fibres

hydro-activated silver

silver complex

Impregnated silver sulfadiazine (SSD)

Acticoat

Silver-coated, silver impregnation, nanocrystalline silver (SEE also Alginate) 1.2% Ionic silver

Silver

Avance A

Silvercel

Actisorb silver 220

Biatain Ag (prev Contreet Ag) Contreet hydrocolloid BioTain Ag

Urgotul SSD

Melgisorb Ag

Mepilex Ag

Aquacel Ag

Allevyn Ag

Examples

Formulation

‘Antimicrobial / Antiseptic’

Table 16.1 Common types of antimicrobial dressings

Smith & Nephew Healthcare http://global.smith-nephew.com Smith & Nephew Healthcare http://global.smith-nephew.com ConvaTec, USA http://www.convatec.com Molnlycke Health Care http://www.molnlycke.com Molnlycke Health Care http://www.molnlycke.com Urgo Medical http://www.urgo.co.uk Coloplast www.coloplast.com Coloplast www.coloplast.com Coloplast www.coloplast.com Systagenix Wound Management http://www.systagenix.com Systagenix Wound Management http://www.systagenix.com Avance

Reference

© Woodhead Publishing Limited, 2011

Suprasorb A + Ag

Alginate containing ionic silver Alginate with nanocrystalline silver

Smith & Nephew Healthcare, UK http://global.smith-nephew.com Mundipharma GmbH, Germany www.mundipharma.net/

HemCon Medical Technologies, Inc. www.hemcon.com/

XCell Cellulose Wound Dressing and XCell Cellulose Wound Dressing Antimicrobial (BWD-PHMB) Iodoflex Iodosorb Repithel

HemCon

Cellulose wound dressing, 0.3% polyhexamethylene biguanide (PHMB)

0.9% cadexomer-iodine

Liposomal formulation with 3% polyvinyl-pyrrolidone iodine (PVP-I) in a polyacrylic acid-based hydrogel Compressed chitosan acetate dressing

Polyhexamethyl biguanide (PHMB)

Iodine

Chitosan acetate

Smith & Nephew Healthcare http://global.smith-nephew.com Lohmann & Rauscher GmbH and Co. KG (Germany) http://www.lohmann-rauscher.com

Bactigras

Lohmann & Rauscher GmbH and Co. KG (Germany) http://www.lohmann-rauscher.com Lohmann & Rauscher GmbH and Co. KG (Germany) http://www.lohmann-rauscher.com Smith & Nephew Healthcare http://global.smith-nephew.com Smith & Nephew Healthcare http://global.smith-nephew.com Coloplast www.coloplast.com ConvaTec http://www.convatec.com

Chlorhexidine impregnated

Comfeel Alginate Dressing Kaltostat

Acticoat Absorbent with SILCRYST Algosteril

Suprasorb A

Alginate alone

Chlorhexidine

Alginate

© Woodhead Publishing Limited, 2011

Burns Silver-coated, silver impregnation, nanocrystalline silver

Clinical condition

Retrospective study on paediatric nonoperative partial-thickness burn

Meta-analysis Review on dressings for superficial and partial thickness burns

Type of study; Formulation

Review of 20 cases, 10 on Aquacel Ag hydrofibre dressing vs. 10 given conventional Xeroflo gauze (meshed gauze impregnated with

26 randomized controlled trials (RCTs) from 1950 to May 2008.

Details of study

Superficial and partial thickness burns may be managed with hydrocolloid, silicon nylon, antimicrobial (silver), polyurethrane film and biosynthetic dressings. Biosynthetic dressings are associated with a decrease in time to healing and reduction in pain during dressing changes. Use of silver sulphadiazine (SSD) for full duration of treatment showed delays in time to wound healing and increased no. of dressing applications using SSD dressings. Limitations of small trials and methodological limitations. Hydrofibre dressing group has reduced length of hospital stay (2.4 vs. 9.6 days) and shortened time to re-epithelialization (10.3 vs. 16.3 days). Also

Outcome

Table 16.2 Clinical evidence on silver and other ‘antimicrobial’ dressings

Saba SC, Tsai R, Glat P. Clinical evaluation comparing the efficacy of Aquacel Ag hydrofibre dressing versus petrolatum gauze

Wasiak J, Cleland H, Campbell F. Dressings for superficial and partial thickness burns. Cochrane Database of Systematic Reviews: 2008; Issue 4, Art. No.: CD002106. DOI:10.1002/14651858. CD002106.pub3

Reference

© Woodhead Publishing Limited, 2011

Leg ulcers Mixed

Polyvinylpyrrolidone iodine (PVP-I) hydrogel vs. SSD

Systematic review of treatments for leg ulcers (venous, arterial or mixed)

RCT in partialthickness burn

6 RCTs of silver dressings (to May 2006) inc. 695 patients. Dressings included Contreet foam, Allevyn hydrocellular, silver releasing hydroalginate dressing (Silvacel), calcium alginate (Algostril), silver-impregnatedactivated charcoal xerodressing (SIAX) and conventional therapy. Outcome measurements at 4 to 6 weeks.

Unblinded RCT with 43 patients in two groups. Endpoint was time to complete wound healing, with intraindividual comparison with SSD, degree of epithelialization.

petrolatum, 3% bismuth tribromophenate) with bacitracin zinc ointment.

No significant benefit for silver dressings (poled RR 1.6, 95% CI 0.68–4.05, p = 0.27) from 2 of the RCTs. Relative reduction in ulcer area at 4 weeks significant (45% vs. 25%, p = 0.034) in one RCT. Inconsistent evidence and generally poor study design, incomplete reporting, and lack of statistical power.

reduced no. in-house dressing changes (2.7 vs. 17.1) and nursing time, reduced pain and analgesics associated with dressing change. Complete healing rate for PVP-I group 9.9 ± 4.5 vs. 11.3 ± 4.9 days (P < 0.015). Cosmetic result, local tolerability and handling favourable with PVP-I.

Chambers H, Dumville JC, Cullum N. Silver treatments for leg ulcers: a systematic review. Wound Rep Reg 2007;15:165–173. Jørgensen B, Price P, Andersen KE, Gottrup F, Bech-Thomsen N, Scanlon E, Kirsner R, Rheinen H, RoedPetersen J, Romanelli M, Jemec G, Leaper DJ, Neumann MH, Veraart J, Coerper S, Agerslev

Homann HH, Rosbach O, Moll W, Vogt PM, Germann G, Hopp M, Langer-Brauburger B, Reimer K, Steinau HU, Homann HH, Rosbach O, Moll W, Vogt PM, Germann G, Hopp M, Langer-Brauburger B, Reimer K, Steinau HU. Ann Plast Surg. 2007 Oct;59(4):423–7.

with antibiotic ointment in partial-thickness burns in a pediatric burn center. J Burn Care & Res 2009;30:380–385.

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Venous leg ulcers

Clinical condition

42 RCTS (to Apr 2006), including hydrocolloids (23), foams (6), miscellaneous (3) vs. standard dressings.

32 RCTs (from 1950 to Sept 2009), including 5 using systemic antibiotics, remaining topical preparations: cadexomer

Systematic review on antibiotics and antiseptics for venous leg ulcers

Details of study

Dressings for healing venous leg ulcers

Type of study; Formulation

Table 16.2 Continued

Insufficient evidence to suggest that any one dressing type was better. No evidence to suggest hydrocolloids are more effective than simple low adherent dressings used beneath compression (relative risk for healing with hydrocolloid 1.09) (95% CI 0.89–1.34). Two trials suggested cadexomer iodine compared to standard care with patients had significantly higher

Outcome

O’Meara S, Al-Kurdi D, Ologun Y, Ovington LG. Cochrane Database of Systematic Reviews: 2010; Issue 1, John

RH, Bendz SH, Larsen JR, Sibbald RG. The silver-releasing foam dressing, Contreet Foam, promotes faster healing of critically colonised venous leg ulcers: a randomised, controlled trial. Int Wound J. 2005;2:64–73. Palfreyman SJ, Nelson EA, Lochiel R, Michaels JA. Cochrane Database of Systematic Reviews: 2006; Issue 3, John Wiley & Sons Ltd, Chichester, UK DOI:10.1002/14651858. CD001103.pub2

Reference

© Woodhead Publishing Limited, 2011

Arterial leg ulcers

Systematic review on wound dressings or topical agents to promote healing and prevention of wounds

VULCAN study – non-blinded RCT costeffectiveness analysis on venous leg ulcers RCT carried out over 30 mths (Mar 2005–7), and compared 6 silver dressings (Aquacel Ag, Acticoat, Acticoat 7, Acticoat Absorbent, Contreet foam, and Urgotul SSD) vs. non-silver, nonantimicrobial, low adherence dressings beneath compression therapy for treatment of leg ulcers, with endpoint of healing at 12 weeks. No trials were found in Trials Registers of the Cochrane Wounds Group (Apr 2002), Peripheral Vascular Disease (Nov 2006) and Central Register of Controlled Trials (Cochrane Library, Issue 4, 2006) to compare dressings for

iodine (10), povidone iodine (2), peroxidebased preparations (3), ethacridine lactate (1), mupirocin (1), chlorhexidine (1).

No RCTs were found to compare dressings for arterial ulcers. No strong evidence for use of topical agents including ketanserin, lavender, or chamomile essential oil.

complete healing at 4–6 weeks (RR 6.72, 95% CI1.56–28.95). No evidence to support routine use of systemic antibiotics. Further studies recommended. No significant difference was obtained. Complete ulcer healing 59.6% for silver vs. 56.7% for control dressings. Cost of silver dressings was much higher than control group.

Nelson EA, Bradley MD. Cochrane Database of Systematic Reviews: 2007; Issue 1, John Wiley & Sons Ltd, Chichester, UK DOI:10.1002/14651858. CD001836.pub2

Michaels JA, Campbell B, King B, Palfreyman SJ, Shackley P, Stevenson M. Randomized controlled trial and cost-effectiveness analysis of silverdonating antimicrobial dressings for venous leg ulcers (VULCAN trial). Br J Surg 2009;96: 1147–1156.

Wiley & Sons Ltd, Chichester, UK DOI:10.1002/14651858. CD003557.pub3

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Diabetic foot ulcers

Clinical condition

RCT on nonischaemic diabetic foot ulcers

Systematic review of silver based wound dressings and topical agents for treating diabetic foot ulcers

Type of study; Formulation

Table 16.2 Continued

134 patients with type 1 or 2 diabetes mellitus with foot ulcers and treated with either Aquacel Ag hydrofibre or calcium alginate, and secondary foam dressing for 8 weeks.

arterial ulcers. Two small studies of topical agents involving use of ketanserin ointment vs. vehicle (polyethylene glycol) alone, and use of lavender vs. German chamomile essential oil. No RCT was found to evaluate type 1 or 2 diabetes with foot ulcers using placebo or a sham or non-silver-based dressing between 1966 and 2005 August.

Details of study

Mean time to complete healing 53 vs. 58 days, p = 0.34; proportion completely healed 31% vs. 22%, p = 0.305. Reduction in ulcer depth greater with silver dressing (0.25 cm vs. 0.13 cm, p = 0.042).

No data available to examine if silver containing dressings are beneficial to diabetic foot ulcer healing.

Outcome

Bergin S, Wraight P. Silver-based wound dressings and topical agents for treating diabetic foot ulcers. Cochrane Database of Systematic Reviews: 2006; Issue 1, Art. No. CD005082. DOI:10.1002/14651858. CD005082.pub2 Jude EB, Apelqvist J, Spraul M, Martini J; Silver Dressing Study Group. Prospective randomised controlled study of Hydrofiber dressing containing ionic silver or calcium

Reference

© Woodhead Publishing Limited, 2011

Acute, chronic, or infected wounds

One RCT on 17 patients using silver dressing (Acticoat) vs. foam dressing (Allevyn) in acute donor site wounds.

Non-blinded RCT on 619 patients with delayed healing ulcers (48% venous ulcers, 19% mixed venous/arterial ulcers, 8% diabetic foot

CONTOP study

99 studies identified between Jan 90 and Jun 06. No high-quality (Level A) RCT. 79 poor quality RCTs (Level C).

Acute donor site wound

Systematic review on dressings for acute and chronic wounds

Median reduction in ulcer area reduced with silver foam dressing (47% vs. 32% in LBP group, p = 0.0019). Ulcer area differed in the groups at baseline

Longer duration for healing in silver dressing group (14.5 ± 6.7 vs. 9.1 ± 1.6 days, p = 0.004).

Little evidence that any of the modern dressings was better than another, or better than saline or paraffin gauze, in terms of general performance criteria. Limitations by poor quality methodology papers.

alginate dressings in non-ischameic diabetic foot ulcers. Diabet Med 2007;24:280–8. Chaby G, Senet P, Vaneau M, Martel P, Guillaume JC, Meaume S, Te´ot L, Debure C, Dompmartin A, Bachelet H, Carsin H, Matz V, Richard JL, Rochet JM, SalesAussias N, Zagnoli A, Denis C, Guillot B, Chosidow O. Dressings for acute and chronic wounds: a systematic review. Arch Dermatol 2007;143:1297–1304. Innes ME, Umraw N, Fish JS, Gomez M, Cartotto RC. The use of silver coated dressings on donor site wounds: a prospective, controlled matched pair study. Burns 2001;27:621–627. Münter K-C, Beele H, Russell L, Crespi A, Grochenig E, Basse P, Alikadic N, Fraulin F, Dahl CA, Jemma AP. Effect of a sustained

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Clinical condition

Systematic review of topical silver for treating infected wounds

Type of study; Formulation

Table 16.2 Continued

ulcers, 7.5% pressure ulcers, 17.5% others inc. burns, donor sites and postoperative wounds). Two groups using sustained silver-releasing foam dressing (Contreet Foam, Coloplast A/S) vs. ‘local best practice, LBP’ (gauze, moist woundhealing products and antimicrobial treatments) for 4 weeks. 3 RCTs (to September 2006) comprising 847 participants, comparing: (1) Silver-containing foam (Contreet) with hydrocellular foam (Allevyn) with leg ulcers. (2) Silver-containing alginate (Silvercel) with alginate (Algosteril) alone. (3) Silver-containing foam dressing (Contreet) with best local practice in chronic wounds.

Details of study

Silver-containing foam dressings did not significantly increase complete or faster ulcer healing as compared with standard foam dressings or best local practice after 4 weeks follow-up. One study found overall size of ulcer reduced more quickly when dressed with a silver-containing dressing.

(median 20 cm2 silver foam dressing vs. 12 cm2 LBP group; ranges 0.1–700 cm2 vs. 0.1–400 cm2; mean 52.9 cm2 vs. 36.6 cm2, p < 0.05). Exudate handling, ease of use, odour and pain improved.

Outcome

Vermeulen H, van Hattem JM, Storm-Versloot MN, Ubbink DT. Topical silver for treating infected wounds. Cochrane Database of Systematic Reviews: 2007; Issue 1, Art. No.: CD005486. DOI:10.1002/14651858. CD005486.pub2

silver-releasing dressing on ulcers with delayed healing: the CONTOP study. J Wound Care 2006;15:199–206.

Reference

© Woodhead Publishing Limited, 2011

8 RCTs selected from 1957 screened, between 1950 and Jun 2007, 1399 patients with non-healing chronic wounds, or wounds with critical colonization or infection. Intervention included silver versus non-silver or traditional wound management.

RCT, open-labelled, phase III study with 26 subjects per arm. Endpoint was upto 2 weeks or complete healing. 45.7% in hydrofibre Ag group vs. 62.5% in povidone iodine gauze group had infected wound (P = ns) at baseline.

Meta-analysis on silver dressings on chronic wounds

Hydrofibre Ag dressing (Aquacel Ag) vs povidone-iodine gauze on open surgical and traumatic wounds

Silver dressings significantly improved wound healing (CI:0.16–0.39, p < 0.001), reduced odour (CI:0.24– 0.52, p < 0.001) and pain related symptoms (CI:0.18– 0.47, p < 0.0001), decreased wound exudates (CI:0.17–0.44, p < 0.0001) and had a prolonged dressing wear time (CI:0.19–0.48, p < 0.028) when compared with alternative management approaches. An overall improvement in quality of life (CI: 0.04–0.33, p = 0.013). Hydrofiber Ag dressing was significantly better than povidone–iodine gauze for overall ability to manage pain (P < 0.001), comfort (P ≤ 0.001), wound trauma on dressing removal (P = 0.001), exudate handling (P < 0.001) and ease of use (P ≤ 0.001). Complete healing at study completion:23% (Hydrofibre Ag) vs. 9% (povidone–iodine gauze) (P = ns). Limitation by short follow-up and small sample size with infections. Jurczak F., Dugre´ T., Johnstone A., Offori T., Vujovic Z., Hollander D., on behalf of the AQUACEL Ag Surgical/ Trauma Wound Study Group. (2007). Randomised clinical trial of Hydrofibre dressing with silver versus povidone-iodine gauze in the management of open surgical and traumatic wounds. Int Wound J. 4(1):66–76.

Lo Shu-Fen, Chang Chee-Jen, Hu Wen-Yu, Hayter Mark and Chang Yu-Ting. The effectiveness of silverreleasing dressings in the management of non-healing chronic wounds: a metaanalysis. J Clin Nursing 2009;18:716–728.

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Burn wounds Burn wounds lose large amounts of fluid through evaporation and exudation, so dressings must absorb fluid but also maintain a high humidity at the wound site to encourage granulation and assist epithelialization. Antimicrobial dressings that can reduce the bioburden of the wound by minimizing the bacterial colonization of wounds might be able to reduce the risk of invasive infection, and improve wound healing. Silver-coated dressings are extensively used in the management of burn wounds (Ross, 1993; Caruso, 2004). These dressings vary in containing compounds of silver nitrate or sulphadiazine, to sustained silver-ion release preparation (White, 2001b) and silver-based crystalline nanoparticles (Klaus, 1999). In general, of the commonly used forms of topical silver applications, silver-coated dressings demonstrated to be effective at killing a broader range of bacteria than the cream base, were less irritating than the silver nitrate solution and were better tolerated (Wright, 1998). In a Cochrane systematic review of dressings for superficial or partial thickness burns (Wasiak, 2008) based on 26 randomized control studies (RCTs), burn wounds dressed with hydrogels, hydrocolloids, silicon coated dressings, biosynthetic dressings and antimicrobial dressings healed more rapidly than those dressed with silver sulphadiazine (SSD) or chlorhexidine impregnated gauze dressings. In addition, there is also an advantage to the number of dressing changes, ease of use and pain experienced, and the ability to absorb and contain exudates (Wasiak, 2008) with these dressings. Overall, the studies showed delays in time to wound healing and increased number of dressing applications in patients treated with SSD dressings. Thus, it might be concluded that if application of silver is to be effective, silver or nanocrystalline silver has to be impregnated or coated onto one of these modern biosynthetic, hydrogel, hydrocolloid or silicon coated dressings. Three of the 26 RCTs compared a silver-impregnated dressing (Acticoat, Smith & Nephew) with SSD (Li, 2006; Muangman, 2006; Varas, 2005) and included a total of 162 patients. Li et al. (2006) included 98 patients with residual burn wounds and found mean healing times significantly shorter in those treated with silver dressings compared with SSD (mean time to wound healing: 12.42 days (SD 5.40) vs 15.79 days (SD 5.60) with SSD). Muangman (2006) and Varas (2005) did not report on time to complete wound healing, but found a reduction in pain associated with silver-impregnated dressing. No statistical significance in outcome measures including hospital length of stay, requirement for surgery, incidence of infection were obtained. Another RCT compared a hydrofibre containing silver (AQUACEL® Ag, ConvaTec) with SSD (Caruso, 2006). Altogether 84 patients were recruited and the group with AQUACEL® Ag dressing had

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a greater rate of re-epithelialization (73.8% vs. 60.0%) at 21 days, and the Ag dressing protocol tended to have lower total treatment costs ($1040 vs. $1180), resulting in cost-effectiveness per burn healed. This group required fewer dressing changes, was associated with less pain, and fewer procedural medications. Adverse events, including infection, were comparable between treatment groups. Overall, although these modern dressings appear to be more effective compared to the use of topical SSD on conventional dressings, there are very limited head-to-head comparisons on silver-based versus non-silverbased modern dressings, or comparative data on the various types of silverbased dressings. Leg ulcers Leg ulcers are a type of chronic wound that may be subclassified into venous, arterial, and diabetic ulcers, depending on the underlying etiologies. These wounds may be colonized by bacteria or show signs of clinical infection, which then delay ulcer healing. A number of meta-analyses on randomized controlled trials (RCTs) and reviews have examined the treatment of leg ulcers (Karlsmark, 2003; Chambers, 2007), or specifically, dressings for venous leg ulcers (Palfreyman, 2006), antibiotics and antiseptics for venous leg ulcers (O’Meara, 2010), dressings and topical agents for arterial ulcers (Nelson, 2007) and diabetic ulcers (Hilton, 2004; Bergin, 2006; Jude, 2007). Overall, there is no or very limited evidence to support the use of antimicrobial dressings or addition of topical antimicrobial or antiseptic agents to promote healing of these ulcers. However, most studies are too small to draw important differences, and further good quality research is required before definitive conclusions can be made. In the treatment of venous ulcers, compression bandages are the mainstay of treatment, and no evidence of additional benefit associated with wound dressings other than simple dressings beneath compression (Palfreyman, 2006). With regards to use of antibiotics and antiseptics, again, there is a lack of reliable evidence in the effectiveness of antibiotics and topical preparations. This was further supported from the results of the VULCAN trial (Michaels, 2009), whereby 213 patients were prospectively recruited into two arms: silver-donating versus non-silver low-adherence dressings in the treatment of venous leg ulcers. No significant differences were found in either the primary or secondary endpoints, but the antimicrobial dressings group incurred significant incremental costs. In general, in view of the increasing problem of bacterial resistance to antibiotics in many current guidelines, antibacterial preparations are only used in cases of clinical infection and not for bacterial colonization.

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There is little evidence from RCTs prior to 2006 in examining silverbased wound dressings and topical agents for treating diabetic foot ulcers. In 2007, a prospective, multicentre study compared the clinical efficacy and safety of Hydrofiber (Aquacel) dressings containing ionic silver with those of calcium alginate (Algosteril, CA) dressings in managing out-patients with Type 1 or 2 diabetes mellitus and non-ischaemic Wagner Grade 1 or 2 diabetic foot ulcers (Jude, 2007). There were 67 patients in each arm, and although the patient characteristics were clearly documented, there were differences to their background, in terms of ulcer characteristics and whether initial antibiotics were prescribed. There was a statistically significant ulcer healing or improvement in the silver dressing subjects (P = 0.058), particularly in the subset initially using antibiotics (P = 0.02). A diagnosis of non-plantar ulcers (P = 0.051) and neuropathic ulcers (P = 0.080) also revealed statistical significant improvement in silver dressing group. A shorter time to healing was obtained in the silver dressings group overall, although this did not reach statistical significance (53 vs. 58 days, P = 0.34). Again, there are limited comparative studies using different silver-based dressings in this group. Acute, chronic or infected wounds Besides burn wounds and leg ulcers, silver-coated dressings have also been used for wound management in a number of other wound types, such as acute wounds, donor sites, and traumatic injuries. Some of the studies evaluated these wounds together under categories of chronic or infected wounds, and are listed in Table 16.2. Overall, all the meta-analyses and reviews highlighted the need for further well-designed randomized controlled trials to evaluate the effectiveness of silver-related dressings or similar antimicrobial dressings.

16.2.2 Laboratory in vitro and in vivo (animal) efficacy Earlier in vitro studies in the 1990s confirmed, in general, that nanocrystalline silver dressings were preferable to topical silver applications, such as silver nitrate solution, and silver sulfadiazine cream in terms of both bactericidal and the spectrum of activities (Wright, 1998; Yin, 1999). A number of studies has confirmed the effectiveness of topical silver against a broad range of bacterial pathogens, and identified their differences in the commonly available dressings (Thomas, 2003; Ip, 2006a). The levels of silver released from available dressings vary from 1 ppm to 100 ppm, and these vary considerably depending on the solvent used during in vitro testing. There is not much animal studies data on comparative studies among different silver-based dressings and non-silver dressings. Two animal studies

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compared silver-based dressings against other antimicrobials, including chlorhexidine acetate (0.5%), fusidic acid 2% (Ulkur, 2005a), and silver sulfadiazine (1%) (Ulkur, 2005b) in a rat model for burn wounds and these are listed in Table 16.3. Results of silver-coated dressings, although effective, were more variable, with the antibiotic fusidic acid being most effective and eradicated MRSA from the eschar, whilst silver sulphadiazine 1% was more effective in reducing P. aeruginosa than silver-coated or chlorhexidine dressings in another similar study. In another animal study, three topical antibacterial dressings, ActicoatTM, chlorhexidine acetate 0.5% and silver sulfadiazine 1%, were compared in the treatment of Acinetobacter baumannii contamination of burns. All treatments were effective and prevented the organism invading the muscle and causing systemic infection, so there were significant differences between the results of the treatment groups and the control group. Mean eschar concentrations did not differ significantly between the silver sulfadiazine and chlorhexidine acetate groups, but there were significant differences between these and the Acticoat group, indicating that Acticoat eliminated A. baumannii from the tissues more effectively (Uygur, 2009). As the promotion of wound healing is the optimal outcome of any dressings, besides the absence of cytotoxic effects and a reduction of bacterial burden, the establishment of a physiological wound environ that promotes healing would be an advantage. In this respect, studies on chronic or nonhealing wounds have shown elevated levels of proteases, like matrix metalloproteinases (MMPs) and polymorphonuclear (PMN) elastase (He, 1999). Excessive elastase leads to reduced amounts growth factors, whilst release of proinflammatory cytokines, the production of reactive oxygen species (ROS) are also present in higher concentrations in chronic wounds (James, 2003). Wright et al. (2002) also demonstrated in a porcine model that nanocrystalline silver product promoted rapid wound healing with a reduction in the matrix metalloproteinase levels and enhanced cellular apoptosis, compared to that of similar (high density polyethylene) dressing but without silver and gauze dressing. It was suggested that nanocrystalline silver may play a role in altering the inflammatory events in wounds and facilitate the early phases of wound healing. A recent study compared the antimicrobial efficacy of an alginate alone, alginate containing ionic silver, and alginate with nanocrystalline silver dressings (Wiegand, 2009). In addition, the biocompatibility of these dressings was demonstrated, in terms of parameters that affect wound healing. Alginate was found to bind elastase, matrix metalloproteases-2, and reduce the concentration of proinflammatory cytokines and inhibit the formation of free radicals. The addition of silver anhanced the antioxidant capacity and inbihit bioburden, so as to create an optimum microenvironment for healing (Wiegand, 2009).

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Details of study

The anti-bacterial activities of five commercially available silver-coated/ impregnated dressings were compared against nine common burn wound pathogens, namely, methicillin sensitive and resistant Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae, Proteus vulgaris, Acinetobacter baumannii and a multi-drug efflux positive Acinetobacter baumannii (BM4454), using a broth culture method. The rapidity and extent of killing of these pathogens under

Formulation

Aquacel Aquacel Ag Acticoat Contreet PolyMem Silver Urgotul SSD

Type of Antimicrobial / Antiseptic

Silver

Table 16.3 In vitro laboratory and in vivo animal studies

All five silver-impregnated dressings investigated exerted bactericidal activity, particularly on Gram negative bacteria, incl. Enterobacter sp, Proteus sp. and E. coli. The spectrum and rapidity of action, however, ranged widely for different dressings. Both Acticoat and Contreet had a broad spectrum of bactericidal activities on both gram positive and negative bacteria. Contreet was characterized by a very rapid bactericidal action and achieved a reduction of ≥10,000 CFU/ml in the first 30 mins for Enterobacter cloacae, Proteus vulgaris,

Outcome

Margaret Ip, * Sau Lai Lui, Vincent KM Poon,1 Ivan Lung, Andrew Burd, Antimicrobial Activities of Silver Dressings – an In Vitro Comparison. JMM 2006b;55:59–63)

Reference

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Silver and chlorhexidine In vivo rat model with full-skin thickness burns wounds contaminated with methicillin-resistant Staphylococcus aureus. Rats were killed at post Day 7 of burns and tissues were

In vivo rat model with contact burns infected post Day3 with Pseudomonas aeruginosa and Staphylococcus aureus. Weekly dressings applied.

Acticoat Silverlon Silvasorb

Acticoat Bactigrass (chlorhexidine acetate 0.5%) Fusidic acid 2%

In vitro testing using 4 silver dressings on 3 organisms; Staphylococcus aureus, E. coli and Candida albicans

Acticoat Actisorb Silver220 Avance Contreet-H

in-vitro conditions were evaluated.

Fusidic acid was most effective and eradicated MRSA from eschar. MRSA was recovered from eschars of the other two groups, whilst spread into muscle was present in 3/8 rats.

Acticoat and Silvasorb treated wounds had significantly lower bacterial counts than Silverlon treated wounds.

Pseudomonas aeruginosa and Acinectobacter baumanii. Other dressings demonstrated a narrower range of bactericidal activities. Antimicrobial activity was more rapid with nanocrystalline silver against Gram positive and Gram negative bacteria and yeast.

Thomas S, McCubbin P. 2003. A comparison of the antimicrobial effects of four silver-containing dressings on three organisms. J Wound Care 2003;12:101–107. Heggers J, Goodheart R, Washington J, McCoy L, Carino E, Dang T, Edgar P, Maness C, Chinkes D. Therapeutic efficacy of three silver dressings in an infected animal model. J Burn Care Rehabil, 2005;26: 53–6. Ulkür E, Oncul O, Karagoz H, Yeniz E, Celiköz B. Comparison of silvercoated dressing (Acticoat), chlorhexidine acetate 0.5% (Bactigrass), and fusidic acid 2% (Fucidin) for topical antibacterial effect in methicillin-resistant

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Type of Antimicrobial / Antiseptic

Burn dressings in a porcine model

Acticoat Bactigrass (chlorhexidine acetate 0.5%) Silverdin (silver sulfadiazine 1%)

Formulation

Table 16.3 Continued

Retrospective review of 8 animal trials using Acticoat (silver) (3), Jelonet (gauze) (3), Solosite gel/Jelonet (gel/gauze) (2). Endpoint was wound healing with re-epithelisation during 6-week period

cultured for MRSA and bacterial counts in muscle and eschar quantified. In vivo rat model with full-skin thickness burns wounds contaminated with Pseudomonas aeruginosa. Rats were killed at post Day 7 of burns and tissues were cultured and bacterial counts in muscle and eschar quantified.

Details of study

Huge variations in time to wound healing. Small sample size and larger animals tend to heal better than smaller ones. Inadequate evidence to suggest beneficial effect of one dressing over another.

Silver sulfadiazine group significantly eliminated P. aeruginosa in exchar wound than the other two groups. All treatment modalities prevented systemic infection and invasion of P. aeruginosa to muscle.

Outcome

Staphylococcicontaminated, full-skin thickness rat burn wounds. Burns 2005a;31:874–877. Ulkür E, Oncul O, Karagoz H, Celiköz B, Cavuslu S. (2005b). Comparison of silver-coated dressing (Acticoat), chlorhexidine acetate 0.5% (Bactigrass), and silver sulfadiazine 1% (Silverdin) for topical antibacterial effect in Pseudomonas aeruginosacontaminated, full-skin thickness rat burn wounds. J Burn Care & Rehabilitation 26:430–433. Wang XQ, Kravchuk O, Kimble RM. (2009). A retrospective review of burn dressings on a porcine burn model. Burns Oct 26; [epub ahead of print].

Reference

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Alginate

Alginate (Suprasorb A) vs alginate + ionic silver (Suprasorb A + Ag) vs alginate + nano silver (Acticoat Absorbent with SILCRYST)

Silver (Aquacel Ag) vs iodine (Iodozyme) dressings on an in vitro biofilm with pathogenic bacteria.

Assessment of 3 dressings on biocompatibility, antimicrobial activity, wound parameters inc. elastase, matrix metalloproteases-2, tumour necrosis factor-alpha, IL-8, free radical formation.

In vitro effects of dressings on mature biofilms of S. aureus and P. aeruginosa.

All 3 dressings gave strong (>3 log microbial growth reduction) antimicrobial activity to P. aeruginosa, E. coli, K. pneumoniae, and S. aureus, except for alginate alone dressing was slightly less active to S. aureus. For C. albicans, activity was strong for Alginate + nano Ag > Alginate + ionic Ag > Alginate dressing alone (0.5–1.0 log reduction).

Both test dressings exerted antimicrobial effect on target species; iodine more effective at reducing bacterial load.

Thorn R.M.S., Austin A.J., Greenman J., Wilkins J.P.G., Davis P.J. (2009). In vitro comparison of antimicrobial activity of iodine and silver dressings against biofilms. Journal of Wound Care 18(8): 343–346. Wiegand C, T. Heinze, U-C Hipler. Comparative in vitro study on cytotoxicity, antimicrobial activity, and binding capacity for pathophysiological factors in chronic wounds of alginate and silvercontaining alginate. Wound Rep Regen 2009;17:511–521.

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Details of study

Chitosan acetate bandage was applied to third degree burns in mice infected with Pseudomonas aeruginosa and Proteus mirablilis.

Formulation

In vivo animal study using chitosan acetate bandage (HemCon) on infected fullthickness excisional wounds

Type of Antimicrobial / Antiseptic

Chitosan

Table 16.3 Continued

For P. aeruginosa infection, survival rate of mice in chitosan acetate bandage was 73.3% vs. that treated with nanocrystalline silver dressing was 27.3% (p = 0.0055) and of untreated mice was 13.3% (p < 0.0002). For P. mirabilis infection, comparable survival rates were 66.7%, 62.5%, and 23.1% respectively.

Outcome

Tianhong D, Tegos G, Burkatovskaya M, Castano AP, Hamblin MR. (2009). Chitosan acetate bandage as a topical antimicrobial dressing for infected burns. Antimicrob Agents Chemother 53:393–400.

Reference

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16.3

439

Types of ‘antimicrobials’

16.3.1 ‘Metal’ with antimicrobial activity Silver Silver has antiseptic, antimicrobial, anti-inflammatory properties (Klasen, 2000b; Kemling, 2002; Orvington, 2004). Silver is biologically active when it is in the soluble form, as Ag+ or Ag0 clusters. Ag+ is the most biologically active, whilst Ag2+ and Ag3+ show some activity but are more likely to form insoluble complexes and be rapidly inactivated by protein binding (Dollery, 1991), and by phosphates, sulphates and chlorides. The antimicrobial activity of silver ions in very low concentrations (10−6 to 10−9 M) has stimulated much research to its mode of action (Russell, 1994; Thurman, 1989; White, 2001a). Silver cations (Ag+) block cellular respiration, and have a strong antimicrobial effect through binding to bacterial walls, causing disruption of the wall and cell death (Lansdown, 2002). Ag+ ions also bind to bacterial enzymes, interchalates with DNA binding specifically with GC groups, and interfere with cell division and replication (Chappell, 1954; Rosenkranz, 1972; Silver, 2003). In dressings, Ag+ ions are released in a number of ways, depending on how silver is incorporated into the dressing. Ag+ ions can be released in dressings through oxidation when the silver atoms come in contact with fluid. The average particle size of nanocrystalline silver is 20–120 nm when deposited onto the surface of a dressing, creating a large surface area to facilitate release of these ions. A continual sustained release of Ag+ from Ag0/Ag+ complexes occurs when exposed to water, and provides approx. 100 parts per million Ag+ over 3–7 days (Dunn, 2004). Silver can also be incorporated as complex silver molecules in the matrix of the dressings, e.g. hydrocolloids or foam, etc., and this alters the rate of release of these ions. Silver (Ag+) has a broad spectrum of activity and inhibits growth of bacteria and yeasts between 8 and 80 ppm, with lower minimal inhibitory concentration (MIC) against Gram negative bacteria than Gram positive bacteria (Hamilton-Miller, 1993). In addition to antibacterial effects, silver has been shown to have immune-modulatory action, e.g. inhibition of matrix metalloproteases (MMPs) that prevent wound healing, and suppression of cytokines IL-12 and TNF-alpha, which could promote wound healing (Wright, 2005). Thus, silver dressings may be bifunctional depending on the concentrations released. There are a number of important parameters that determine the efficacies, spectrum of effects, and limitations of silver, as an ‘antimicrobial’. Silver appears to carry a low risk of development of resistance. However, previous studies have demonstrated that bacteria, e.g. Pseudomonas, Salmonella, ESBL-Enterobacteria, from burns wounds developed resistance to silver

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(McHugh, 1975; Hendry, 1979; Percival, 2005; Ip, 2006b). The silver resistance determinant has been shown to be carried on a conjugative plasmid and proven transferable (McHugh, 1975). Although the emergence of resistance remains rare, there are concerns that may further develop because of misuse and overuse. The quantity and rate of silver released from a dressing may also influence the overall antimicrobial effect. These effects could be demonstrated in vitro and in animal models, but may not be directly extrapolated to human wounds. Halide, e.g. Cl−, ions have been shown to have profound effects on silver and alter its ‘bioavailability’ by acting as both a precipitating agent, as well as, soluble forms of silver complexes (Gupta, 1998). In vivo, silver ions are bound up by chloride in wound exudates. The concentration and rate of ‘bioavailable’ silver ions that are released from the surface of the dressing to the wound exudates will thus have to be considered. Poon and Burd (2004) demonstrated that silver was toxic to keratinocytes and fibroblasts and affected wound healing. The Ag+ ions also effect on host proteins of the wound and may thus compromise healing and recovery of wounds. Last but not least, systemic toxicity and safety of the agent, as well as accumulation in skin and other tissues also need to be considered. Previously, systemic argyria had been described secondary to topical use of silver nitrate (Marshall, 1977).

16.3.2 Antibiotics and antiseptics Iodine Iodine or iodophors, e.g. povidone-iodine, have been widely used as an antiseptic for the prevention and treatment of wounds. It is a highly efficient microbicide with a wide antimicrobial spectrum and its efficiency against clinically and epidemiologically significant new pathogens, such as methicillin-resistant Staphylococcus aureus and Enterococcus sp. has been validated (Fleischer, 1997). Iodine is an oxidizing agent, and its bactericidal activity involves the inorganic form of I2 (Carroll, 1955), and essentially no development of resistance by microorganisms has been determined. Povidone-iodine (PVP-I) is a stable chemical complex of polyvinylpyrrolidone (povidone, PVP) and elemental iodine, is less toxic, and had been used in infected wounds and treatment of burn injuries (Fleischer, 1997). However PVP-I preparations have been show to desiccate the wound surface (Steen, 1993). Thus, formulation of the PVP-I in a liposome hydrogel to improve drug delivery properties and create a moisturizing wound environment, has been produced. Studies have shown better anti-infective efficacy and improved re-epithelialization on wounds in comparison with chlorhexidine gauze (Vogt, 2006).

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16.3.3 Others Alginate Fibre dressings such as the calcium alginate dressings are absorbent, biodegradable and are derived from seaweed (Wiegand, 2009). Alginate is often obtained from three genera of marine brown algae, Phaeophyceae (Macrocystis pyrifera, Laminaria digitata, and Laminaria saccharina) (Thomas, 2000; Thomas et al., 2000). A strong antimicrobial activity (>3 log bacterial growth reduction) against Pseudomonas aeruginosa, E. coli, and Klebsiella pneumoniae was demonstrated in an alginate only dressing, whereas inhibition was incomplete with S. aureus, and only slight with Candida albicans (Wiegand, 2009). The alginate molecule is a polysaccharide consisting of (D)-mannuronic and (L)-guluronic acid, and ionized with calcium and sodium ions. Various alginate dressings are available that possess different chemical and physical properties, depending on the proportion and arrangement of these acid residues and content of calcium and sodium ions (Thomas, 2000; Thomas et al., 2000). Some examples of alginate dressings are: Algosteril (Johnson &Johnson), Comfeel Alginate Dressing (Coloplast), CarrasorbH(Carrington Laboratories), Kaltostat (ConvaTec). In some, alginate dressings are incorporated with nanocrystalline silver, e.g. Suprasorb A (+Ag) (Lohmann & Rauscher GmbH and Co. KG), and Acticoat Absorbent with SILCRYST (Smith & Nephew Healthcare). Alginate dressings are widely used in the treatment of exuding wounds. In addition to antibacterial activity, alginate takes up wound exudates, through creation of a gel surface that absorbs moisture and maintains a moist environment (Winter, 1962). It also has haemostatic properties (Piacquaio, 1992) and has been shown to reduce pain associated with wounds (Lallau, 2002). Thus, alginate dressings may help to maintain a moist microenvironment conducive to healing, whilst limiting wound secretions and minimising bacterial contamination. Chitosan Chitosan acetate has potentially wide biological beneficial properties including homeostasis, antimicrobial activity, stimulation of healing, tissue engineering scaffolds and drug delivery (Dai, 2009). It is the soluble form of chitin, which is a biopolymer consisting of poly-N-acetylglucosamine and widespread in nature, particularly in marine arthropod shells (Dai, 2009). Chitosan is polycationic in nature and possesses natural antimicrobial properties. Its biological activity of chitosan derivatives, the postulated modes of action, and the antimicrobial effects against fungi, bacteria, and viruses and as an elicitor of plant defense mechanisms have been comprehensively reviewed by Rabea et al. (2003). The antimicrobial action varies by the type

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of chitosan (e.g. plain or derivative); degree of chitosan polymerization; host natural nutrient constituency; substrate chemical and/or nutrient composition; and environmental conditions, e.g. in moisture (Cuero, 1999). In general, chitosan antimicrobial action is more immediate on fungi and algae, followed by bacteria; and the chitosan site of action is at the microbial cell wall. One dressing, HemCon bandage, is a compressed chitosan acetate dressing that was developed as a hemostatic agent (Kheirabadi, 2005) and with its antimicrobial properties, would potentially be useful as a wound dressing, and is being evaluated in infected burns (Dai, 2009). Recently, the combination of nanosilver to a chitin preparation also produced a novel chitin/nanosilver scaffold that may have promising wound dressing applications (Madhumathi, 2010).

16.4

Future trends

16.4.1 Potential new molecules Naturally occurring peptides have shown antimicrobial activity. Recently, synthetic mimics of antimicrobial peptides, such as polymethacrylate derivatives have been produced. Such molecules, e.g. ABA triblock copolymers (A = poly(2-hydroxypropyl methacrylate) or PHPMA and B = 2-(methacryloyloxy)ethyl phosphorylcholine or PMPC) self-assemble to form copolymer gels in aqueous solution and have been found to be biocompatible and reduced growth of S. aureus and P. aeruginosa growth in vitro by 45% and by 38% in an infected skin model (Bertal, 2009). The mode of action of these polymers was hypothesized to be due to leakage of cell contents through penetration of the bacterial membranes by the hydrophobic PHPMA chains. These copolymers also exhibited antimicrobial activity when immobilized on surfaces, and thus may offer a promising approach to antimicrobial dressings. Biofilm formation is often involved in the persistence of chronic wounds. Staphylococci are common pathogens in wound infections. The quorumsensing inhibitor RNAIII-inhibiting peptide (RIP) inhibits staphylococcalbiofilm formation by inhibiting the phosphorylation of its target protein, TRAP, leading to suppression of virulence factors produced (AnguitaAlonso, 2007). It has been previously demonstrated that both TRAP- and agr-negative strains are deficient in biofilm formation in vivo, indicating the importance of quorum sensing to biofilms in the host. RIP injected systemically into rats has been found to have strong activity in preventing methicillin-resistant Staphylococcus aureus biofilm formation and infections in grafts (Balaban, 2007). Thus, peptides such as RIP may also have an application in wound dressings or wounds as a prophylactic or therapeutic agent

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in biofilm control. As one understands further with the mechanisms of biofilm formation in other bacteria, specific inhibitors of biofilm formation may potentially be prepared to target the specific organisms concerned in wound management.

16.4.2 New technology in ‘antimicrobial’ formulation Multifunctinal photopolymerized semi-interpenetrating network (sIPN) system has been shown to be effective in the wound healing of donor site in a swine model (Faucher, 2010). The system has been shown to be an effective and safe delivery vehicle for drugs, e.g. growth factor, silver sulfadiazine, in vitro, in a swine model. The advantages of sIPN include spray-on application or in situ photopolymerization, and efficacy of these combinations in promoting healing and preventing infection will require further studies. Last but not least, nanotechnology advancement and new ‘thin film’ assembly, or application of new carrier molecules, may further improve the efficacy of transfer of silver ions or other ‘antimicrobial’ molecules in wound dressings. Infection or an increased bacterial load on the surface of a wound enhances a proinflammatory environment, thus creating an unfavourable environment for host repair mechanisms to foster healing of wounds. Thus it is postulated that mechanisms that eradicate bacterial load and reverse this condition will be beneficial in wound management. Further understanding of these mechanisms will therefore help improve healing. It is anticipated that antimicrobial dressings will continue to play an important role in the management of wound care, and in particular, to chronic and infected wounds. Prospective comparative studies of these dressings on specific wound types may delineate further the specific applications for a particular type of antimicrobial dressing to maximize the benefits and improve the efficacies of such dressings.

16.5

References

Anguita-Alonso P, Giacometti A, Cirioni O, Ghiselli R, Orlando F, Saba V, Scalise G, Sevo M, Tuzova M, Patel R and Balaban N. (2007). RNAIII-inhibiting-peptideloaded polymethylmethacrylate prevents in vivo Staphylococcus aureus biofilm formation. Antimicrob Agents Chemother 51(7):2594–6. Balaban N, Cirioni O, Glacometti A , Ghiselli R, Braunstein JB, Silvestri C, Mocchegiani F, Saba V and Scalise G. (2007). Treatment of Staphylococcus aureus biofilm infection by the quorum-sensing inhibitor RIP. Antimicrob Agents Chemother 51(6):2226–9. Bergin S and Wraight P. (2006). Silver-based wound dressings and topical agents for treating diabetic foot ulcers. Cochrane Database of Systematic Reviews: 2006; Issue 1, Art. No. CD005082. DOI:10.1002/14651858.CD005082.pub2.

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Bertal K, Shepherd J, Douglas CWI, Madsen J, Morse A, Edmondson S, Armes SP, Lewis A and MacNeil S. (2009). Antimicrobial activity of novel biocompatible wound dressings based on triblock copolymer hydrogels. Journal of Materials Science 44:6233– 46. Carroll B. (1955). The relative germicidal activity of triiodide and diatomic iodine. J Bact 69(4):413–17. Caruso DM, Foster KN, Hermans MH and Rick C. (2004). Aquacel Ag in the management of partial-thickness burns: results of a clinical trial. J Burn Care Rehabil 25:89–97. Caruso DM, Foster KN, Blome-Eberwein SA, Twomey JA, Herndon DN, Luterman A, Silverstein P, Antimarino JR and Bauer GJ. (2006). Randomized clinical study of Hydrofiber dressing with silver or silver sulfadiazine in the management of partial-thickness burns. Journal of Burn Care and Research 27(3):298–309. Chaby G, Senet P, Vaneau M, Martel P, Guillaume JC, Meaume S, Te´ot L, Debure C, Dompmartin A, Bachelet H, Carsin H, Matz V, Richard JL, Rochet JM, SalesAussias N, Zagnoli A, Denis C, Guillot B and Chosidow O. (2007). Dressings for acute and chronic wounds: a systematic review. Arch Dermatol 143:1297– 1304. Chambers H, Dumville JC and Cullum N. (2007). Silver treatments for leg ulcers: a systematic review. Wound Rep Reg 15:165–173. Chappell JB and Greville GD. (1954). Effect of silver ions on mitochondrial adenosine triphosphatase. Nature 174:930–1. Cuero RG. (1999). Antimicrobial action of exogenous chitosan. EXS 87:315–33. Dai M, Zheng X, Xu X, Kong X, Li X, Guo G, Luo F, Zhao X, Wei YQ, Qian Z. (2009). Chitosan-alginate sponge: preparation and application in curcumin delivery for dermal wound healing in rat. J Biomed Biotechnol 595126. Darouiche, RO. (1999). Anti-infective efficacy of silver-coated medical prostheses. Clin Infect Dis 29:1371–7. Dollery C. (1991). Silver sulphadiazine. In Therapeutic Drugs, Churchill Livingstone. Dunn K and Edwards-Jones V. (2004). The role of ActicoatTM with nanocrystalline silver in the management of burns. Burns 30(Suppl 1):S1–9. Faucher LD, Kleinbeck KR and Kao WJ. (2010). Multifunctional photopolymerized semiinterpenetrating network (sIPN) system containing bupivacaine and silver sulfadiazine is an effective donor site treatment in a swine model, J Burn Care Res 31:137– 45. Fleischer W and Reiner K. (1997). Povidone-iodine in antisepsis – state of the art. Dermatology 195(Suppl 2):3–9. Fong J and Wood F. (2006). Nanocrystalline silver dressings in wound management: a review. Int J Nanomedicine 1:441–9. Fox CL. (1968). Silver sulphadiazine: a new topical therapy for Pseudomonas aeruginosa in burns. Arch Surg 96:184–8. Goodman MP and Fronek A. (1992). Consensus paper on venous leg ulcers. The Alexander House Group. J Dermatol Surg Oncol 18:592–602. Gupta A, Maynes M and Silver S. (1998). Effects of halides on plasmid-mediated silver resistance in Escherichia coli. Appl Environ Microbiol 64:5042–5. Hamilton-Miller JM, Shah S and Smith C. (1993). Silver sulphadiazine; a comprehensive in vitro reassessment. Chemotherapy 39:405–9.

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He C, Hughes MA, Cherry GW and Arnold F. (1999). Effects of chronic wound fluid on the bioactivity of platelet-derived growth factor in serum-free medium and its direct effect on fibroblast growth. Wound Repair Regen 7:97–105. Heggers J, Goodheart R, Washington J, McCoy L, Carino E, Dang T, Edgar P, Maness C and Chinkes D. (2005). Therapeutic efficacy of three silver dressings in an infected animal model. J Burn Care Rehabil 26:53–6. Hendry AT and Stewart IO. (1979). Silver-resistant Enterobacteriaceae from hospital patients. Canada J Microbiol 25:915–21. Hilton JR, Williams DT, Beuker B, Miller DR and Harding KG. (2004). Wound dressings in diabetic foot disease. Clin Infect Dis 39:(Suppl 2):S100–3. Homann HH, Rosbach O, Moll W, Vogt PM, Germann G, Hopp M, LangerBrauburger B, Reimer K, Steinau HU, Homann HH, Rosbach O, Moll W, Vogt PM, Germann G, Hopp M, Langer-Brauburger B, Reimer K and Steinau HU. (2007). Ann Plast Surg 59:423–7. Innes ME, Umraw N, Fish JS, Gomez M and Cartotto RC. (2001). The use of silver coated dressings on donor site wounds: a prospective, controlled matched pair study. Burns 27:621–7. Ip M, Lui SL, Poon VKM, Lung I and Burd A. (2006a). Antimicrobial activities of silver dressings – an in vitro comparison. JMM 55:59–63. Ip M, Lui SL, Chau SSL, Lung I and Burd A. (2006b). The prevalence of resistance to silver in a burns unit. J Hosp Infect 63:342–57. James TJ, Hughes MA, Cherry GW and Taylor PT. (2003). Evidence of oxidative stress in chronic venous ulcers. Wound Repair Regen 11:172–6. Jørgensen B, Price P, Andersen KE, Gottrup F, Bech-Thomsen N, Scanlon E, Kirsner R, Rheinen H, Roed-Petersen J, Romanelli M, Jemec G, Leaper DJ, Neumann MH, Veraart J, Coerper S, Agerslev RH, Bendz SH, Larsen JR, Sibbald RG. (2005). The silver-releasing foam dressing, Contreet Foam, promotes faster healing of critically colonised venous leg ulcers: a randomised, controlled trial. Int Wound J 2:64–73. Jude EB, Apelqvist J, Spraul M, Martini J; Silver Dressing Study Group. (2007). Prospective randomised controlled study of Hydrofiber dressing containing ionic silver or calcium alginate dressings in non-ischameic diabetic foot ulcers. Diabet Med 24:280–8. Jurczak F, Dugre´ T, Johnstone A, Offori T, Vujovic Z, Hollander D, on behalf of the AQUACEL Ag Surgical/Trauma Wound Study Group. (2007). Randomised clinical trial of Hydrofiber dressing with silver versus povidone-iodine gauze in the management of open surgical and traumatic wounds. Int Wound J 4:66– 76. Karlsmark T, Agerslev RH, Bendz SH, Larsen JR, Roed-Petersen J and Andersen KE. (2003). Clinical performance of a new silver dressing, Contreet Foam, for chronic exuding venous leg ulcers. J Wound Care 12:351–4. Kemling R and DeSanti L. (2002). The rate of re-epithelization across meshed skin grafts is increased with exposure to silver. Burns 28:264–6. Kheirabadi BS, Acheson EM, Deguzman R, Sondeen JL, Ryan K L, Delgado A, Dick EJ and Holcomb JB. (2005). Hemostatic efficacy of two advanced dressings in an aortic hemorrhage model in swing. J Trauma 59:25–34. Klasen HJ. (2000a). Historical review of the use of silver in the treatment of burns. Part I. Early uses. Burns 26:117–30.

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Klasen HJ. (2000b). A historical review of the use of silver in the treatment of burns. II. Renewed interest for silver. Burns 26:131–8. Klaus T, Joerger R, Olsson E. and Granqvist CG. (1999). Silver-based crystalline nanoparticles, microbially fabricated. Proc Natl Acad Sci U S A 96:13611– 14. Lallau JD, Bresson R, Charpentier P, Coliche V, Erlher S, Ha Van G, Magalon G, Martini J, Moreau Y, Pradines S, Rigal F, Wemeau JL and Richard JL. (2002). Efficacy and tolerance of calcium alginate versus Vaseline gauze dressings in the treatment of diabetic foot lesions. Diabetes Metab 28:223–9. Lansdown AB. (2002). Silver 1: its antibacterial properties and mechanism of action. J Wound Care 11:125–30. Li XL, Huang YS, Peng YZ, Liao ZJ, Zhang GA and Liu Q. (2006). Multicenter clinical study of Acticoat (nanocrystalline silver dressing) for the management of residual burn wounds. Chinese Journal of Burns 22(1):15–18. Lo Shu-Fen, Chang Chee-Jen, Hu Wen-Yu, Hayter Mark and Chang Yu-Ting. (2009). The effectiveness of silver-releasing dressings in the management of non-healing chronic wounds: a meta-analysis. J Clin Nursing 18:716–28. Madhumathi K, Kumar PT, Abhilash S, Sreeja V, Tamura H, Manzoor K, Nair SV and Jayakumar R. (2010). Development of novel chitin/nanosilver composite scaffolds for wound dressing applications. J Mater Sci Mater Med 2009 Oct 3 [Epub ahead of print]. Manafi A, Hashemlou A, Momeni P, Moghimi HR. (2008). Enhancing drugs absorption through third-degree burn wound eschar. Burns 34:698–702. Marshall JP and Schneider RP. (1977). Systemic argyria secondary to topical silver nitrate. Arch Dermatol 113:1077–9. McHugh GL, Moellering RC, Hopkins CC and Swartz MN. (1975). Salmonella typhimurium resistant to silver nitrate, chloramphenicol and ampicillin. Lancet 1:235–40. Michaels JA, Campbell B, King B, Palfreyman SJ, Shackley P and Stevenson M. (2009). Randomized controlled trial and cost-effectiveness analysis of silverdonating antimicrobial dressings for venous leg ulcers (VULCAN trial). Br J Surg 96:1147–56. Moyer CA. (1965). A treatment of burns. Trans Stud, Coll Physicians 33:53– 103. Moyer Ca, Brentano L, Gravens Dl, Margraf HW and Monafo WW. (1965). Treatment of large human burns with 0.5% silver nitrate solution. Arch Surg 90:812–67. Muangman P, Chuntrasakul C, Silthram S, Suvanchote S, Benjathanung R, Kittidacha S and Rueksomtawin S. (2006). Comparison of efficacy of 1% silver sulfadiazine and Acticoat for treatment of partial-thickness burn wounds. Journal of the Medical Association of Thailand 89(7):953–8. Münter K-C , Beele H, Russell L, Crespi A, Grochenig E, Basse P, Alikadic N, Fraulin F, Dahl CA and Jemma AP. (2006). Effect of a sustained silver-releasing dressing on ulcers with delayed healing: the CONTOP study. J Wound Care 15:199–206. Nelson EA, Bradley MD. (2007). Cochrane Database of Systematic Reviews: 2007; Issue 1, John Wiley & Sons Ltd, Chichester, UK DOI:10.1002/14651858.CD001836. pub2.

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O’Meara S, Al-Kurdi D, Ologun Y, Ovington LG. (2010). Cochrane Database of Systematic Reviews: 2010; Issue 1, John Wiley & Sons Ltd, Chichester, UK DOI:10.1002/14651858.CD003557.pub3. Orvington L. (2004). The truth about silver. Ostomy Wound Manage 50 (Suppl 9A):1S–10S. Palfreyman SJ, Nelson EA, Lochiel R and Michaels JA. (2006). Cochrane Database of Systematic Reviews: Issue 3, John Wiley & Sons Ltd, Chichester, UK, DOI:10.1002/14651858.CD001103.pub2. Percival SL, Bowler PG and Russel D. (2005). Bacterial resistance to silver in wound care. J Hosp Infect 60:1–7. Piacquaio D and Nelson DB. (1992). Alginates. A new dressing alternative. J Dermatol Surg Oncol 18:992–5. Poon VK and Burd A. (2004). In vitro cytotoxity of silver: implication for clinical wound care. Burns 30:140 –7. Rabea EI, Badawy MET, Stevens CV, Smagghe G and Steurbaut W. (2003). Chitosan as antimicrobial agent: Applications and mode of action. Biomacromolecules 4:1457–65. Rosenkranz HS and Rosenkranz S. (1972). Silver sulfadiazine: interaction with isolated deoxyribonucleic acid. Antimicrob Agents Chemother 2:373– 83. Ross DA, Phipps AJ and Clarke JA. (1993). The use of cerium nitrate-silver sulphadiazine as a topical burns dressing. Br J Plast Surg 46:582–4. Russell AD and Hugo WB. (1994). Antimicrobial activity and action of silver. Progress in Medical Chemistry 31:351–71. Saba SC, Tsai R and Glat P. (2009). Clinical evaluation comparing the efficacy of Aquacel Ag hydrofiber dressing versus petrolatum gauze with antibiotic ointment in partial-thickness burns in a pediatric burn center. J Burn Care & Res 30:380–5. Silver S. (2003). Bacterial silver resistance: molecular biology and uses and misuses of silver compounds. FEMS Microbiol Rev 27:341–53. Sharp CA and McLaws M. (2001). Wound dressings for surgical sites. Cochrane Database of Systematic Reviews, Issue 2. DOI:10.1002/14651858. Steen M. (1993). Review of the use of povidone-iodine (PVP-1). in the treatment of burns. Postgraduate Med J 69 (Suppl 3):S84–92. Thomas A, Harding KG and Moore K. (2000). Alginates from wound dressings activate human macrophages to secrete tumour necrosis factor-alpha. Biomaterials 21:1797–802. Thomas S. (2000). Alginate dressings in surgery and wound management – part 1. J Wound Care 9:56–60. Thomas S and McCubbin P. (2003). A comparison of the antimicrobial effects of four silver-containing dressings on three organisms. J Wound Care 12: 101–7. Thorn RMS, Austin AJ, Greenman J, Wilkins JPG and Davis PJ. (2009). In vitro comparison of antimicrobial activity of iodine and silver dressings against biofilms. Journal of Wound Care 18:343–6. Thurman RB and Gerba CP. (1989). The molecular mechanisms of copper and silver ion disinfection of bacteria and viruses. Critical Review of Environmental Control 4:295–315.

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Tianhong D, Tegos G, Burkatovskaya M, Castano AP and Hamblin MR. (2009). Chitosan acetate bandage as a topical antimicrobial dressing for infected burns. Antimicrob Agents Chemother 53:393–400. Ulkür E, Oncul O, Karagoz H, Yeniz E and Celiköz B. (2005a). Comparison of silver-coated dressing (Acticoat), chlorhexidine acetate 0.5% (Bactigrass), and fusidic acid 2% (Fucidin) for topical antibacterial effect in methicillin-resistant Staphylococci-contaminated, full-skin thickness rat burn wounds. Burns 31:874–7. Ulkür E, Oncul O, Karagoz H, Celiköz B and Cavuslu S. (2005b). Comparison of silver-coated dressing (Acticoat), chlorhexidine acetate 0.5% (Bactigrass), and silver sulfadiazine 1% (Silverdin) for topical antibacterial effect in Pseudomonas aeruginosa-contaminated, full-skin thickness rat burn wounds. J Burn Care & Rehabilitation 26:430–3. Uygur F, Oncul O, Evinc R, Diktas H, Acar A and Ulkur E. (2009). Effects of three different topical antibacterial dressings on Acinetobacter baumanniicontaminated full-thickness burns in rats. Burns 35:270–3. Varas RP, O’Keefe T, Namias N, Pizano LR, Quintanan OD, Tellachea MH, Rashid Q and Gillion Ward C. (2005). A prospective, randomized trial of acticoat versus silver sulfadiazine in the treatment of partial thickness burns: which method is less painful? Journal of Burn Care and Rehabilitation 26(4):344–7. Vermeulen H, van Hattem JM, Storm-Versloot MN and Ubbink DT. (2007). Topical silver for treating infected wounds. Cochrane Database of Systematic Reviews: 2007; Issue 1, Art. No.: CD005486. DOI:10.1002/14651858.CD005486.pub2. Vogt PM, Reimer K, Hauser J, Rossbach O, Steinau HU, Bosse B, Muller S, Schmidt T and Fleischer W. (2006). PVP-iodine in hydrosomes and hydrogel: a novel concept in wound therapy leads to enhanced epitelialization and reduced loss of skin grafts. Burns 32(6):695–705. Wang XQ, Kravchuk O and Kimble RM. (2009). A retrospective review of burn dressings on a porcine burn model. Burns Oct 26; [epub ahead of print]. Wasiak J, Cleland H, Campbell F. Dressings for superficial and partial thickness burns. Cochrane Database of Systematic Reviews: 2008; Issue 4, Art. No.: CD002106. DOI:10.1002/14651858.CD002106.pub3. White R, Cooper R and Kingsley A. (2002). A topical issue: the use of antibacterials in wound pathogen control. In White R, ed. Trends in wound care. UK: Bath Press. p16–17. White RJ. (2001a). An historical overview of the use of silver in wound management. Brit J Nursing 10:3–8. White RJ. (2001b). Actisorb silver 220: The silver supplement. Brit J Nursing 11: 3–8. Wiechula R. (2003). The use of moist wound-healing dressings in the management of split-thickness skin graft donor sites: a systematic review. Intl J Nursing Practice 9:S9–S17. Wiegand C, Heinze T and Hipler U-C. (2009). Comparative in vitro study on cytotoxicity, antimicrobial activity, and binding capacity for pathophysiological factors in chronic wounds of alginate and silver-containing alginate. Wound Rep Regen 17:511–21. Winter GD. (1962). Foramtion of the scab and the rate of epithelisation of superficial wounds in the skin of the young domestic pig. Nature 193:293–4.

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Woodward (2005). Silver dressings in wound healing: what is the evidence? Primary Intention 13:153–60. Wright, JB, Lam K and Burrell RE. (1998). Wound management in an era of increasing bacterial antibiotic resistance: A role for topical silver treatment. Am J Infect Control 26:572–7. Wright JB, Lam K, Buret AG, Olson ME and Burrell RE. (2002). Early healing events in a porcine model of contaminated wounds: effects of nanocrystalline silver on matrix metalloproteinases, cell apoptosis, and healing. Wound Repair Regen 10:141–51. Wright JB, Bhol KC and Schechter PT. (2005). Topical nanocrystalline cream suppresses proinflammatory cytokines and induces apoptosis of inflammatory cells in murine model of allergic contact dermatitis. Br J Dermatol 152:1235–42. Yin H, Langford R and Burrell R. (1999). Comparative evaluation of the antimicrobial action of Acticoat: antimicrobial barrier dressing. J Burn Care Rehabil 20:195–9.

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17 Avotermin: emerging evidence of efficacy for the improvement of scarring J. B U S H, K. S O, T. M A S O N, N. L. O C C L E S T O N, S. O ’ K A N E and M. W. J. F E R G U S O N, Renovo Group Plc, UK

Abstract: This chapter discusses the medical need for effective therapies for the reduction of scarring, followed by a brief discussion of the differences between adult and fetal wound healing which led to interest in the role of the Transforming Growth Factor β (TGFβ) family of cytokines in scar formation. The pre-clinical and clinical development of avotermin (TGFβ3) as a potential therapeutic for the reduction of scar appearance is described. Key words: scar, scarless wound healing, transforming growth factor beta 3 (TGFβ3), avotermin.

17.1

There is a medical need for therapies that reduce scarring following surgery

A recent survey performed in the USA confirmed that many patients are disappointed with their scar resulting from a surgical procedure, irrespective of gender, age or ethnicity.1 Understandably, patients are very conscious about visible scars. However, many patients reported scars on non-visible body sites that caused dissatisfaction and that they wished were less noticeable.1 A patient’s perception of the severity of their scar can be influenced not only by the objective appearance of the scar, but also by other factors including the surgical technique used and the patient’s sensitivity to the resulting scar.1,2 Many patients would value any opportunity to improve or minimise scarring following surgery.1 Indeed it was estimated that there are approximately 44 million surgical procedures performed in the US (Independent research: Mattson Jack Group) and approximately 42 million surgical procedures performed in the EU per annum (Independent research for Renovo: MedTech Insights and TforG) that could benefit from scar reduction therapy. 450 © Woodhead Publishing Limited, 2011

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Current treatments for scar management are unsatisfactory

The optimal outcome of wound repair following trauma, injury or surgery is complete restoration of normal skin. Adult wound healing has evolved to rapidly replace missing tissue with repaired tissue, consisting predominantly of fibronectin and collagen types I and III in order to prevent infection. The repaired tissue provides an immediate barrier to foreign bodies and infectious agents, irrespective of optimal function or appearance.3 However, in the context of modern surgery, which is performed under sterile conditions, this immediate barrier is unnecessary and therefore scarring can be considered an inappropriate response. Dermal scarring can have significant adverse consequences including restriction of movement and psychological trauma. A number of different approaches have been used in an effort to manage scarring post-surgery ranging from non-invasive (silicone gel sheeting, pressure garments, hydrating creams and ointments) to invasive (steroid injections, lasers, dermabrasion, surgery) techniques4–9 (Table 17.1). Unfortunately, many of these approaches are uncomfortable or burdensome for the patients, and many require a high level of patient compliance. Furthermore, prospective, robust clinical trials to demonstrate the efficacy of scar therapies are lacking, with the majority of published studies providing only level 4 evidence.10 The lack of clear definitions of criteria for scar improvement, coupled with the heterogenous nature of scars themselves, make it difficult to interpret and compare data from these studies. Consequently, no single treatment or regimen has been universally adopted as the standard of care to manage scarring post surgery. The high level of dissatisfaction with scar therapies among patients is reflected by the high number of patients who undergo scar revision surgery, estimated to be over 150,000 per annum in the USA (http://www.yourplasticsurgeryguide.com/trends/asps-2007.htm). Indeed many more patients are believed to request scar revision surgery

Table 17.1 Commonly used approaches to manage scarring post-surgery Approaches currently used to manage scarring post-surgery Non-invasive

Invasive

Silicone gel sheeting Pressure garments Hydrating creams/ointments

Steroid injections Lasers Dermabrasion Scar-revision surgery

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Table 17.2 Agents in development for the reduction of dermal scarring Company

Agent

Status

Agents in preclinical development for the reduction of dermal scarring First String polypeptide α-connexin Preclinical Excaliard antisense inhibitors of Smads, Connective Preclinical Tissue Growth Factor Phylogica PYC-35B Preclinical Sirnaomics STP-705 Preclinical Agents in clinical development for the reduction of dermal scarring Capstone AZX-100: 24 amino acid peptide analogue of Therapeutics heat shock protein 20, an intracellular actinrelaxing molecule CoDa NexagonTM: an anti-connexin oligonucleotide, shown to increase rate of wound healing Therapeutics Renovo Ilodecakin (Prevascar): recombinant human interleukin 10 Avotermin (Juvista®): recombinant human TGFβ3

Phase II

Phase I Phase II Phase III

but are refused as the clinician believes improvement is unlikely with surgery alone.

17.3

New biological approaches are in development for the prophylactic improvement of scarring

An increase in our understanding of the processes involved in scarring at the molecular, cellular and tissue levels has facilitated the development of new pharmaceutical approaches to prevent or treat scarring. While the majority of these approaches are still being investigated in the laboratory, a few have progressed to human clinical trials (Table 17.2).9 To date there is no approved pharmaceutical product in the US or the EU indicated for the reduction, improvement or prevention of dermal scarring.

17.3.1 The TGFβ isoforms play a key role in scar formation Skin wounds in adult skin result in scarring which has been defined as ‘a macroscopic disturbance of the normal structure and function of the skin architecture that results from a healed wound’.11 Scar severity differs between people and body locations. By contrast, skin wounds on early mammalian embryos have been shown to heal perfectly with no signs of scarring. The transition between scar-free healing (e.g. in early embryonic

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wounds), to scar-forming healing, (e.g. in adults) is characterised by a change in the organisation of the dermal extracellular matrix from a normal basket weave orientation to the deposition of parallel bundles of fine collagen fibres that form a scar.11 There are a large number of differences between early fetal and adult wounds, the majority of which appear to be irrelevant to healing and scar formation. However, much effort has gone into identifying factors that play a causative role in the scar-free healing phenotype.12 In particular, the Transforming Growth Factor beta (TGFβ) isoforms have been shown to play a key role in determining the scarring outcome. The high ratio of TGFβ3 to TGFβ1 and β2 in embryonic wounds that heal without a scar compared with adult wounds that scar,13 led to interest in the role of this family of cytokines in scar formation. More recently, a significantly higher ratio of TGFβ3 to the other TGFβ isoforms has been shown in the adult oral mucosa, also known to heal with minimal scarring, compared with dermal wounds.14 Conversely, TGFβ1 and β2 are elevated in adult wounds that heal with a scar compared with embryonic wounds that heal without a scar.15 The addition of TGFβ1 to a rat fetal wound that would normally heal without a scar results in scar formation,16 whereas in adult rats, scarring can be reduced by the inhibition of TGFβ1 or TGFβ2 using antibodies or the addition of TGFβ3.17,18 Furthermore, mice embryos that are genetically null for TGFβ3 heal with a scar in comparison with wild type littermates (with two normal copies of the TGFβ3 genes), which exhibit scar-free healing.19 Collectively, data from these experimental manipulations suggest that TGFβ3 plays an important role in scar-free healing and that the application of this cytokine to adult wounds may reduce the magnitude and accelerate the resolution of the scarring response, resulting in a phenotype that more closely resembles that of normal skin3 (Fig. 17.1)

17.3.2 Preclinical studies have demonstrated the efficacy and safety of avotermin (TGFβ3) for the improvement of scar appearance An extensive, preclinical programme has investigated the feasibility of therapeutically manipulating the scarring response using human recombinant TGFβ3, avotermin (Juvista®: Renovo, UK) to improve the appearance of scars.20 This preclinical programme was facilitated by the high level of amino acid homology between humans and animals for TGFβ3 and the surface receptors through which it exerts its biological effects (TGFβ receptors type I and type II). A standardised rat model was used to investigate the efficacy of avotermin for the improvement of scarring. Intradermal

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Mechanism of action of avotermin (recombinant) human TGFβ3): overview Scar-forming healing

Magnitude of response

(a)

TGFβ3: scar improvement

(b)

(c) Embryonic scar-free healing

Time

Inflammatory phase Proliferative phase ECM deposition and remodelling phase

17.1 Effect of TGFβ3 on the duration and magnitude of the scar-forming healing response. There are typically three overlapping phases involved in healing and scarring: inflammatory phase (light grey), proliferative phase (medium grey) and deposition and remodelling phase (dark grey). TGFβ3 reduces both the magnitude and the duration of each of the phases, resulting in a permanent change in tissue architecture such that collagen within the dermis is arranged in a more ‘basket weave’ orientation (b), unlike the closely packed, parallel bundles of collagen that are characteristic of adult scar-forming healing (a). Consequently, the application of TGFβ3 to adult wounds results in a phenotype that more closely resembles that of normal skin (c).

injection of avotermin (50 and 100 ng/100 μL/linear cm) to cutaneous incisional wounds significantly reduced scarring compared with controls in adult rats.18 The macroscopic improvements in scarring achieved with avotermin were accompanied by histological improvements in the architecture of the neodermis, including a more normal basket weave arrangement of collagen.2,18,19 The rat model was also used to optimise the dose, frequency and formulation of avotermin, demonstrating that two injections of avotermin (50 and 100 ng/100 μL/linear cm) administered at the time of wounding and 24 hours later, resulted in the greatest improvements in scarring compared with controls. A comprehensive preclinical safety program to support the development of avotermin was also completed. Specific safety studies in a clinically relevant pig model also demonstrated that intradermal administration of avotermin, at concentrations > 12 times higher than those shown

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to be efficacious in people, is well tolerated, does not adversely affect wound healing or wound tensile strength, has low systemic bioavailability and is rapidly cleared with no systemic toxicity.21

17.3.3 Avotermin is the first in a new class of prophylactic medicines in clinical development to improve scar appearance Following encouraging efficacy and safety data from the preclinical studies, an extensive phase I/II clinical trial programme was executed to evaluate the safety and optimise both the administration of avotermin and the design of clinical studies to assess its scar improving effects. The phase I/II trial programme included a series of prospective, doubleblind, within-subject placebo-controlled, randomised clinical trials in human volunteers and patients. A number of parameters that can influence the appearance of scars (e.g. age, race, sex, anatomical location, etc.) have been identified. To overcome scar variability due to these parameters, a withinsubject trial design, which allowed the effect of the drug to be compared with placebo across anatomically matched pairs of scars, was used in the phase I/II clinical programme.22 This within-subject design controlled for genetic and environmental factors affecting wound healing and scarring between individuals. By exploring a number of ways of assessing scars that result from experimental wounds, robust endpoints for the assessment of scarring have been developed and validated. In addition to assessing standard, objective endpoints, e.g. scar redness, pigmentation, width, height, volume, surface area,23,24 more holistic assessments of scars have been developed. A visual analogue scale was used along with scar ranking to assess scars either on the patients (assessed by either the investigator or the patient themselves) or using standardised digital images (assessed by panels of clinicians and lay people). The visual analogue scale and scar ranking assessments have been shown to be robust and sensitive methods for accurately assessing dermal scarring.25 During the phase I/II programme, an improved scar assessment tool was developed, the Global Scar Comparison Scale (GSCS). The GSCS incorporates the well established principles of a visual analogue scale, with the benefits of scar ranking to provide a more accurate and sensitive measure of treatment effect with clinical relevance. Both the within-patient design and use of the GSCS, which has now been validated in a number of phase II studies, received favourable feedback from the European Medicines Agency (EMA) and are currently being used to assess avotermin further in phase III studies. In all clinical studies, avotermin was administered around the time of surgery as an intradermal injection, either along the planned line of incision

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or down both margins of a closed wound, at a volume of 100 μl per wound margin. The data collected from more than 1100 subjects who have been exposed to avotermin during the extensive phase I/II programme demonstrate that avotermin is well tolerated with a favourable safety profile. Furthermore, no adverse effects on normal healing have been reported. To date, seven double-blind, placebo-controlled, prospective efficacy trials have met their primary endpoint, demonstrating a statistically significant improvement in scarring with avotermin. (These trials are registered with ClinicalTrials.gov: NCT00847925, NCT00847795, NCT00432211, NCT00629811, NCT00627536, NCT00594581 and NCT00430326.) Intradermal avotermin has a broad efficacious dose range with doses of 50 to 500 ng/100 μL per linear cm of wound margin, administered around the time of surgery, significantly improving scar appearance26,27 (Fig. 17.2). Although effective following a single application, optimal efficacy is achieved using a twice dosing regimen, the first dose given at the time of wounding and the second dose 24 hours later. The rationale for dosing at the time of wounding is to influence the initial cascade of molecular and cellular processes involved in wound healing and scarring that are triggered immediately after wounding. A subsequent administration, 24 hours later, has been shown to provide further improvements in scarring compared with placebo, which are apparent after only 6 weeks and are maintained beyond one year.26 Dosing schedules involving more than two administrations have been assessed and found to be sub-optimal due to potential injury caused by repeated injections at the wound site and inconvenient due to multiple clinic visits. Although the majority of the phase II studies investigating the safety and efficacy of avotermin have been conducted in volunteers, two recent studies performed in patients demonstrate that the improvements in scarring translate to a clinical setting. Two double-blind, placebo-controlled, randomised, phase II trials in patients demonstrated the scar-improving efficacy and safety of intradermal avotermin administered at a dose of 500 ng/100 μl per linear cm of wound margin on a single occasion following bilateral varicose vein surgery,28 or administered at a dose of 200 ng/100 μl per linear cm of wound margin on two occasions (immediately after wound closure and 24 hours later) in patients undergoing scar revision surgery29 (Fig. 17.3). The mechanism of action associated with the macroscopic benefits in scarring reported by assessment panels, investigators and the patients undergoing scar revision surgery in this study was confirmed by histological improvements in the architecture of the scars. Key learning from the extensive phase I/II clinical trial programme has been used to optimise the design of a phase III study to further evaluate the potential benefits of administering avotermin to improve scar appearance (ClinicalTrial.gov NCT00742443). This study is a randomised double-blind,

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Avotermin: evidence of efficacy for improvement of scarring (a)(i)

(b)(i)

(a)(ii)

(b)(ii)

(a)(iii)

(b)(iii)

457

17.2 Photographic images showing the improvement in scar appearance with avotermin vs. placebo and standard care. Two patients (one with paler skin (b)) with wounds treated with avotermin 200 ng/100 μl/linear cm of wound margin immediately before surgery and 24 hrs later (top), placebo (middle) and standard care (bottom) at Month 12.

within-patient, placebo-controlled trial to investigate the efficacy of avotermin in conjunction with scar revision surgery for the improvement of disfiguring scars. The effects of avotermin, administered immediately after wounding and 24 hours later, on subsequent scar formation will be assessed by an independent clinical scar assessment panel. The panel will assess digital photographs of scars at 12 months after surgery using the GSCS. The trial is fully recruited with patients who have undergone scar revision surgery for disfiguring linear scars and data are awaited with interest.

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Advanced wound repair therapies (a)

(b)

17.3 Photographic images showing the improvements in scarring following scar revision surgery with avotermin vs. placebo at Month 7 (a) and Month 12 (b). Sections of mature linear scars were randomised to receive placebo (left) or avotermin 200 ng/100 μL (right) per linear cm immediately following wound closure and 24 hrs later.

17.4

Conclusions and future trends

As many patients suffer physical and psychological trauma as a result of scarring, there is a medical need for a prophylactic therapy given at the time of surgery to improve the appearance of scars. Current treatments for scarring have limited efficacy and are not supported by data from prospective,

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robust clinical trials. Consequently there is no well-established standard of care, and the management of scarring is often inadequate. While a number of biological agents designed to manipulate the scarring process are in development, avotermin is the most advanced, in phase III clinical trials, for the improvement of scar appearance following scar revision surgery. An extensive preclinical and clinical programme has shown that avotermin promotes the regeneration of normal skin and improves scar appearance. The ongoing phase III programme is designed to show that avotermin supplements good surgical technique, resulting in less noticeable scars that more closely resemble the surrounding skin following scar revision surgery. Avotermin is the first in a new class of prophylactic therapeutics in development for the improvement of scarring and could have a significant impact on the outcome of scarring for patients in the future.

17.5

References

1. Young V L. Insights into patient and clinician concerns about scarring. Hutchison JB, editor. Plast Reconstr Surg 2009;124(1):256–65. 2. Young VL, Bush J, O’Kane S. A new approach for the prophylactic improvement of surgical scarring: avotermin (TGF beta 3). Clin Plast Surg 2009;36(2):307–13, viii. 3. Ferguson MW, O’Kane S. Scar-free healing: from embryonic mechanisms to adult therapeutic intervention. Philos Trans R Soc Lond B Biol Sci 2004; 359(1445):839–50. 4. Meier K, Nanney LB. Emerging new drugs for scar reduction. Expert Opin Emerg Drugs 2006;11(1):39–47. 5. Mustoe TA, Cooter RD, Gold MH et al. International clinical recommendations on scar management. Plast Reconstr Surg 2002;110(2):560–71. 6. Reish RG, Eriksson E. Scars: a review of emerging and currently available therapies. Plast Reconstr Surg 2008;122(4):1068–78. 7. Reish RG, Eriksson E. Scar treatments: preclinical and clinical studies. J Am Coll Surg 2008;206(4):719–30. 8. Tierney E, Mahmoud BH, Srivastava D, Ozog D, Kouba DJ. Treatment of surgical scars with nonablative fractional laser versus pulsed dye laser: a randomized controlled trial. Dermatol Surg 2009;35(8):1172–80. 9. Occleston NL, O’Kane S, Goldspink N, Ferguson MWJ. New therapeutics for the prevention and reduction of scarring. Drug Discovery Today 2008;13(21–22): 973–81. 10. Durani P, Bayat A. Levels of evidence for the treatment of keloid disease. J Plast Reconstr Aesthet Surg 2008;61(1):4–17. 11. Ferguson MW, Whitby DJ, Shah M, Armstrong J, Siebert JW, Longaker MT. Scar formation: the spectral nature of fetal and adult wound repair. Plast Reconstr Surg 1996;97(4):854–60. 12. McCallion RL, Ferguson MWJ. Fetal Wound Healing and the Development of Antiscarring Therapies for Adult Wound Healing. 2nd ed. New York: Plenum Press; 1996:561–600. 13. O’Kane S, Ferguson MWJ. Transforming growth factor betas and wound healing. Int J Biochem Cell Biol 1997;29(1):63–78.

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14. Schrementi ME, Ferreira AM, Zender C, Dipietro LA. Site-specific production of TGF-beta in oral mucosal and cutaneous wounds. Wound Repair Regen 2008;16(1):80–6. 15. Chen HB, Rud JG, Lin K, Xu L. Nuclear targeting of TGF-beta-activated Smad complexes. J Biol Chem 2005;280(22):21329–36. 16. Lin RY, Sullivan KM, Argenta PA, Meuli M, Lorenz HP, Adzick NS. Exogenous transforming growth factor-beta amplifies its own expression and induces scar formation in a model of human fetal skin repair. Ann Surg 1995;222(2):146–54. 17. Shah M, Foreman DM, Ferguson MW. Control of scarring in adult wounds by neutralising antibody to transforming growth factor beta. Lancet 1992;339(8787): 213–4. 18. Shah M, Foreman DM, Ferguson MWJ. Neutralisation of TGF-beta 1 and TGFbeta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci 1995;108:985–1002. 19. Occleston NL, Laverty HG, O’Kane S, Ferguson MWJ. Prevention and reduction of scarring in the skin by transforming growth factor beta 3 (TGFβ3): from laboratory discovery to clinical pharmaceutical. J Biomater Sci Polym Ed, 2008;19(8):1047–63. 20. Durani P, Occleston N, O’Kane S, Ferguson MW. Avotermin: a novel antiscarring agent. Int J Low Extrem Wounds 2008;7(3):160–8. 21. Laverty H, Occleston N, Jones R et al. Effects of transforming growth factor beta 3 in a clinically relevant model of full thickness incisional wounding in the Gottingen minipig. Wound Rep Reg 2008;16:A29. 22. Bush JA, McGrouther DA, Young VL, Herdon DN, Longaker MT, Mustoe TA, Ferguson MWJ. Recommendations on clinical proof of efficacy for potential scar prevention and reduction therapies. Wound Rep Reg (In Press). 23. Bond J, Duncan JAL, Sattar A et al. The maturation of the human scar: an observational study. Plast Reconstr Surg 2008;121(2):1650–8. 24. Bond J, Duncan J, Mason T et al. Scar redness in man: how long does it persist following incisional and excisional wounding? Plast Reconstr Surg 2008;121(2):487–96. 25. Duncan JA, Bond JS, Mason T et al. Visual analogue scale scoring and ranking: a suitable and sensitive method for assessing scar quality? Plast Reconstr Surg 2006;118(4):909–18. 26. Ferguson MW, Duncan J, Bond J et al. Prophylactic administration of avotermin for improvement of skin scarring: three double-blind, placebo-controlled, phase I/II studies. Lancet 2009;373(9671):1264–74. 27. Bush J, Duncan JAL, Bond JS et al. Scar-improving efficacy of avotermin administered in to the wound margins of skin incisions as evaluated by a randomized, double-blind, placebo-controlled, phase II clinical trial. Plast Reconstr Surg 2010;126(5):1604–15. 28. McCollum PT, Bush JA, James G et al. Avotermin for scar improvement evaluated by a randomised double-blind placebo-controlled dose ranging phase II clinical trial in bilateral varicose vein surgery. Br J Song (In Press). 29. So K, McGrouther DA, Bush J et al. Avotermin for scar improvement following scar revision surgery: a randomised, double-blind, within-patient, placebocontrolled phase II clinical trial. Plast Reconstr Surg (In Press).

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N. J. T U R N E R and S. F. BA DY L A K, University of Pittsburgh, USA

: Wound management has traditionally involved controlling the underlying cause and allowing the body to heal the wound naturally, or utilizing skin grafts to replace lost tissue. The emergence of regenerative medicine coupled with an increased understanding of the cellular and biochemical factors involved in wound repair have provided new therapeutic options which aim to alter the wound microenvironment from one that promotes inflammation and scar tissue formation to one that facilitates rapid regeneration with minimal scarring. In this chapter we will review the wound repair process and the influence of the wound microenvironment on the healing process. We will also discuss regenerative medicine strategies that target and manipulate the microenvironment to enhance healing. : wound repair, skin, cells, tissue engineering.

Loss of skin integrity as a result of illness or injury presents a major healthcare problem associated with significant disability or even death. In 2006, approximately 12 million wounds were treated in emergency departments in the United States, including over 500,000 burns, and approximately 8 million open wounds and lacerations (Cherry, Hing et al. 2008). In addition, 23.6 million people in the US were diagnosed with diabetes in 2007 (Centers for Disease Control and Prevention 2008) of which, between 15 and 20% are likely to develop chronic, non-healing foot wounds (Reiber, Boyko et al. 1995). The traditional approach to wound management has involved controlling the underlying cause, such as infection, ischemia or diabetes, and allowing the body to heal the wound naturally or, as in burns cases, utilizing skin grafts either from autologous healthy skin or allogeneic cadaveric donor graft tissue. The emergence of tissue engineering and regenerative medicine, coupled with an increased understanding of the cellular and biochemical factors involved in wound repair, has provided additional therapeutic options. For example, it is now considered advantageous to provide a regenerative medicine strategy that provides the biochemical cues that alter the wound microenvironment from one that promotes inflammation and scar tissue formation to one that facilitates rapid regeneration with

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Flaps must obviously be taken from sites adjacent to or near the wound and thus can produce good aesthetic results since differences in skin pigment and composition between the donor and recipient sites is minimal. However, the use of flaps can result in multiple scars and, particularly in the face, physiognomic alterations that may not be acceptable to the patient. A graft, as opposed to a flap, is the simplest way to cover superficial skin loss, and consists of a section of skin which is completely detached from the donor site and moved to cover the injury site. Grafts are classified according to the thickness of the explants as either split-thickness or full-thickness. Split thickness grafts comprise the epidermis plus a proportion of the underlying dermis and can be up to 0.6 mm thick. Full thickness grafts include the epidermis plus the entirety of the underlying dermis. Grafts are also classified according to their source; Autografts, where the donor and recipient are the same; homografts or allografts, where the donor and recipients are different individuals of the same species; and xenografts where the donor and recipient are different species. The benefit of both homografts and xenografts is the ability to preserve and store the graft material for future use. Autografts are generally capable of restoring complete, permanent skin continuity since no adverse immune response occurs. Homografts, most commonly derived from cadaveric skin, are generally used in the treatment of major burn injuries or for the temporary coverage of injuries where sufficient autograft tissue is unavailable. Typically, the upper dermal and epidermal layers must be removed within approximately three weeks as these are the most immunologically incompatible parts of the graft. A vascularized dermis remains that provides a surface for subsequent autologous skin grafts. Similarly, xenogeneic skin grafts are associated with a rapid rejection response to the epidermis. As a result xenogeneic grafts are typically de-epidermidized and used only to provide protection to a wound until a more suitable grafting option become available. The success of skin grafts is determined, in part, by their thickness. Full thickness grafts are most successful when the recipient area is small and the graft can be nourished by adjacent tissue. In addition, full thickness grafts are less inclined to retract and they retain elasticity due to the presence of the dermal layer. Split thickness grafts integrate with the wound site more easily than full thickness grafts and can be used to cover much larger injuries. These grafts may even be meshed to increase the surface area coverage. However, split thickness grafts are associated with significant scarring and retraction due to the absence of the dermis and the repaired skin is generally thinner and more fragile than uninjured tissue. Conversely, split-thickness grafts have become the gold standard treatment for the repair of full thickness dermal burns and other conditions with extensive skin loss, although full thickness grafts are still preferred for skin losses on the face and fingers.

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The development of cellular therapies for wound repair dates back more than 30 years with the publication of the first successful protocols for the culture of keratinocytes (Rheinwald and Green 1975). Eventually, small sheets of cells two to three layers thick (Green, Kehinde et al. 1979) and known as cultured epithelial autografts or allografts were developed. Since these early efforts, a better understanding of the wound healing process, coupled with advances in cell culture techniques and the development of in vitro bioreactor systems, has led to the creation of more complex tissue engineered constructs including engineered dermal constructs and complete 3-dimensional constructs that are promoted as complete skin substitutes. Table 18.3 lists a selection of cellular engineered skin replacements.

Cultured epithelial autografts were first used as an alternative to splitthickness skin grafts in treating burns patients (O’Conner, Mulliken et al. 1981), and thermal injuries have remained the major clinical target for these cultured sheets of epithelial cells. Preparation of autologous epithelial grafts primarily utilizes the protocol developed by Green in 1979 (Green, Kehinde et al. 1979) in which a small skin biopsy is taken and the keratinocytes isolated and expanded in culture. This process uses a murine fibroblast feeder layer to support the proliferation of the keratinocytes and takes approximately 2 to 3 weeks to obtain a complete epithelial graft. While the cultured graft is prepared, the patients’ wounds are typically dressed or covered with allogeneic donor skin. The design of bioreactors and the development of automated culture processes have facilitated the development of commercial autologous epidermal grafts EpicelTM (Genzyme Biosurgery) and EpidexTM (Euroderm GmbH). EpicelTM was first introduced in 1988 and consists of autologous keratinocytes grown on a feeder layer of mouse fibroblasts, along with bovine serum in the culture medium. Until 1996 this product was classified as a banked human tissue. However, due to the use of animal cells and products EpicelTM became the first xenotransplantation device approved by the FDA in 1999 and is contraindicated for patients with known allergies to murine or bovine materials. EpidexTM takes an alternative approach and consists of autologous cells that are derived from the outer sheath of actively growing hair follicles. These autologous cells are multipotent stem cells located in the bulge of the hair follicle and through a controlled culture process are induced to differentiate into mature keratinocytes in vitro. The culture problems associated with growing cells

et al

et al

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thus providing a functional, vascularized bed that can facilitate the restitution of the underlying dermal structures and promote reepithelialization. As such, the FDA has classified these products as interactive wound dressings with the biggest market targeted to be the treatment of chronic wounds. These dermal constructs use allogeneic fibroblasts from neonatal foreskin which have a high proliferative capacity and relatively low immunogenicity. The cells are cultured within a 3-dimensional, bioresorbable carrier matrix that allows for the deposition of ECM proteins and soluble growth factors by the fibroblast cells. Examples of this technology include Dermagraft® (Advanced Biohealing Inc) and Cyzact® (Intercytex Ltd). Dermagraft® was originally developed by Advanced Tissue Sciences and Smith & Nephew as a wound dressing for chronic non-healing wounds, especially diabetic foot ulcers. The manufacturing process involves the culture of neonatal fibroblasts on a biodegradable polyglactin mesh for approximately 3 weeks and then cryopreservation, giving the product a reasonably long shelf life. The fibroblasts within Dermagraft® secrete a number of growth factors including vascular endothelial growth factor, insulin like growth factor, transforming growth factor β1 and matrix proteins involved in wound healing (Mansbridge 1998; Mansbridge, Liu et al. 1999; Gath, Hell et al. 2002). However, the product has proven awkward to handle due to its fragility and long thawing and rinsing procedure. Limited regulatory (Anon. 2006) approval significantly limited the profitability of these products. Cyzact® (Intercytex) aimed to address the poor handling properties of other engineered dermal grafts by using dermal fibroblasts cultured within a human fibrin gel matrix which was designed to be applied to non-healing wounds, primarily venous leg ulcers, at regular intervals to promote healing. Unfortunately, as has been the case with many of the recently developed wound dressings for chronic wounds, Cyzact® failed to show any significant improvement in the rate of wound healing when compared to the current standard of care, compression bandages, and is no longer being produced.

The third type of cellular engineered skin graft combines both cultured epidermal sheets and engineered dermal grafts to form a bilayer graft that approximates a full thickness skin graft, termed a living skin equivalent. Like other allogeneic products these have limited survival in vivo and therefore cannot be considered a skin replacement. The first devices of this type consisted of a contracted collagen matrix impregnated with dermal fibroblasts and covered by an epidermal sheet (Bell, Ehrlich et al. 1981). Subsequently, this model was modified, replacing the contracted collagen matrix with a type I collagen gel and the mature dermal fibroblasts with neonatal foreskin fibroblasts. This product, Apligraf® (Organogenesis),

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based on incidence of complete wound closure). Certainly, cost and ease of manufacturing will be significantly lower than most first-generation products. However, the practical robustness of the strategy, particularly in VLU is uncertain (see further). Time will tell if this is a productive strategy. KeraPacTM developed by KeraCure (Chicago, IL) is an example of using cells as a true “interactive dressing”. Unlike the Intercytex strategy of using the fibroblast as the active agent, KeraPacTM uses the epidermal keratinocyte. The strategy simplifies the delivery of keratinocyte factors to the wound in a way that is also contained and easily removed. The device consists of keratinocytes grown on non-porous microcarriers, encased in a non-woven fabric pouch, much like a tea bag. The product is placed on the wound and is then removed after several days. KeraCure began a 300 Patient, randomized, open label Phase II study for the treatment of DFU in 2006 (clinicaltrials.gov Identifier NCT00330265). Although the trial was estimated to complete by March 2009, the trial is still open and no results have been reported to date. KeraCure is a small, venture-backed company and the slowed progress may be attributed, at least in part, to lack of adequate financial backing for the clinical studies. Unlike ICXTM, KeraPacTM would be regulated as a combination device rather than a biologic. Cellerix (Madrid, Spain) is developing a bilayered skin construct for epidermolysis bullosa, an indication that both Transcyte® and OrCelTM are currently approved as a temporary treatment and the investigational use of graftskin has shown some clinical benefit (Fivenson et al., 2003). The strategy is a unique combination of several components in other therapies. Cx501, currently in Phase II trials for EB, consists of allogeneic fibroblasts in human fibrin with an autologous layer of epidermal keratinocytes. The rationale is that use of autologous keratinocytes will offer the advantage of persistent repair without the possibility of chronic rejection (graftskin clinical data and bench studies have shown that acute rejection of allogeneic keratinocytes is not an issue) with allogeneic fibroblasts (shown by ATS to persist for some period of time) that can contribute to the formation of normal basement membrane, lacking in the EB patient. Like the Intercytex strategy, the use of human fibrin avoids the use of xenogeneic collagen. But unlike other second generation strategies, this strategy is more complex in production than most first generation. Also of note is that Cx501 targets a smaller, Orphan Drug indication where patients are in substantial need. There are no clinical reports to date. There are several other therapies and companies that have come and gone in the last twenty years, but in the end, the title of the blog post sums up the experiences to date. Some examples in this section may yet advance to commercialization; however there are several lessons that both first- and second-generation products can teach us about strategy, outcomes and business models.

Sciences, in particular, were having severe financial difficulties, and both

However, these products are primarily marketed for their ability to maintain a moist wound environment rather than for any bioactive properties. A related product is Biobrane® (Smith & Nephew) which consists of a silicone sheet with a partially embedded nylon fabric embedded on the surface (Tavis, Thornton et al. 1980). This nylon fabric is then coated with porcine dermal collagen. Blood and sera from the wound clot within the nylon fabric attaching the dressing to the wound while the collagen promotes fibroblast growth and maintains a hydrated environment. This product has been targeted towards the treatment of partial thickness burns (Hansbrough, Zapata-Sirvent et al. 1984; Gerding, Imbembo et al. 1988; Whitaker, Worthington et al. 2007) and for healing donor skin graft sites and skin slough disorders (McHugh, Robson et al. 1986; Arevalo and Lorente 1999). Trancyte™ (Advanced Biohealing Inc) attempted to address the poor handling properties of Dermagraft® and utilized primarily the same culture methods as Dermagraft®. Cultured fibroblasts are allowed to deposit an organized ECM within the construct, but with Trancyte™ the fibroblasts are physically removed from the final graft to leave an acellular product. The final graft is then covered by a silicone covered nylon mesh for stability and to provide a pseudoepithelium. The product has been used for the temporary coverage of burn wounds in place of cadaver skin (Hansbrough 1997; Purdue, Hunt et al. 1997; Noordenbos, Dore et al. 1999; Kumar, Kimble et al. 2004). Originally developed by Smith & Nephew, Transcyte™ met with limited regulatory approval (Anon. 2006) and was sold to Advanced Biohealing Inc in 2006 but is currently not actively being marketed.

Engineered dermal replacements based on natural ECM materials derived from decellularized tissues share a number of similarities in their mode of action in promoting wound healing; however, these products differ greatly in their source material and preparation and processing. These differences

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based on incidence of complete wound closure). Certainly, cost and ease of manufacturing will be significantly lower than most first-generation products. However, the practical robustness of the strategy, particularly in VLU is uncertain (see further). Time will tell if this is a productive strategy. KeraPacTM developed by KeraCure (Chicago, IL) is an example of using cells as a true “interactive dressing”. Unlike the Intercytex strategy of using the fibroblast as the active agent, KeraPacTM uses the epidermal keratinocyte. The strategy simplifies the delivery of keratinocyte factors to the wound in a way that is also contained and easily removed. The device consists of keratinocytes grown on non-porous microcarriers, encased in a non-woven fabric pouch, much like a tea bag. The product is placed on the wound and is then removed after several days. KeraCure began a 300 Patient, randomized, open label Phase II study for the treatment of DFU in 2006 (clinicaltrials.gov Identifier NCT00330265). Although the trial was estimated to complete by March 2009, the trial is still open and no results have been reported to date. KeraCure is a small, venture-backed company and the slowed progress may be attributed, at least in part, to lack of adequate financial backing for the clinical studies. Unlike ICXTM, KeraPacTM would be regulated as a combination device rather than a biologic. Cellerix (Madrid, Spain) is developing a bilayered skin construct for epidermolysis bullosa, an indication that both Transcyte® and OrCelTM are currently approved as a temporary treatment and the investigational use of graftskin has shown some clinical benefit (Fivenson et al., 2003). The strategy is a unique combination of several components in other therapies. Cx501, currently in Phase II trials for EB, consists of allogeneic fibroblasts in human fibrin with an autologous layer of epidermal keratinocytes. The rationale is that use of autologous keratinocytes will offer the advantage of persistent repair without the possibility of chronic rejection (graftskin clinical data and bench studies have shown that acute rejection of allogeneic keratinocytes is not an issue) with allogeneic fibroblasts (shown by ATS to persist for some period of time) that can contribute to the formation of normal basement membrane, lacking in the EB patient. Like the Intercytex strategy, the use of human fibrin avoids the use of xenogeneic collagen. But unlike other second generation strategies, this strategy is more complex in production than most first generation. Also of note is that Cx501 targets a smaller, Orphan Drug indication where patients are in substantial need. There are no clinical reports to date. There are several other therapies and companies that have come and gone in the last twenty years, but in the end, the title of the blog post sums up the experiences to date. Some examples in this section may yet advance to commercialization; however there are several lessons that both first- and second-generation products can teach us about strategy, outcomes and business models.

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In comparison to adult human tissue, which is approximately 8–10% type III collagen (Smith, Holbrook et al. 1986), the foetal bovine dermis ECM in Primatrix™ is approximately 30% type III collagen (Ramshaw 1986). In addition to providing elasticity to the ECM, type III collagen has been shown to promote migration of fibroblasts (Postlethwaite, Seyer et al. 1978) and to be an essential regulator of ECM deposition and organization (Fleischmaher, MacDonald et al. 1990; Liu, Wu et al. 1997). Unlike EZ-DermTM and PermacolTM, which are cross-linked, Primatrix™ incorporates into the wound and rapidly degrades and has shown success for the treatment of acute, full thickness wounds (Wanitphakdeedecha, Chen et al. 2008). Similar to PrimatrixTM, Alloderm® (Lifecell), GraftJacket® (Wright Medical Technology) and DermaMatrix™ (Synthes) are decellularized dermal constructs. Unlike Primatrix™, these products are derived from human dermis with GraftJacket® being cross-linked to maintain the collagen architecture, while Alloderm® and DermaMatrix™ are non-crosslinked. Since these materials are derived from human tissue, careful screening is required to ensure the source tissue is free from transmissible viruses such as Hepatitis B and C, HIV and syphilis. Clinically, these products have been indicated in the treatment of non-healing, non-ischemic ulcers and other dermal wounds, demonstrating the ability to significantly reduce healing time, wound volume and depth (Brigido 2006). GraftJacket® increased the probability of successful wound healing of diabetic foot ulcers within 12 weeks by 2.7 fold with a significant increase in the total number of successfully healed wounds (Reyzelman, Crews et al. 2009). Acellular therapies for wound repair have shown significant advantages over cellular therapies in terms of ease of handling, cost and shelf life. However, the wide array of source material, coupled with differences in decellularization, sterilization and preservation, has created a complex range of products which although similar in nature vary greatly in their ability to promote tissue regeneration. Although clinical studies have been generally positive, results have been mixed, with some studies showing significant benefits and others showing no beneficial effect. The full potential of these ECM scaffolds to promote constructive remodelling will not be realized until we have a complete understanding of the biology of ECM.

Tissue engineering has been heralded as having the potential to revolutionize the treatment of numerous medical problems including the healing of skin wounds. Despite over 30 years of published research on the development of skin replacements, to date there are no effective engineered skin replacements that completely replicate the anatomy, physiology, biological

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Grimes, M., J. T. Pembroke, et al. (2005). “The effect of choice of sterilisation method on the biocompatibility and biodegradability of SIS (small intestinal submucosa).” Biomed Mater Eng (1–2): 65–71. Gurtner, G. C., S. Werner, et al. (2008). “Wound repair and regeneration.” Nature (7193): 314–21. Hachiya, A., P. Sriwiriyanont, et al. (2005). “An in vivo mouse model of human skin substitute containing spontaneously sorted melanocytes demonstrates physiological changes after UVB irradiation.” J Invest Dermatol (2): 364–72. Halstenberg, S., A. Panitch, et al. (2002). “Biologically engineered protein-graft-poly (ethylene glycol) hydrogels: a cell adhesive and plasmin-degradable biosynthetic material for tissue repair.” Biomacromolecules (4): 710–23. Hansbrough, J. (1997). “Dermagraft-TC for partial-thickness burns: a clinical evaluation.” J Burn Care Rehabil (1 Pt 2): S25–8. Hansbrough, J. F., S. T. Boyce, et al. (1989). “Burn wound closure with cultured autologous keratinocytes and fibroblasts attached to a collagen-glycosaminoglycan substrate.” JAMA (15): 2125–30. Hansbrough, J. F., R. Zapata-Sirvent, et al. (1984). “Clinical experience with Biobrane biosynthetic dressing in the treatment of partial thickness burns.” Burns Incl Therm Inj (6): 415–9. Harper, C. (2001). “Permacol: clinical experience with a new biomaterial.” Hosp Med (2): 90–5. Hodde, J., A. Janis, et al. (2007). “Effects of sterilization on an extracellular matrix scaffold: part I. Composition and matrix architecture.” J Mater Sci Mater Med (4): 537–43. Hodde, J. P., S. F. Badylak, et al. (1996). “Glycosaminoglycan content of small intestinal submucosa: a bioscaffold for tissue replacement.” Tissue Eng (3): 209–17. Hodde, J. P., D. M. Ernst, et al. (2005). “An investigation of the long-term bioactivity of endogenous growth factor in OASIS Wound Matrix.” J Wound Care (1): 23–5. Hodde, J. P., R. D. Record, et al. (2001). “Vascular endothelial growth factor in porcine-derived extracellular matrix.” Endothelium (1): 11–24. Hodde, J. P., R. D. Record, et al. (2002). “Retention of endothelial cell adherence to porcine-derived extracellular matrix after disinfection and sterilization.” Tissue Eng (2): 225–34. Hopkins, C., R. Walker, et al. (2009). “Permacol in augmentation rhinoplasty: how we do it.” Clin Otolaryngol (1): 68–75. Hsu, P. W., C. J. Salgado, et al. (2008). “Evaluation of porcine dermal collagen (Permacol) used in abdominal wall reconstruction.” J Plast Reconstr Aesthet Surg (11): 1484–9. Iocono, J. A., H. P. Ehrlich, et al. (1998). “Hyaluronan induces scarless repair in mouse limb organ culture.” J Pediatr Surg (4): 564–7. Jarman-Smith, M. L., T. Bodamyali, et al. (2004). “Porcine collagen crosslinking, degradation and its capability for fibroblast adhesion and proliferation.” J Mater Sci Mater Med (8): 925–32. Jimenez, P. A. and S. E. Jimenez (2004). “Tissue and cellular approaches to wound repair.” Am J Surg (5A): 56S–64S. Karpelowsky, J. S. and A. J. Millar (2009). “Porcine dermal collagen (Permacol®) for chest and abdominal wall reconstruction in thoraco-omphalopagus conjoined twin separation.” Pediatr Surg Int (3): 315–18.

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Mostow, E. N., G. D. Haraway, et al. (2005). “Effectiveness of an extracellular matrix graft (OASIS Wound Matrix) in the treatment of chronic leg ulcers: a randomized clinical trial.” J Vasc Surg (5): 837–43. Moustafa, M., C. Simpson, et al. (2004). “A new autologous keratinocyte dressing treatment for non-healing diabetic neuropathic foot ulcers.” Diabet Med (7): 786–9. Muangman, P., L. H. Engrav, et al. (2006). “Complex wound management utilizing an artificial dermal matrix.” Ann Plast Surg (2): 199–202. Muhart, M., S. McFalls, et al. (1999). “Behavior of tissue-engineered skin: a comparison of a living skin equivalent, autograft, and occlusive dressing in human donor sites.” Arch Dermatol (8): 913–8. Netzlaff, F., C. M. Lehr, et al. (2005). “The human epidermis models EpiSkin, SkinEthic and EpiDerm: an evaluation of morphology and their suitability for testing phototoxicity, irritancy, corrosivity, and substance transport.” Eur J Pharm Biopharm (2): 167–78. Neveux, Y., J. M. Rives, et al. (1995). “Clinical interest of cutaneous models reproduced in vitro for severe burn treatment: histopathological and ultrastructural study.” Cell Biol Toxicol (3–4): 173–8. Nguyen, D. Q., T. S. Potokar, et al. (2010). “An objective long-term evaluation of Integra (a dermal skin substitute) and split thickness skin grafts, in acute burns and reconstructive surgery.” Burns (1): 23–8. Noordenbos, J., C. Dore, et al. (1999). “Safety and efficacy of TransCyte for the treatment of partial-thickness burns.” J Burn Care Rehabil (4): 275–81. O’Conner, N. E., J. B. Mulliken, et al. (1981). “Grafting of burns with cultured epithelium prepared from autologous epidermal cells.” Lancet (8211): 75–8. Otto, W. R., J. Nanchahal, et al. (1995). “Survival of allogeneic cells in cultured organotypic skin grafts.” Plast Reconstr Surg (1): 166–76. Parenteau, N. L., P. Bilbo, et al. (1992). “The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function.” Cytotechnology (1–3): 163–71. Phillips, T. J. (1998). “New skin for old: developments in biological skin substitutes.” Arch Dermatol (3): 344–9. Postlethwaite, A. E., J. M. Seyer, et al. (1978). “Chemotactic attraction of human fibroblasts to type I, II, and III collagens and collagen-derived peptides.” Proc Natl Acad Sci U S A (2): 871–5. Pu, L. L. (2005). “Small intestinal submucosa (Surgisis) as a bioactive prosthetic material for repair of abdominal wall fascial defect.” Plast Reconstr Surg (7): 2127–31. Purdue, G. F., J. L. Hunt, et al. (1997). “A multicenter clinical trial of a biosynthetic skin replacement, Dermagraft-TC, compared with cryopreserved human cadaver skin for temporary coverage of excised burn wounds.” J Burn Care Rehabil (1 Pt 1): 52–7. Raeber, G. P., M. P. Lutolf, et al. (2005). “Molecularly engineered PEG hydrogels: a novel model system for proteolytically mediated cell migration.” Biophys J (2): 1374–88. Ramshaw, J. A. (1986). “Distribution of type III collagen in bovine skin of various ages.” Connect Tissue Res (4): 307–14.

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Tyrone, J. W., J. E. Mogford, et al. (2000). “Collagen-embedded platelet-derived growth factor DNA plasmid promotes wound healing in a dermal ulcer model.” J Surg Res (2): 230–6. Valentin, J. E., A. M. Stewart-Akers, et al. (2009). “Macrophage participation in the degradation and remodeling of extracellular matrix scaffolds.” Tissue Eng Part A (7): 1687–94. VandeVondele, S., J. Voros, et al. (2003). “RGD-grafted poly-L-lysine-graft(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion.” Biotechnol Bioeng (7): 784–90. Vanstraelen, P. (1992). “Comparison of calcium sodium alginate (KALTOSTAT) and porcine xenograft (E-Z DERM) in the healing of split-thickness skin graft donor sites.” Burns (2): 145–8. Vowden, P., M. Romanelli, et al. (2006). “The effect of amelogenins (Xelma) on hard-to-heal venous leg ulcers.” Wound Repair Regen (3): 240–6. Wanitphakdeedecha, R., T. M. Chen, et al. (2008). “The use of acellular, foetal bovine dermal matrix for acute, full-thickness wounds.” J Drugs Dermatol (8): 781–4. Welch, M. P., G. F. Odland, et al. (1990). “Temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction.” J Cell Biol (1): 133–45. Welss, T., D. A. Basketter, et al. (2004). “In vitro skin irritation: facts and future. State of the art review of mechanisms and models.” Toxicol In Vitro (3): 231–43. Whitaker, I. S., S. Worthington, et al. (2007). “The use of Biobrane by burn units in the United Kingdom: a national study.” Burns (8): 1015–20. Wood, J. D., A. Simmons-Byrd, et al. (2005). “Use of a particulate extracellular matrix bioscaffold for treatment of acquired urinary incontinence in dogs.” J Am Vet Med Assoc (7): 1095–7. Yu, J., Y. Zeng, et al. (2004). “Quantitative analysis of collagen fibre angle in the submucosa of small intestine.” Comput Biol Med (6): 539–50.

19 Commercialization of engineered tissue products N. L. PA R E N T E AU, Parenteau BioConsultants, LLC, USA

Abstract: Health care is increasingly evidence-based as products compete for limited dollars. Advanced wound therapies are particularly at risk because of the paucity of Level I evidence to support their effectiveness and economic advantage over good wound care. This chapter looks for lessons from the commercialization of advanced products and product concepts over the last 30 years. For advanced wound therapies to be adopted, they will have to demonstrate clinical and cost effectiveness capable of driving practice from the persistent approach of wound care and management to one of active wound healing. Key words: chronic wounds, skin equivalent, dermal template, dermal replacement, epithelial grafting, commercialization, venous leg ulcer, diabetic foot ulcer.

19.1

Introduction

Beginning about thirty years ago, advanced therapies for skin had a heyday. In the span of about 15 years, Yannas and colleagues developed the first bioengineered dermal regeneration template (DRT)(Yannas et al., 1982), Green and colleagues developed the first cultured epithelial autografts (CEA)(Green et al., 1979), saving lives of severely burned patients (Gallico et al., 1984), and Bell envisioned the possibility of producing a living skin equivalent containing both epidermis and dermis (Bell et al., 1981). All three ideas would make it to the clinic, and later, the market as Integra Dermal Regeneration Template® (DRT) (Integra Life Sciences, Plainsboro, NJ), EpiCel® (BioSurface later acquired by Genzyme, Cambridge MA) and Apligraf® (Organogenesis Inc., Canton MA) respectively. Around the same time, Livesey and colleagues, developed a way to decellularize and freeze-dry dermis in a way that retained the architecture of native dermis (Livesey et al., 1995). Their human product, Alloderm® (LifeCell later acquired by Kinetic Concepts Inc., San Antonio, TX) entered the market as a universal dermal transplant, governed by tissue banking regulations. Naughton and colleagues at Advanced Tissue Sciences (ATS, LaJolla, CA) envisioned growing dermal fibroblasts on synthetic mesh to form a 495 © Woodhead Publishing Limited, 2011

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cell-synthesized dermal replacement (Hansbrough et al., 1992). Versions of this product concept made it to the market first as Transcyte®, a temporary covering for first and second degree burn wounds and then as Dermagraft® for the treatment of diabetic foot ulcers (DFU) (Advanced Tissue Sciences/ Smith & Nephew, later acquired by Advanced BioHealing, Westport, CT) (Naughton et al., 1997). Growth factors were not to be left out during this period as a gel of purified platelet-derived growth factor (PDGF) becalpermin was developed for the treatment of diabetic foot ulcers by Johnson and Johnson (New Brunswick, NJ) and marketed as Regranex® beginning in 1997. This early work embraced aspects of tissue transplantation, advanced biomaterials, stem cell biology, cell therapy, and tissue engineering – all now part of regenerative medicine. Although often equated, the first generation advanced wound therapies targeted skin repair in different ways, and their strategies for reaching the market differed. As will be detailed, first-generation businesses suffered some significant challenges and there has been some criticism that first generation regenerative medicine products, of which these skin applications represent the lion’s share, were based on science to a fault and so faced production, financial and practical difficulties when commercialized (Mason, 2007). In an attempt to avoid some of the perceived pitfalls and limitations of the early products, second-generation product development focused on what were seen as innovations that would make the products easier to produce and use, and in doing so lower the costs of producing the products to create more favorable product margins. Unfortunately, these assumptions led several efforts down development paths that failed in the clinic, while others have not yet produced an approved product. Some of the latter may be due to the wariness of investors and commercial partners to get into the business of advanced therapies after the business difficulties seen in first-generation companies and later, the slow market adoption of the products. So it is important to the success of future therapies to understand what went right, what went wrong, and what could have been done better, as well as what more can be done going forward. In particular, factors that help or hinder the medical and commercial acceptance of these products will be increasingly important as health care, and demands of the world’s health care systems evolve.

19.2

Engineered templates and scaffolds

When Yannas and colleagues envisioned an engineered template, the primary goal was to implant a scaffold that could persist to delay wound contraction and thus modify tissue granulation and repair, yet not be encapsulated (Yannas, 2001). Previous experience with collagen sponges and gels showed that collagen lacking telopeptides, cleaved during the extraction

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process, were quickly resorbed. Chemical cross-linking would make the collagen sponge last longer but heavily cross-linked collagen could induce a foreign body response leading to increased inflammation and encapsulation, making collagen constructs no better substitutes than plastic. What was needed was a matrix that could last, yet be ultimately remodeled. Ideally, the template could, by virtue of its physical structure, help direct and thus modify the ingrowing tissue to achieve a better quality of repair where scarring and contraction were limited. Yannas and colleagues tested several design parameters in developing a dermal regeneration template (DRT) that would: • • • •

suppress or delay wound contraction induce synthesis of ‘physiological’ tissue suppress inflammation compared to what was experienced with crosslinked collagen alone exhibit a controlled degradation that would allow the DRT to persist long enough as a template to achieve the above but ultimately remodel into dermis. They identified several variables that impacted the function of the DRT:

• • •

pore diameter, pore architecture and pore volume cross-link density molecular composition.

One of the most significant innovations was the inclusion of glycosaminoglycan (GAG) as a copolymer. They found that shark chondroitin 6sulphate cross-linked to the collagen offered several advantages over a cross-linked collagen scaffold alone. The inclusion of GAG suppressed inflammation, in particular platelet aggregation, compared to collagen alone and it controlled the degradation of the collagen sponge allowing the DRT to act as a template for the synthesis of new tissue. The proper pore diameter, size and architecture promoted ingrowth of cells and tissue rather than encapsulation of the implant. The product gained US Food and Drug Administration (FDA) approval through a pre-market approval application (PMA) in 1996. Integra DRT® became the first, and to date, the only engineered biodegradable template for the synthesis of dermal tissue. A commercial challenge of using native polymers is aseptic processing and terminal sterilization. Most forms of sterilization cause denaturation. Heavy cross-linking might allow terminal sterilization, but it would defeat the objectives of the DRT. The first version of the DRT was aseptically manufactured, initially a significant production challenge. Later, a terminally sterilized version Integra-TS® was developed and is now available in the US and Europe. Addition of a silastic membrane served as a temporary artificial epidermis, providing a barrier to fluid loss and protection from infection. In a

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second procedure, the silastic membrane is removed and replaced by a thin autograft to establish permanent biologic wound closure once the new dermal bed is established. Integra was initially approved for use as a DRT in burns for ‘life-threatening full-thickness or deep partial-thickness thermal injuries’ where sufficient autograft was either not available or not desirable because of the health status of the patient. Subsequently in 2002, its approved use was expanded to include burn scar revision. In both applications, biological wound closure is ultimately achieved with the grafting of a thin autograft to establish the epidermis as a second procedure. Although not approved for chronic wound indications, the scar revision application is the first step in considering the use of the DRT in other deep wounds, particularly those with exposed bone and tendon (an application currently contraindicated for other skin substitutes). Experience in treating deep chronic wounds is limited thus far (Campitiello et al., 2005). It should be noted that the DRT is not used to promote wound closure in the chronic wound but, staying true to form, is used as a way to establish subcutaneous and dermal tissue. The wounds are ultimately closed using a thin, expanded autograft. It is not unreasonable to expect that, if results for deep wounds show promise, other methods of providing biologic wound closure, like the application of cultured keratinocytes or companion skin equivalent grafting, might be possible alternatives to autograft. However, because of cost, it is also reasonable to expect that these methods would be reserved for the deepest and most problematic wounds. That said, there is a clear unmet medical need for advanced regenerative therapies for those suffering pressure sores that are not only difficult to manage, but pose a very real lifethreatening risk of infection.

19.3

Processed tissues

Acellular human cadaver dermis (AHCD, also referred to as acellular dermal matrix ADM) was positioned as a processed, banked human tissue for transplantation. In this way it was not subject to clinical trials or US regulation beyond those governing the safety of tissue banks as the FDA considers it ‘minimally processed’ banked human tissue. This was a rapid path to the market for the commercial product, Alloderm®, particularly compared to an engineered scaffold, which would require demonstration of efficacy through pivotal clinical trials and a PMA. The first indication was as a dermal graft for burns, which supported the use of thinner autografts to supply an epithelium. This indication is not dissimilar to that for the DRT. The benefits of the native tissue were thought to be that by retaining its native structure and other matrix biomolecules, it would, in theory, naturally possess both adequate stability as a dermal template and superior

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non-inflammatory remodeling potential like rapid and directed revascularization due to the retention of native vascular channels and adhesion molecules. Because it is freeze-dried, it can be stored off-the-shelf, ready when needed. The drawback is that it does not provide any form of wound closure, temporary or permanent. Like the original DRT, the product is also aseptically processed and each lot, i.e. skin harvested from each cadaver much be tested for human pathogens as any transplant would be. This adds significant cost to processing. Although publications imply that AHCD may have active biological properties, referring to it as a ‘dermal regenerative tissue matrix’, and some even equating the matrix to ‘bioengineered skin grafts’, the action of the implant beyond a role as a temporary scaffold or template is not firmly established. The human composition, natural stability, and normal architecture are the primary desirable components as a dermal scaffold. The use of AHCD has expanded to use in periodontal surgery (Thoma et al., 2009), abdominal hernia repair (Misra et al., 2008), orthopedic (Ho and Miller, 2007), wound healing applications (Reyzelman et al., 2009) and breast reconstruction (Sbitany et al., 2009). Many of these additional applications are the result of physician innovation (personal communication)1. However, major US payers, e.g. CIGNA, Blue Cross Blue Shield, and Aetna, currently regard the only clinically supported and clearly reimbursable use of the human universal dermal transplant to be breast reconstruction, where the processed tissue assists the surgeons in reconstructing the breast at the time of mastectomy in a process that improves cosmetic outcome and limits the need for further surgical procedures (Sbitany et al., 2009). All other applications are still considered investigational or experimental, which means that its use in these indications may be out of pocket, or at the very least, reviewed on a case-by-case basis. This, in practical terms, limits use to thought leaders and company-sponsored studies, unless, like in reconstruction after breast cancer, there is an obvious benefit to the patient and savings to the medical system. Another example of this kind of commercial support is in the use of negative pressure wound therapy (NPWT), also referred to as vacuumassisted closure (V.A.C.®, Kinetic Concepts Inc.(KCI), San Antonio, TX). NPWT was originally envisioned as a treatment for chronic wounds (Morykwas and Argenta, 1997), yet it has gained particular acceptance as a useful technology for the management of large, traumatic and difficult-to-treat wounds. Although the benefits of NPWT therapy in wound healing are not well-established, the benefits of V.A.C.® to the medical system are supported by reduction in the amount and type of nursing care with its associated costs 1 Non-confidential interviews were conducted with key executives from several companies as research for this chapter. Information shared during the interviews is noted as a personal communication.

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is a cost-savings to hospitals and payers (Gregor et al., 2008). The additional business benefit of V.A.C.® therapy is that, while it involves an initial investment in hardware, the system (and its associated consumables) are more likely to be used thereafter because of that hardware. It is of interest to note that V.A.C.® has been used to facilitate the use of DRT (Park et al., 2009) and that AHCD leader, LifeCell was acquired by KCI, the producers of V.A.C.®. NPWT may improve the clinical performance of dermal scaffolds and in turn, the healing benefit of NPWT may be advanced by the incorporation of a more biologically compatible “dressing.” Although AHCD and similar xenogeneic dermal products make it to the marketplace, it is increasingly clear that payers and clinicians need objective evidence of their value. This leaves a long-standing standing need for quality studies that justify clinical benefit. So although the regulatory route to market may permit broader, more flexible clinical use of a product and allow a company to broadly market it for repair applications, the actual use of the product is still limited by lack of clinical evidence that meets Level I standards of at least one prospective, randomized, controlled trial (US Preventative Services Task Force). AHCD is licensed to Wright Medical (Arlingtion, TN) and marketed as GraftJacket® for use in orthopedic and wound healing applications, although, as already noted, its use for these indications faces reimbursement pressure without additional clinical studies. In 2009, Reyzelman et al. published a prospective, randomized, multi-center study on the use of GraftJacket® to speed wound closure of diabetic foot ulcers. Ninety-three patients were treated in 11 sites. Following debridement, patients were randomized into treatment and control groups. The treatment groups received one dermal graft at the time of debridement and the physicians were allowed to then treat the wounds as they saw fit thereafter until wound closure or the end of the study. The standard of care varied within the control group where patients received a variety of wound dressings at the discretion of the treating physicians. Fortyone of 46 patients completed the trial in the treatment group and 37 of 39 patients completed the study in the standard of care arm. Results demonstrated a healing benefit to the use of a dermal graft at 12 weeks where the odds of healing were 2.7 times higher using a dermal graft than not using one, although the data is limited by the small number of patients, study design and potential treatment bias. Whether this study is sufficient in quality and size to substantiate the product’s reimbursement as a treatment for diabetic foot ulcers is unclear, particularly when compared to the much larger and more rigorously controlled Phase III trials conducted for the regulated tissue and growth factor therapies like Apligraf®, Dermagraft® and Regranex®, approved for the same indication. Also, without sufficient clinical evidence, cost-benefit based on actual clinical benefit is difficult to substantiate. A wound healing application like the use of the dermal graft in deep wounds with exposed bone or tendon (currently contraindicated for Apligraf® and Dermagraft®) © Woodhead Publishing Limited, 2011

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may be a well-suited application. However, the challenge will be determining an acceptable clinical endpoint. Wound closure still remains the ultimate clinical goal and the endpoint accepted by regulators. This suggests that a clinically meaningful endpoint may lie within combination studies or demonstrating an advantage to a second procedure, similar to DRT in burns. This is a strategy being pursued with engineered DRT as the use of the product expands beyond burn wounds. The human cadaver source for native dermal tissue is being replaced by xenogeneic sources that offer the advantages of cost, supply and an opportunity for greater consistency of the product. For example, Primatrix® (TEI Biosciences, Boston, MA) is processed fetal bovine dermis and Strattice® (LifeCell (KCI)) is a processed porcine dermis product developed as the next generation to Alloderm®. Other non-dermal collagen membranes like processed porcine intestinal submucosa (Oasis®, Cook Biomedical, Bloomington, IN) is also used as a collagen dressing capable of acting as a “regenerative” scaffold although it is primarily used as a collagen dressing. Unlike Alloderm, the xenogeneic tissues are regulated under a 510K pathway in the US, where approval relies on demonstrating substantial equivalence to prior collagen dressings. As such, they run into the difficulty of maintaining a position of substantial equivalence to earlier dressings while wanting the product to be perceived as much more. Companies walk a fine line between claiming active promotion of wound healing versus facilitating it and substantiating how and why it facilitates healing. While tissue banking and 510K product routes look more advantageous for getting product to the marketplace, both impact commercialization of the products and the ways companies can support the use of these products. While the 510k products can claim a wider scope of potential uses than the Class III products or biologics that must prove efficacy through pivotal trials before entering the market, demonstrating the clinical value of a 510k product can be a hurdle that persists for a very long time. With the use of health care dollars being ever more scrutinized, the need to substantiate the treatment value will likely only increase for all products. This could put current and future 510K products at a disadvantage if they are not supported by quality evidence-based studies on clinical utility as well as cost-effectiveness.

19.4

Cell-based products

19.4.1 Cultured epithelial sheets When Green and colleagues developed a method to expand cultured keratinocytes, they envisioned using the thin sheets of a few layers of epidermal cells as cultured epithelial autografts (CEA) (Green et al., 1979) that would take on a wound and permanently restore the epidermis (Compton et al., © Woodhead Publishing Limited, 2011

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1989). This was life saving for severely burned patients where autografting was not an option (Gallico et al., 1984). Culture expansion allowed the treatment of the entire body starting with only a small, postage-stamp sized biopsy. Unfortunately, epidermal coverage did little to mitigate severe dermal scarring and wound contraction. Take of CEA in deep wounds was difficult but it improved significantly with prior engraftment of the wounds with cadaver skin (Hickerson et al., 1994). CEA were then grafted upon removal of the cadaver epidermis. The technology (EpiCel®) was offered as a service by Biosurface Technologies, which was later acquired by Genzyme (Cambridge, MA). Both allogeneic and autologous CEA were tested for their ability to close chronic wounds and the benefit of CEA application was reported in several small studies, often with investigators culturing the CEA on site (Phillips and Gilchrest, 1990, 1991). However, the CEA, allogeneic or autologous, have never made it to broad commercial use for wound repair. There are several likely reasons for this – including the need for further development in product design and delivery. One of the most notable process issues was the use of mouse 3T3 feeder cells for cell propagation. Although initially used as a transplant, the FDA later approved EpiCel® under the Humanitarian Device Exemption program in 2007 due to the small number of candidate patients. The approval was for the product grown with the mouse 3T3 feeder cells. In severe burns, maintenance of proliferative capacity is key and many believed that the only way to achieve that was through feeder cell support. One could argue that removal of the feeder layer would have been an innovation well worth pursuing, particularly for applications beyond severe burns; however, the costs of taking on that challenge, including overcoming patent hurdles, were not minor considerations – particularly for a humanitarian application. What the experience with CEA in chronic wounds did do for chronic wounds was offer additional evidence of healing potential that supported the already reported clinical benefit of skin grafts; if one could design a product that delivered an epidermis, there was a good chance it would demonstrate a clinical benefit.

19.4.2 A cultured dermal replacement The use of a mesh to culture cells allowed cell culture to become 3-dimensional. Naughton and colleagues, using techniques originally researched for bone marrow stroma, envisioned growing dermal fibroblasts on resorbable mesh where the cells could grow and synthesize extracellular matrix ultimately forming a cultured dermal replacement (Naughton, 1999). They first used the technology to develop the Transcyte® dressing as a temporary skin substitute. It consists of fibroblasts grown on a non-resorbable, porcine collagen-coated nylon mesh with a polymer membrane and is used to tem-

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porarily cover surgically excised full-thickness and partial thickness burns. The product is stored frozen and the cells are non-viable at the time of use. In more superficial wounds capable of healing by secondary intention, the product sloughs as the wound heals; in deeper wounds, it is surgically excised before allografting, not unlike cadaver skin. The scientific rationale behind its use is that the extracellular matrix and associated factors in the cell-produced matrix assist in healing. In a multi-center clinical trial, Transcyte® compared favorably to human cadaver skin for temporary coverage of excised burn wounds. It was equivalent or better to allograft with respect to take of subsequent autograft and was easier to remove than cadaver skin while maintaining an adequate wound bed (Purdue and Still, 1997). In partial-thickness burns, it was reported to be as good or better than standard care with faster re-epithelialization, less need for dressing changes and less need for autografting (Kumar et al., 2004). Transcyte® was approved by the FDA through the PMA process in 1997. The use of resorbable mesh and extended bioreactor culture created a human dermal matrix containing viable, allogeneic fibroblasts derived from foreskin that could serve as a dermal implant or tissue engineered dermal replacement. Unlike the DRT and AHCD, the product (Dermagraft®) was used for the promotion of wound healing in diabetic foot ulcers (DFU), a much larger wound healing application. The pivotal clinical trial in DFU allowed multiple applications to improve the wound bed and provide matrix proteins and growth factors that, in theory, could stimulate healing and speed wound closure (Naughton et al., 1997). While Dermagraft® showed a trend towards better healing, the FDA was not convinced of its efficacy, which relied heavily on retrospective analysis, and issued a non-approvable letter in 1998 requiring an additional trial. Some of the practical issues that may have plagued the early trial were lack of consistency, and control of viability following the cryopreservation process. Variation in how much of the product was used and when also may have been factors. After refinements to the clinical protocol and product specifications, a second study using eight weekly applications of the product, demonstrated sufficient efficacy to support approval in 2001 for the treatment of DFU. Production and commercialization of the product was through a joint venture with Smith & Nephew (Hull, UK). Unfortunately, the costs of the joint venture and ongoing operations were unsustainable. ATS was forced to file for bankruptcy in 2002 and the company was liquidated. The products continued to be manufactured and sold by Smith and Nephew which, upon failure of Dermagraft® to demonstrate efficacy in a venous leg ulcer trial, sold the rights to a small start-up Advanced BioHealing (Westport, CT) in 2006. Today, Advanced BioHealing is increasing the use and acceptance of Dermagraft® for the treatment of DFU, with equal emphasis on good wound care (personal communication). Advanced BioHealing is also

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looking at additional applications for the dermal matrix. Unlike processed dermis, its use in additional applications will require one or more prospective, randomized, pivotal trials and formal regulatory approval.

19.4.3 The bi-layered skin equivalent In the early 1980s, Bell and colleagues discovered that fibroblasts mixed within a collagen gel would contract the gel into a more tissue-like collagen lattice. He termed it a ‘tissue equivalent’. Bell envisioned that a tissue equivalent of dermal fibroblasts (dermal equivalent) could serve as a suitable substrate for epidermal cells to form a bilayered skin equivalent (Bell et al., 1981). After overcoming issues of maintaining sidedness, collagen processing and sterilization, feeder free large-scale culture of epidermal progenitor cells and production, Bell’s vision developed into the product Apligraf® (Parenteau, 1999). Although cryopreservation technology was developed for the product in the 1990s, the living product began clinical trials with a fresh product and is still shipped ready to use with a two-week shelf life. Very early, Organogenesis pursued a small human study for the use of the skin equivalent in burns. What was found was that the bilayered skin equivalent (generic name, graftskin) was not robust enough to persist as cadaver skin would. Interestingly, graftskin did survive if used in conjunction with meshed autograft (Waymack et al., 2000) implying that graftskin lacked something that the cadaver skin had to control inflammation or that signaling from the cadaver skin was sufficient to quell inflammatory response by cells in the cultured construct (see further). To this day, this difference in graftskin response on the burn wound is still not well understood. However, based on positive use of skin grafts to stimulate healing in hard to heal chronic wounds, and the reports of CEA potential in chronic wounds, the company targeted graftskin for use in chronic wounds. A graftskin pivotal trial in venous leg ulcers (VLU) was successful and the product (Apligraf®) became the first living therapy to demonstrate clinical efficacy in a prospective, randomized trial (Falanga and Sabolinski, 1999) and the first product ever approved via a traditional FDA approval process in 1998. A second pivotal trial in DFU was also successful (Veves et al., 2001) and led to approval of Apligraf® in this indication in 2000. It should be noted that to date, Apligraf® is still the only biologically active therapy to have shown pivotal efficacy in the treatment of VLU. Despite clinical and regulatory success, the business suffered from unworkable financials with their marketing partner Novartis Pharmaceuticals (Basel, Switzerland). The combination of deal terms combined with the cost of manufacture, early reimbursement hurdles, and the slow entry into a difficult first market (VLU) lead to an untenable situation for Organo-

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genesis, which they and their partner were unable to resolve within the partnership. Organogenesis took back the rights to Apligraf®, restructured under bankruptcy in 2002, and emerged as a private company. Today, Organogenesis is a profitable and growing private biotechnology company still fueled by its flagship product, Apligraf®. The product is now reimbursed for use in both VLU and DFU although market penetration is still developing at about 25%, with estimated revenues for 2009 at just over $100 million (personal communication). Organogenesis recently submitted a PMA application for the use of a modified form of graftskin (CelTx®) for gingival repair.

19.5

Lessons from the first generation

What stands out in the early regulated products was the degree of scientific characterization, strategy and innovation that went into them. The Yannas product had a very deliberate design, ATS engineers produced novel bioreactors that permitted large-scale fabrication of the dermal substitute on resorbable mesh, and the matrix was also extensively characterized. Organogenesis not only developed needed innovations in collagen processing and cell culture but also analyzed the biology of the construct extensively to support what it was as well as its behavior, as well as the immunology of the allogeneic cells. All these efforts were not only vital to the eventual success of the products but also to their approval. A concern is that next generation products lack sufficient, or the right kind of, science and data, leaving the efforts open to wrong design decisions as well as lack of support throughout the regulatory process.

19.6

The second generation of advanced therapies

A recent headline on a regulatory blog read, “Will [name of company] be the first regenerative medicine product approved by the FDA in over 12 years?” The headline sums up the experience in advancing second-generation products. So what has been different in the last 15 years compared to the first? Some have argued that money is one factor. The early products were developed in public companies during a time when biotechnology investing was positive (both ATS and Organogenesis were public companies; LifeCell and Integra Life Sciences are public companies), but cost of developing a tissue engineered product, for example, is still far lower than drugs and other biologics. Others might argue that the failure of the early products to make it as profitable commercial products out of the gate hurt others with similar products in their pipeline. While this may be true, several next generation products were well on their way through development and clinical testing before it was clear that Organogenesis and Advanced Tissue

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Sciences, in particular, were having severe financial difficulties, and both Integra Life Sciences and LifeCell did not suffer similar circumstances and were able to grow their businesses, the former by diversifying, and the latter by expanding the use of its product into areas like gingival repair that did not hinge on reimbursement, and finding an unmet medical need in breast reconstructive surgery. In both cases, however, the wound healing application was not instrumental to the company’s commercial success, lending further support to the weak commercial potential of active wound healing applications. Nevertheless, the unmet medical need and market opportunity remained and was interpreted, particularly by several next generation developers, as a fault of the first generation products rather than the business decisions or business model. Even before the business difficulties of the leaders were known, next generation developers looked at ways to improve upon what had been done. There were and still are what would be considered in the business vernacular, ‘me too’ products. OrCelTM bilayered cellular matrix (BCM) (Ortec International, later renamed Fibrocell, New York, NY) was a bilayered construct of dermal fibroblasts in a collagen sponge overlaid with keratinocytes. While BCM was similar in composition to graftskin, the character of the keratinocyte layers, a key functional component of graftskin (Wilkins et al., 1994) was unclear in the BCM product. A potential advantage was that it was cryopreserved, which would allow it to be inventoried. The company first gained approval of BCM for the treatment of epidermolysis bullosa under a Humanitarian Device Exception. A non-cryopreserved version of OrCelTM was also approved for donor site wounds through the PMA process in 2001. In 2006 the company received approval for use of the cryopreserved product in epidermolysis bullosa, again through a Humanitarian Device Exemption. The company conducted clinical trials in VLU, and later DFU. However, the first pivotal VLU trial did not lead to FDA approval and the company was required to perform a confirmatory trial. The second study was completed in 2006 and the application was resubmitted to the FDA with no approval issued to date. The company filed for bankruptcy in 2008. Another product following the bilayered skin equivalent concept is StrataDermTM (Stratatech, Madison, WI). This product has been developed in a small private company, funded in large part by government grants. StrataDerm appears very similar to Apligraf® with the exception that the keratinocytes are derived from a proprietary cell line, rather than normal keratinocyte cell strains (Schurr et al., 2009). The production advantage is a ready and consistent cell bank of keratinocytes. It could also translate to substantial savings in eliminating some of the safety testing each time a new strain is developed. It also allows for genetic manipulation, which Stratech is pursuing for second-generation products. The drawback is the use of a transformed cell line. While the line is non-tumorigenic and appears to yield

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a mature epidermis with all the properties of normal tissue, the stem cell and progenitor cell regulation of this population is, by definition, abnormal. Despite the challenge and cost of having to periodically develop and test new cell strains, the benefit of being able to demonstrate normal senescence of the cell population is a valuable indicator of normalcy and the ultimate safety and genetic stability of the allogeneic cell strains. Perpetual renewal could represent a formidable safety hurdle for its developers – even when used temporarily (the company has reported on a small PhaseI/II study using StrataDermTM as a substitute for cadaver allograft for burns where it performed comparably (Schurr et al., 2009)). Exposure to inflammatory and regenerative challenge encountered in the patient could be a cause for concern. The benefit of using a perpetual line is that the exact nature of its self-renewal can be thoroughly examined to answer these questions and substantiate the safety of the line. Failure to do so will likely mean lack of approval, even if effective in clinical testing. One might also expect competition from the more established companies like Organogenesis and Advanced BioHealing, both of whom use normal human cells. Genetically modified second-generation constructs will have their own set of additional efficacy and safety hurdles. Although the prior two examples embraced the concept of a bilayered skin equivalent, the Intercytex (Manchester, UK) strategy was to deliver fibroblasts in a fibrin matrix (ICXTM). Use of fibroblasts in fibrin, two readily available human components, is simpler and less costly to produce compared to Dermagraft® or the combination of collagen and fibroblasts in a dermal sponge of dermal equivalent and it eliminates the use of bovine collagen. Fibrin can serve as a natural, highly biocompatible delivery vehicle. However, a pivotal trial in VLU reported no significant difference over standard of care. Although Intercytex was developing a second generation construct consisting of cell-produced dermis with addition of epidermal cells (ICX-SKNTM) (Flasza et al., 2007), failure of the pivotal trial to show significance in frequency or time to complete wound closure forced Intercytex to close its doors and liquidate its assets. The cell technologies were acquired by HealthPoint (San Antonio, TX), a company with a wide range of wound care products with an interest in developing active therapies. According to information obtained on clinicaltrials.gov, HealthPoint’s first product (HP802-247), now in Phase II clinical trials consists of both fibroblasts and keratinocytes applied to the wound suspended in fibrin (Identifier NCT 00852995). They are testing different concentrations of cells per microliter of fibrin in VLU. Of interest is the clinical endpoint of reduction in wound area at 16 weeks rather than the standard endpoint of complete wound closure. This is an example of a cell-based therapy used as a drug rather than a construct, measuring a clinical endpoint more often associated with dressings (Apligraf®, Dermagraft® and Regranex® approvals were

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based on incidence of complete wound closure). Certainly, cost and ease of manufacturing will be significantly lower than most first-generation products. However, the practical robustness of the strategy, particularly in VLU is uncertain (see further). Time will tell if this is a productive strategy. KeraPacTM developed by KeraCure (Chicago, IL) is an example of using cells as a true “interactive dressing”. Unlike the Intercytex strategy of using the fibroblast as the active agent, KeraPacTM uses the epidermal keratinocyte. The strategy simplifies the delivery of keratinocyte factors to the wound in a way that is also contained and easily removed. The device consists of keratinocytes grown on non-porous microcarriers, encased in a non-woven fabric pouch, much like a tea bag. The product is placed on the wound and is then removed after several days. KeraCure began a 300 Patient, randomized, open label Phase II study for the treatment of DFU in 2006 (clinicaltrials.gov Identifier NCT00330265). Although the trial was estimated to complete by March 2009, the trial is still open and no results have been reported to date. KeraCure is a small, venture-backed company and the slowed progress may be attributed, at least in part, to lack of adequate financial backing for the clinical studies. Unlike ICXTM, KeraPacTM would be regulated as a combination device rather than a biologic. Cellerix (Madrid, Spain) is developing a bilayered skin construct for epidermolysis bullosa, an indication that both Transcyte® and OrCelTM are currently approved as a temporary treatment and the investigational use of graftskin has shown some clinical benefit (Fivenson et al., 2003). The strategy is a unique combination of several components in other therapies. Cx501, currently in Phase II trials for EB, consists of allogeneic fibroblasts in human fibrin with an autologous layer of epidermal keratinocytes. The rationale is that use of autologous keratinocytes will offer the advantage of persistent repair without the possibility of chronic rejection (graftskin clinical data and bench studies have shown that acute rejection of allogeneic keratinocytes is not an issue) with allogeneic fibroblasts (shown by ATS to persist for some period of time) that can contribute to the formation of normal basement membrane, lacking in the EB patient. Like the Intercytex strategy, the use of human fibrin avoids the use of xenogeneic collagen. But unlike other second generation strategies, this strategy is more complex in production than most first generation. Also of note is that Cx501 targets a smaller, Orphan Drug indication where patients are in substantial need. There are no clinical reports to date. There are several other therapies and companies that have come and gone in the last twenty years, but in the end, the title of the blog post sums up the experiences to date. Some examples in this section may yet advance to commercialization; however there are several lessons that both first- and second-generation products can teach us about strategy, outcomes and business models.

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Delivering value in advanced therapies

19.7.1 The role of science in product strategy and use Jon Northrup has argued that to meet the challenge of today’s pharmaceutical development, companies must make the connection between fundamental scientific data and lead optimization (Northrup, 2006). This need for connectivity between the science and the product is not just good advice for pharmaceuticals but should be an integral part of the design and use of advanced wound care therapies as well. In theory, biomaterials can be designed to modify tissue response in several ways: • • • • •

by modifying the inflammatory response by providing a scaffold for cell adhesion and infiltration by delivering molecules spatially oriented to direct cell infiltration and interaction by being able to deliver an agent over time to modulate ongoing processes by changing the physical and biochemical framework in such a way to alter the overall repair and/or regenerative tissue response.

Promogran® (Johnson and Johnson, New Brunswick, NJ) a collagen/ oxidized cellulose dressing, is an example of a product that blurs the interface between a dressing and an active therapy with claims to modify matrixmetalloprotease activity in the wound. But there is more to advancing new biomaterials. Innovation is not just product based. The therapy must also be used in an advanced, or more informed way. Since even seemingly simple biomaterials can have a biological effect, it pays to understand the underlying mechanism of the biomaterial’s interaction with the wound so that the material can be used in the right wounds, in the right way and at the right time. Chitin is used for illustration. Chitin (a polymer of N-acetyl-D-glucosamine) and its polymer chitosan are components of bacteria, yeast and fungal cell walls, although it is more commonly known as the primary component of the exoskeleton of insects and crustaceans. It has similarities to cellulose, already used as a material to pack deep wounds. While the first impulse is to treat chitin as a structural biomaterial or scaffold, there is evidence that chitin derivatives can chemically mediate biological response. Chitin and chitosan have been reported to enhance wound healing since the 1970s. There was evidence that chitin derivatives could stimulate neutrophil and macrophage infiltration and granulation, both through stimulation of angiogenesis as well as fibroblast migration (Shi et al., 2006). Their influence on inflammation is not surprising if we consider that these polysaccharides are components of bacteria, but

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there appears to be more to it. Chitosan appears to activate macrophages through interaction with the mannose receptor (Mori et al., 2005). Activation of inflammation is proposed to bring with it increased amounts of growth factors such as PDGF and transforming growth factor beta, which can speed wound granulation. However, a substantial amount of the material is needed to see a significant effect on wound granulation in preclinical studies, where granulation is not impaired (Kojima et al., 2004). Could it, however, be effective in the chronic wound where there is some impairment? Given its route of action, we might anticipate chitin to be of greater benefit where monocyte activation is diminished, as is the case in some DFU. In contrast, we might be concerned that the use of the material might be counter-productive in conditions of chronic inflammation where the macrophage activation is not compromised, like the VLU. Going a bit further, if we consider the fact that macrophage activation in the wound periphery of a diabetic patient is not impaired, but the problem appears to be one of access of the macrophages to the wound, it becomes clear that we need more information on why macrophage response is diminished in the diabetic wound proper in order to be confident in chitosan’s potential utility in this application. We need to appreciate the primary cause of lack of macrophage activation since macrophages can be activated by many elements, which are already present in the wound. Will chitosan stimulation deliver essentially more of the same and be ineffective? Or will it deliver something substantially different? Without making these connections between what is scientifically known and the medical condition of the patient, it is tempting to jump to the conclusion that Chitosan should be of use in any condition where a granulation tissue is lacking. But drilling down to the biology and mechanism changes the perspective. Chitosan will not be expected to promote granulation tissue unless it is able to stimulate increased macrophage infiltration. If inadequate macrophage activation does not contribute to the pathology, then the treatment might even be counterproductive. Establishing this type of connectivity, even for a seemingly “simple” biomaterial can mean the difference between developing a truly interactive, high value treatment or yet another wound dressing that sometimes facilitates wound healing. A true regenerative scaffold or template is not a dressing or filler, it is an implant and it has biological effect. Advanced therapies can and should fall into that category. While collagen was a prominent component of first generation products, fibrin gained favor in several second-generation strategies like ICXTM and HP802-247. If one looks at how fibrin is used by the body in acute wound healing, it becomes clear that fibrin is a natural wound dressing. It is a wound sealant and tissue support material. Although fibrin breakdown products may have some signaling capacity, fibrin is basically a noninflammatory native matrix that supports inflammatory cell infiltration,

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platelet adhesion and activation. If that is the goal, then fibrin is the molecule of choice. However, there are things fibrin is not: it is not a substrate for keratinocytes (Kubo et al., 2001), nor is it a provisional matrix for granulation or dermal repair. Fibrin is highly cell-compatible, making it a good delivery vehicle as we have seen in second-generation products, but could substituting a substance that regenerative tissues actually utilize like hyaluronic acid, collagen and fibronectin add something more or make an advanced therapy more robust? Even though keratinocytes do adhere to collagens and fibronectin, developing a product using collagen or fibronectin to speed wound closure is likely to meet with disappointment if the reason why the epidermis stopped in the first place is ignored. Epidermal migration is governed by mechanisms within the epidermis; it does not require fibroblasts or a special underlying substrate to achieve wound closure (Hardin-Young and Parenteau, 2004). For example, lack of fibrin does not stop wound closure in fibrin-free mice (Drew et al., 2001). In fact, very little can stop wound closure experimentally in the normal animal. Epidermal keratinocytes are able to degrade extracellular matrix and synthesize their own migratory substrate, if given the right set of signals, which often emanate from the epidermis itself (Parenteau and Hardin-Young, 2006). Adding a substrate without also doing something to address the message to the epidermis is like paving the road but omitting the gas for the car. One might argue that too little is known about this signaling, necessitating a more empirical approach, but if one looks not only to the wound healing literature but also to what is known about other degenerative diseases, fibrosis and even cancer, there are many clues that can guide product strategy during the development process. What is certain is that even though the exact details of non-healing are still to be learned, it does not have to prevent us from using what we do know to design more effective, biologically and mechanistically robust advanced therapies. A closer look at mechanisms and underlying pathology of the different chronic wounds illustrate the point.

19.7.2 Connecting the technology with the medical need Cell signaling, particularly those involved in fibrosis, or what could be thought of as a chronic attempt at stabilization (Parenteau and HardinYoung, 2007), can certainly have a negative impact on the parenchyma, in this case, epithelialization. If it didn’t, we wouldn’t have chronic wounds, so a correction of signaling, which is what debridement often does and what Apligraf is thought to do in the chronic wound (Sabolinski et al., 1996), is capable of activating the epidermis to close the wound. While an epidermis may fail to migrate and close a chronic wound, it will not be for lack of factors per se but rather the message those factors convey to cells already

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in a state of chronic inflammation. The therapy must either impart a new message over and above the background, or change the tissue to impart the new message. This is in essence what clinicians do when they use sharp debridement to remove the fibrotic tissue and stimulate an acute response (Schultz et al., 2003). Unfortunately, the pathology is often able to overcome even the native response, suggesting that the therapy will also have to be persistent, if not also very potent to make a sizable difference. Both the DFU and VLU are stuck in a chronic inflammatory state that is unable to resolve and progress to a normal, homeostatic state (Diegelmann and Evans, 2004). However, chronic wounds do have some important differences (Loots et al., 1998) that can impact the effectiveness of advanced wound therapies, compared in Table 19.1. Chronic venous leg ulcers There are several things that happen prior to the existence of a VLU. When return blood flow in the leg veins is compromised, there is distention of the leg veins and inflammatory damage. This damage creates changes in the microvasculature. Neutrophils and mast cells adhere to the endothelium and macrophages infiltrate the walls of the damaged veins and valves. The inflammatory response then extends beyond a reaction to the stressed microvascluature into the extavascular space. Activated endothelium allows extravasation of leukocytes into the perivascular space and there is leakage of fibrinogen and the accumulation of fibrin around the damaged vessels. Lipodermatosclerosis is the term used to describe the changes in the vasculature and connective tissue that ensues. Change in the microvasculature causes a prolonged response to inflammation and an impetus to resolve it and reach homeostasis. Growth factors and remodeling enzymes are upregulated in an attempt to progress towards regeneration and repair. The tissues retain a robust production of vascular endothelial growth factor (VEGF), a major driver of granulation, even as it progresses to a VLU (Lauer et al., 2000). With repeatedly unsuccessful attempts to resolve inflammation, advance repair and stabilize, the connective tissue becomes increasingly fibrotic and thickened (Diegelmann and Evans, 2004). Damage to the microvascular bed localizes in the papillary dermis, closest to the epidermis. Although VEGF is abundant, angiogenesis is no longer stimulated. Other growth factors that would normally support regeneration decline and the VLU settles into an abnormal, corrupt state (Danon et al., 1989). Analysis of wound fluid suggests that the VLU environment will be inhibitory to fibroblast proliferation and migration, and will promote apoptosis (Seah et al., 2005). Although wound fluid is more a composite view of the physiological state of the tissue rather than the cause for non-healing, this environment will challenge any cellular implant and indicates what the

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Table 19.1 Similarities and differences to keep in mind when targeting healing in different types of chronic wounds A. Chronic venous ulcers Root cause: Although the epidermis does not migrate, the fault is likely elsewhere. The underlying pathology is microvascular in origin manifesting as secondary pathology (lipodermatosclerosis) in the wound bed before an open wound even occurs. Component:

Inflammatory

Microvascular

Connective tissue

Epidermal

While the inflammatory response contributes to the development of the condition, the normal inflammatory response is ultimately blunted by a chronic feedback loop and pathological changes in the ECM. The tissue inflammatory response elsewhere in the body is unimpaired. Angiogenesis takes its cues from the inflammatory response. The blunted inflammatory response results in blunted angiogenesis. Changes in the connective tissue exacerbate the difficulty to mount a vascular response. Vascular leakage and chronic inflammatory cell infiltration cause a corruption of the ECM. The CVU ECM becomes prematurely, yet ineffectively stabilized in a corrupt repair response including an altered fibroblast proliferative and biosynthetic response. Corrupt or “exhaustive” signaling by a lipodermatosclerotic substratum may drown out or otherwise prevent the epidermis from activating mechanisms of wound closure (signaling inherent within the epidermis).

B. Pressure ulcers Root cause: Chronic injury prevents the normal repair response to advance and resolve. Component:

Inflammatory

Microvascular

Connective tissue

Chronic physical injury and inadequate resolution results in a blunted ability to respond anew over time. Re-growth of the damaged microvasculature is thwarted by inadequate connective tissue support and a blunted inflammatory response. Unable to repair adequately due to chronic injury and the presence of chronic inflammation.

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Table 19.1 Continued Epidermal

The epidermis is unable to re-establish coverage due to the chronic physical challenge. Over time, chronic signaling and lack of substratum add to the challenge.

C. Diabetic foot ulcers Root cause: Diabetic neuropathy creates a pressure sore (above) where healing is further complicated by diabetic changes in the microvasculature and ECM. Component:

Inflammatory

Microvascular

Connective tissue

Epidermal

There is an impaired ability to respond. Changes in the vascular wall promote increased adhesion but do not translate to increased inflammation – resulting in an overall reduction in functional macrophages. Microvascular pathology involves reduced function of the endothelium and perivascular cells. Endothelial surface changes promote increased leukocyte adhesion but a thickened basement membrane changes the relationship between vascular cells and leukocytes, which must transverse the vascular wall. Advanced glycosylated endproducts modify the extracellular matrix changing its degradation characteristics. Fibroblast-matrix interaction with the glycosylated matrix is corrupted resulting in changes in apoptosis and matrix biosynthesis. A chronic fibrotic response adds to the pathology. The trauma and an inadequate substratum result in difficult conditions for epidermal migration. The epidermis at the wound margin is often highly calloused and shows evidence of hyperproliferation.

Adapted from Parenteau and Hardin-Young (2006). ECM = Extracellular matrix, CVU = chronic venous ulcer.

implant will have to overcome. If one limits further damage with proper compression therapy, could a cell implant now have a greater chance of normalizing signaling? The fibrotic wound bed is still likely to be significantly potent and hostile – capable of overpowering many applied cells. If one surgically debrides the VLU, removing the thick, fibrotic, corrupt wound bed to initiate an acute response, can one now augment what might be a weak acute response by adding normal cells? In this case, the cell therapy

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now has to be biologically robust and persistent enough to withstand acute inflammatory factors like tumor necrosis factor alpha that sharp debridement would trigger (Parenteau and Hardin-Young, 2007). If that is the case, could a therapy be more effective using other forms of debridement? Would epidermal cells work better than fibroblasts, or are both fibroblasts and keratinocytes needed? These questions point out two things: 1. That the clinical protocol can have a very real impact on the therapy. 2. Making the connection between the product and the biological process at work is important. A chronic wound is not simply an acute wound that fails to heal. In VLU, it is the end result of an underlying pathological condition. Lipodermatosclerosis causes changes in the dermal bed before the epidermis fails. If one looks at the wound periphery of a non-healing VLU, it looks like chronically inflamed skin. Studies of wound peripheries show no impairment in keratinocyte proliferation, cytokine response or enhanced apoptosis; even expressing keratins 6 and 16, used by migrating epithelium (Stojadinovic et al., 2005). The epidermis does not appear fundamentally impaired but more likely conditioned. One hypothesis is that interferon gamma is involved (Simka, 2006), another hypotheses implicate connective tissue growth factor and transforming growth factor beta (Parenteau, 2006, Amendt et al., 2002, Parenteau and Hardin-Young, 2006) – prominent factors in fibrosis and stabilization. The epidermis appears to be in an inflammatory state but lacks the impetus to migrate (normally present in the early stages of acute healing and distinct from later stages of healing). It appears that chronic venous insufficiency blunts the epidermal response that will activate migration during the first stages of healing. It is as if the epidermis is able to wither away without mounting sufficient signaling to convey damage has occurred. The underlying dermis is also unable to relay signaling to change that. It is in effect, as though the damage to and loss of the epidermis cannot be discerned above the noise of the chronic disease in either the dermis or epidermis. This implies that a powerful signal will be necessary to overcome the tissue pathology, which may explain why achieving success with an active therapy in VLU has been particularly difficult. In VLU, adding normal fibroblasts may augment signaling, but an epidermis will be as, or more likely to have within it signaling capable of activating the patient’s epithelium, particularly since scientific evidence suggests that the host epidermis is not fundamentally impaired. The epidermis of GS is mature and when wounded, the epidermis undergoes the normal wound healing process to heal itself (Falanga et al., 2002). When it is meshed and applied to the wound surface, it undergoes that acute response. So while it may not take on the hostile wound bed, its own struggle to survive and heal is transmitted to the patient’s tissue. Now, instead of an inadequate signal

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of epidermal injury, the surrounding tissue receives the message of an acute wound, from the GS. Clinical observation of new granulation tissue formation in even long-standing VLU following even non-surgical debridement indicate that this change in signaling also extends to the dermal bed (Sabolinski et al., 1996) although it is unclear how much is conveyed by the dermal fibroblasts or the epidermis, or the combination of both. The failure of OrCelTM, composed of collagen, fibroblasts and keratinocytes, to successfully complete a VLU trial suggests that it is not as simple as delivering the component parts, but is also subject to how it is put together, its persistence, its robustness, and how it is used clinically (Parenteau, 2009). Diabetic foot ulcers In the diabetic wound, fibroblasts and macrophages appear plentiful at the wound margins but their migration and function are impeded in the diabetic wound bed (Galkowska et al., 2005). Some of the fibroblast deficit is attributed to interaction with advanced glycosylated endproducts (AGE)-modified collagen (Lerman et al., 2003, Twigg et al., 2001, Alikhani et al., 2005). The diabetic environment appears to be one of general impairment in the ability to respond, first to the physiological and physical needs of the foot and second to the need for repair, once injured. It is a pressure ulcer with physiological complications. When the foot is then injured, a result of the diabetic neuropathy, the acute response may be inadequate to heal the wound rapidly, if at all. Debridement to remove fibrotic tissue may help mount an acute response, but in the DFU, the wound may also benefit from the addition of normal fibroblasts as well as a naïve extracellular matrix provided, the patient can provide adequate vascularization. Circulation may be impaired in the long-standing diabetic patient (Veves et al., 1998). The use of most advanced therapies test for adequate circulation beforehand and patients with inadequate circulation are presently contraindicated for most therapies. It should also be noted that the threat of infection is of particular concern in the diabetic patient at risk of amputation, making the speed of wound closure an important clinical endpoint in addition to number of patients healed. Pressure ulcers The pressure ulcer is similar to the diabetic foot ulcer in being a result of constant mechanical insult to the tissue (Diegelmann and Evans, 2004) but unlike the diabetic foot ulcer, there is no additional underlying pathology affecting the skin. The challenge of the pressure ulcer is in overcoming the chronic inflammation and fibrosis that has developed, usually with debridement with off-loading, and then finding a way to restore connective tissue

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loss as well as achieve wound closure in a durable way. This appears to be an ideal opportunity for the addition of a scaffold, template or other ECM technology that can support new tissue formation. The question alluded to earlier in the discussion of scaffolds and templates is how one can best use them and achieve wound closure. The use of and need for cells in these patients may be less acute than in the VLU or DFU but may add a benefit in time to repair and healing. In open wounds under constant threat of infection, biologic wound closure and the speed of healing will be vital, as it is with the diabetic patient. Certainly, if one could induce formation of a durable, compliant connective tissue that fully supported the health of new epidermis, we would have a medical win. What becomes evident in attempting to connect the theoretical with clinical considerations and the practical experience thus far, is that design based on biological connectivity, delivery, and timing are important, and perhaps pivotal considerations in the ability to turn a positive trend into a significant clinical result capable of regulatory approval and commercial acceptance. So far, Dermagraft® is the only successful fibroblast-based therapy for DFU based on the use of eight weekly applications. Only Apligraf® has succeeded to date in demonstrating clinical efficacy in VLU although Advanced BioHealing is making another attempt in VLU with what they hope will be better use of Dermagraft® and better trial design (personal communication).

19.8

Advanced therapies in the marketplace

What can be learned from the advanced therapies that have made it to the market? One of the most salient points is that they still have a long way to go in gaining market acceptance. For example, company sources admit that Apligraf after more than 10 years on the market and despite being the only approved advanced therapy for VLU, yet reaches only approximately 25% of the US market with revenues just over $100 M in 2009 (personal communication). The alternative therapies also struggle to convince physicians to go beyond their own standard of care. There are perhaps several factors that impact market penetration: • •

•

The cost of the advanced therapies are more than wound dressings. The persistent mentality beyond thought leaders that chronic wounds are principally managed with nursing care rather than healed and that good wound management has as good a chance at healing a wound capable of it than anything else. The regulators and payers have limited the use of the advanced therapies to wounds that do not heal by traditional means lending support to the idea of continued use of older methods.

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•

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The availability of alternatives that are supported by capital equipment expenditure like hyperbaric oxygen, electrical stimulation or VAC therapy where the machines and in the case of hyperbaric oxygen, investment in a center has to be justified and paid for. Last but not least, the economics of care and reimbursement.

Medicare policies in the US made it difficult to use advanced therapies at first. Medicare prospective payer policies covered wound management but the use of advanced wound care ran the risk of being denied if it was considered premature. Problems with healing and ongoing nursing care had to be carefully documented to justify the use of more expensive products (Motta, 1998, Peck and Gross, 1999). This made it easier to default to good wound care rather than deal with issues of reimbursement. To overcome this hurdle, companies became pro-active in helping doctors navigate the reimbursement system and understand when their products could be used. A 2009 systematic review of the economics of human cell-derived wound care products (Apligraf®, Dermagraft® and Regranex®) for VLU and DFU concluded that health care providers and coverage decision makers should not focus on the high cost of the biotechnology product but on the total cost of care (Langer and Rogowski, 2009). They concluded that these treatments might be cost-effective if restricted to those that fail to respond to good wound care. They went on to advise that the effectiveness as well as the cost-effectiveness of the products needed further study as no study met all quality criteria, most notably, quality independent analysis. It is important then that advanced therapies be supported by independent analysis of cost-benefit as well as continued scientific and post-marketing clinical studies that can stand as Level 1 evidence to further justify how and when these therapies can be used to achieve the best result for the patient, and the best economy for the healthcare system.

19.9

Conclusion

There are several lessons to be learned from the experiences so far. First, that good science is not optional. Economies and ease of process are only exercises without efficacy. Second, that successful application of the science is not the concern of how well science is done but of how well it is used. The connectivity between what is known scientifically and medically must be integrated into the strategy, design and use of advanced products. And finally, that making it to market is not the same as making it in the market. Successful commercialization will require demonstrating value to the patient and cost-savings to the medical system. To do that, therapies will have to target effectiveness significantly beyond what can be achieved through good wound care while targeting treatments to where they best fit

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and are most needed. The business of wound care has to evolve beyond the economics of wound management to wound healing. If a case can and should be made for the use of advanced therapy or a combination of therapies for the healing of a life-threatening pressure ulcer, then it will be up to the developers and clinicians to take an active role in making the case with quality evidence-based medicine. Advanced therapies have languished in their potential long enough. While we might debate the importance of science versus business in this effort, it is clear that both need to be done well. Businesses have to have a realistic eye on costs of manufacture and what it will take to penetrate the market; structuring partnerships and their businesses accordingly. Advanced therapies must demonstrate efficacy substantially beyond good wound management – in a way that impacts patient care, morbidity and quality of life. To accomplish this, future products must become combined innovative in design and production with robust connectivity to the biology and medical need. To gain market penetration, efforts will have to be validated with quality clinical studies and sound economic analysis – irrespective of the regulatory route to market. If advanced wound therapies are to reach their maximum potential and regain interest from investors and large corporate partners, they will have to be good enough to change the paradigm of wound care – driving practice and perception from a wound management mentality to one of active wound healing.

19.10 References Alikhani, Z., Alikhani, M., Boyd, C. M., Nagao, K., Trackman, P. C. & Graves, D. T. (2005) Advanced glycation end products enhance expression of pro-apoptotic genes and stimulate fibroblast apoptosis through cytoplasmic and mitochondrial pathways. J Biol Chem, 280, 12087–95. Amendt, C., Mann, A., Schirmacher, P. & Blessing, M. (2002) Resistance of keratinocytes to TGFbeta-mediated growth restriction and apoptosis induction accelerates re-epithelialization in skin wounds. J Cell Sci, 115, 2189–98. Bell, E., Ehrlich, H. P., Buttle, D. J. & Nakatsuji, T. (1981) Living tissue formed in vitro and accepted as skin-equivalent tissue of full thickness. Science, 211, 1052–4. Campitiello, E., Della Corte, A., Fattopace, A., D’Acunzi, D. & Canonico, S. (2005) The use of artificial dermis in the treatment of chronic and acute wounds: regeneration of dermis and wound healing. Acta Biomed, 76 Suppl 1, 69–71. Compton, C. C., Gill, J. M., Bradford, D. A., Regauer, S., Gallico, G. G. & O’Connor, N. E. (1989) Skin regenerated from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting. A light, electron microscopic and immunohistochemical study. Lab Invest, 60, 600–12. Danon, D., Kowatch, M. A. & Roth, G. S. (1989) Promotion of wound repair in old mice by local injection of macrophages. Proc Natl Acad Sci U S A, 86, 2018–20. Diegelmann, R. F. & Evans, M. C. (2004) Wound healing: an overview of acute, fibrotic and delayed healing. Front Biosci, 9, 283–9.

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Drew, A. F., Liu, H., Davidson, J. M., Daugherty, C. C. & Degen, J. L. (2001) Woundhealing defects in mice lacking fibrinogen. Blood, 97, 3691–8. Falanga, V. & Sabolinski, M. (1999) A bilayered living skin construct (APLIGRAF) accelerates complete closure of hard-to-heal venous ulcers. Wound Repair Regen, 7, 201–7. Falanga, V., Isaacs, C., Paquette, D., Downing, G., Kouttab, N., Butmarc, J., Badiavas, E. & Hardin-Young, J. (2002) Wounding of bioengineered skin: cellular and molecular aspects after injury. J Invest Dermatol, 119, 653–60. Fivenson, D. P., Scherschun, L., Choucair, M., Kukuruga, D., Young, J. & Shwayder, T. (2003) Graftskin therapy in epidermolysis bullosa. J Am Acad Dermatol, 48, 886–92. Flasza, M., Kemp, P., Shering, D., Qiao, J., Marshall, D., Bokta, A. & Johnson, P. A. (2007) Development and manufacture of an investigational human living dermal equivalent (ICX-SKN). Regen Med, 2, 903–18. Galkowska, H., Wojewodzka, U. & Olszewski, W. L. (2005) Low recruitment of immune cells with increased expression of endothelial adhesion molecules in margins of the chronic diabetic foot ulcers. Wound Repair Regen, 13, 248–54. Gallico, G. G., 3rd, O’Connor, N. E., Compton, C. C., Kehinde, O. & Green, H. (1984) Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med, 311, 448–51. Green, H., Kehinde, O. & Thomas, J. (1979) Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci U S A, 76, 5665–8. Gregor, S., Maegele, M., Sauerland, S., Krahn, J. F., Peinemann, F. & Lange, S. (2008) Negative pressure wound therapy: a vacuum of evidence? Arch Surg, 143, 189–96. Hansbrough, J. F., Cooper, M. L., Cohen, R., Spielvogel, R., Greenleaf, G., Bartel, R. L. & Naughton, G. (1992) Evaluation of a biodegradable matrix containing cultured human fibroblasts as a dermal replacement beneath meshed skin grafts on athymic mice. Surgery, 111, 438–46. Hardin-Young, J. & Parenteau, N. L. (2004) Progenitor cell properties and the engineering of tissues. Curr Neurovasc Res, 1, 241–9. Hickerson, W. L., Compton, C., Fletchall, S. & Smith, L. R. (1994) Cultured epidermal autografts and allodermis combination for permanent burn wound coverage. Burns, 20 Suppl 1, S52–5; discussion S55–6. Ho, J. Y. & Miller, S. L. (2007) Allografts in the treatment of athletic injuries of the shoulder. Sports Med Arthrosc, 15, 149–57. Kojima, K., Okamoto, Y., Kojima, K., Miyatake, K., Fujise, H., Shigemasa, Y. & Minami, S. (2004) Effects of chitin and chitosan on collagen synthesis in wound healing. J Vet Med Sci, 66, 1595–8. Kubo, M., Van de Water, L., Plantefaber, L. C., Mosesson, M. W., Simon, M., Tonnesen, M. G., Taichman, L. & Clark, R. A. (2001) Fibrinogen and fibrin are antiadhesive for keratinocytes: a mechanism for fibrin eschar slough during wound repair. J Invest Dermatol, 117, 1369–81. Kumar, R. J., Kimble, R. M., Boots, R. & Pegg, S. P. (2004) Treatment of partial-thickness burns: a prospective, randomized trial using Transcyte. ANZ J Surg, 74, 622–6. Langer, A. & Rogowski, W. (2009) Systematic review of economic evaluations of human cell-derived wound care products for the treatment of venous leg and diabetic foot ulcers. BMC Health Serv Res, 9, 115.

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Lauer, G., Sollberg, S., Cole, M., Flamme, I., Sturzebecher, J., Mann, K., Krieg, T. & Eming, S. A. (2000) Expression and proteolysis of vascular endothelial growth factor is increased in chronic wounds. J Invest Dermatol, 115, 12–8. Lerman, O. Z., Galiano, R. D., Armour, M., Levine, J. P. & Gurtner, G. C. (2003) Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia. Am J Pathol, 162, 303–12. Livesey, S. A., Herndon, D. N., Hollyoak, M. A., Atkinson, Y. H. & Nag, A. (1995) Transplanted acellular allograft dermal matrix. Potential as a template for the reconstruction of viable dermis. Transplantation, 60, 1–9. Loots, M. A., Lamme, E. N., Zeegelaar, J., Mekkes, J. R., Bos, J. D. & Middelkoop, E. (1998) Differences in cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers versus acute wounds. J Invest Dermatol, 111, 850–7. Mason, C. (2007) Regenerative medicine 2.0. Regen Med, 2, 11–8. Misra, S., Raj, P. K., Tarr, S. M. & Treat, R. C. (2008) Results of AlloDerm use in abdominal hernia repair. Hernia, 12, 247–50. Mori, T., Murakami, M., Okumura, M., Kadosawa, T., Uede, T. & Fujinaga, T. (2005) Mechanism of macrophage activation by chitin derivatives. J Vet Med Sci, 67, 51–6. Morykwas, M. J. & Argenta, L. C. (1997) Nonsurgical modalities to enhance healing and care of soft tissue wounds. Journal of the Southern Orthopaedic Association, 6, 279–288. Motta, G. J. (1998) Solving the mysteries of wound care reimbursement. Nursing Homes, Nov–Dec issue. Naughton, G. (1999) The Advanced Tissue Sciences story. Sci Am, 280, 84–5. Naughton, G., Mansbridge, J. & Gentzkow, G. (1997) A metabolically active human dermal replacement for the treatment of diabetic foot ulcers. Artif Organs, 21, 1203–10. Northrup, J. (2006) The Pharmaceutical Sector. In Burns, L. R. (Ed.) The Business of Healthcare Innovation. Cambridge, Cambridge University Press. Parenteau, N. (1999) Skin: the first tissue-engineered products. Sci Am, 280, 83–4. Parenteau, N. & Hardin-Young, J (2006) Cutaneous Healing, Fair Haven, Parenteau BioConsultants, LLC. Parenteau, N. & Hardin-Young, J. (2006) Achieving Medical and Commercial Success in Wound Repair and Regeneration. Cutaneous Healing, Fair Haven, Parenteau BioConsultants, LLC. Parenteau, N. & Hardin-Young, J. (2007) The Biological Mechanisms Behind Injury and Inflammation: How They Can Affect Product Performance, and Healing. Wounds, 19, 87–96. Parenteau, N. L. (2009) Commercial development of cell-based therapeutics: strategic considerations along the drug to tissue spectrum. Regen Med, 4, 601–11. Park, C. A., Defranzo, A. J., Marks, M. W. & Molnar, J. A. (2009) Outpatient reconstruction using integra* and subatmospheric pressure. Ann Plast Surg, 62, 164–9. Peck, R. L. & Gross, M. (1999) Wound care post-PPS: are these vendors showing the way? – Wound care programs in nursing and long term care facilities in wake of the medicare prospective payament system, Nursing Homes/Long Term Care Management 48(4). Phillips, T. J. & Gilchrest, B. A. (1990) Cultured epidermal grafts in the treatment of leg ulcers. Adv Dermatol, 5, 33–48; discussion 49.

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Phillips, T. J. & Gilchrest, B. A. (1991) Cultured epidermal allografts as biological wound dressings. Prog Clin Biol Res, 365, 77–94. Purdue, G. P., Jr., Still, J. M., Jr. et al. (1997) A multicenter clinical trial of a biosynthetic skin replacement, Dermagraft-TC, compared with cryopreserved human cadaver skin for temporary coverage of excised burn wounds. J Burn Care Rehabil, 62–67. Reyzelman, A., Crews, R. T., Moore, J. C., Moore, L., Mukker, J. S., Offutt, S., Tallis, A., Turner, W. B., Vayser, D., Winters, C. & Armstrong, D. G. (2009) Clinical effectiveness of an acellular dermal regenerative tissue matrix compared to standard wound management in healing diabetic foot ulcers: a prospective, randomised, multicentre study. Int Wound J, 6, 196–208. Sabolinski, M. L., Alvarez, O., Auletta, M., Mulder, G. & Parenteau, N. L. (1996) Cultured skin as a ‘smart material’ for healing wounds: experience in venous ulcers. Biomaterials, 17, 311–20. Sbitany, H., Sandeen, S. N., Amalfi, A. N., Davenport, M. S. & Langstein, H. N. (2009) Acellular dermis-assisted prosthetic breast reconstruction versus complete submuscular coverage: a head-to-head comparison of outcomes. Plast Reconstr Surg, 124, 1735–40. Schultz, G. S., Sibbald, R. G., Falanga, V., Ayello, E. A., Dowsett, C., Harding, K., Romanelli, M., Stacey, M. C., Teot, L. & Vanscheidt, W. (2003) Wound bed preparation: a systematic approach to wound management. Wound Repair Regen, 11 Suppl 1, S1–S28. Schurr, M. J., Foster, K. N., Centanni, J. M., Comer, A. R., Wicks, A., Gibson, A. L., Thomas-Virnig, C. L., Schlosser, S. J., Faucher, L. D., Lokuta, M. A. & AllenHoffmann, B. L. (2009) Phase I/II clinical evaluation of StrataGraft: a consistent, pathogen-free human skin substitute. J Trauma, 66, 866–73; discussion 873–4. Seah, C. C., Phillips, T. J., Howard, C. E., Panova, I. P., Hayes, C. M., Asandra, A. S. & Park, H. Y. (2005) Chronic wound fluid suppresses proliferation of dermal fibroblasts through a Ras-mediated signaling pathway. J Invest Dermatol, 124, 466–74. Shi, C., Zhu, Y., Ran, X., Wang, M., Su, Y. & Cheng, T. (2006) Therapeutic potential of chitosan and its derivatives in regenerative medicine. J Surg Res, 133, 185–92. Simka, M. (2006) A potential role of interferon-gamma in the pathogenesis of venous leg ulcers. Med Hypotheses, 67, 639–44. Stojadinovic, O., Brem, H., Vouthounis, C., Lee, B., Fallon, J., Stallcup, M., Merchant, A., Galiano, R. D. & Tomic-Canic, M. (2005) Molecular pathogenesis of chronic wounds: the role of beta-catenin and c-myc in the inhibition of epithelialization and wound healing. Am J Pathol, 167, 59–69. Thoma, D. S., Benic, G. I., Zwahlen, M., Hammerle, C. H. & Jung, R. E. (2009) A systematic review assessing soft tissue augmentation techniques. Clin Oral Implants Res, 20 Suppl 4, 146–65. Twigg, S. M., Chen, M. M., Joly, A. H., Chakrapani, S. D., Tsubaki, J., Kim, H. S., Oh, Y. & Rosenfeld, R. G. (2001) Advanced glycosylation end products up-regulate connective tissue growth factor (insulin-like growth factor-binding protein-related protein 2) in human fibroblasts: a potential mechanism for expansion of extracellular matrix in diabetes mellitus. Endocrinology, 142, 1760–9. Veves, A., Akbari, C. M., Primavera, J., Donaghue, V. M., Zacharoulis, D., Chrzan, J. S., Degirolami, U., Logerfo, F. W. & Freeman, R. (1998) Endothelial dysfunction

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and the expression of endothelial nitric oxide synthetase in diabetic neuropathy, vascular disease, and foot ulceration. Diabetes, 47, 457–63. Veves, A., Falanga, V., Armstrong, D. G. & Sabolinski, M. L. (2001) Graftskin, a human skin equivalent, is effective in the management of noninfected neuropathic diabetic foot ulcers: a prospective randomized multicenter clinical trial. Diabetes Care, 24, 290–5. Waymack, P., Duff, R. G. & Sabolinski, M. (2000) The effect of a tissue engineered bilayered living skin analog, over meshed split-thickness autografts on the healing of excised burn wounds. The Apligraf Burn Study Group. Burns, 26, 609–19. Wilkins, L. M., Watson, S. R., Prosky, S. J., Meunier, S. F. & Parenteau, N. L. (1994) Development of a bilayered living skin construct for clinical applications. Biotechnol Bioeng, 43, 747–56. Yannas, I. V. (2001) 8.2.4 Biological activity of certain ECM analogs. In Yannas, I. V. (Ed.) Tissue and Organ Regeneration in Adults. New York, Springer-Verlag. Yannas, I. V., Burke, J. F., Orgill, D. P. & Skrabut, E. M. (1982) Wound tissue can utilize a polymeric template to synthesize a functional extension of skin. Science, 215, 174–6.

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20 Biologically derived scaffolds K. N U M ATA, RIKEN Institute, Japan and D. L. K A P L A N, Tufts University, USA

Abstract: This chapter discusses biomaterial scaffolds processed from biologically-derived polymers, such as polyhydroxyalkanoate, silk, collagen, elastin, resilin, keratin, chitosan, chitin, cellulose, and hyaluronan, for tissue engineering and regenerative medicine. Recent studies concerning the preparation and application of these biologically derived polymers for biomaterial scaffolds are summarized in this chapter. Key words: biopolymer, protein, polysaccharide, scaffold, tissue engineering.

20.1

Introduction

Biopolymers, polymers originated from biological sources, are usually divided into four types: 1) nucleotide, 2) protein and poly(amino acid), 3) polysaccharide and 4) poly(hydroxyalkanoate). Biopolymers of natural origin have been investigated for the preparation and application of biomaterials for a range of applications, including scaffolds in tissue engineering, because of their biocompatibility, cellular adhesive features and biodegradability. However, biomaterial scaffolds from biopolymers often require improved mechanical properties, control of porosity and optimized processing for practical use in tissue engineering, and regenerative medicine. Many studies concerning the preparation and application of biopolymer-based scaffolds have been conducted, and properties have been achieved that are on par or improved over synthetic polymer-based scaffolds (Table 20.1). In this chapter, these studies of biologically derived polymers for biomaterial scaffolds are summarized.

20.2

Polyhydroxyalkanoate (PHA)-derived scaffolds

Poly(hydroxyalkanoate)s (PHAs), one of the eco-friendly, biobased, and biodegradable polymeric materials, are polyesters synthesized by a variety of bacteria as an intracellular storage material of carbon and energy (Doi, 1990; Doi and Steinbüchel, 2001; Lenz and Marchessault, 2005; Sudesh et al., 2000). Monomer units of PHA are shown in Fig. 20.1. 524 © Woodhead Publishing Limited, 2011

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1 2 −1 −2 51 – 137–175 175–210 –

150 135 127 120 60 –

–

–

–

– – – – – –

170 240 –

Elastin (bovine ligament) Resilin (dragonfly tendon) Keratin/chitosan Keratin/glycerol Keratin/glycerol/chitosan Cellulose

Reference materials Polypropylene Polystyrene Kevlar

38 50 3600

2 4 27–34 1 9–14 100–120

400 7 2.7

150 190 4–9 28 ~28 20–30

13

25 ± 5

800 ± 400 120

22 ± 4

20 100 400 850 1000 430

5 64

Elongation at break (%)

400 ± 100

25 20 21 20 50 33

43 32

Tensile strength (Mpa)

– – (Zimmerman and Gordon, 1988).

(Sun et al., 2010).

(Martin and Williams, 2003). (Chanprateep and Kulpreecha, 2006). (Drummy et al., 2005; Sirichaisit et al., 2003). (Sirichaisit et al., 2003; Wang et al., 2004). (Pollock and Shadwick, 1994). (Aaron and Gosline, 1981). (Weisfogh, 1961). (Tanabe et al., 2002).

(Doi et al., 1995; Iwata et al., 2005; Kusaka et al., 1997; Kusaka et al., 1999).

Reference

3HB: 3-Hydroxybutyrate, 3HH: 3-Hydroxyhexanoate, 3HV: 3-Hydroxyvalerate, 4HB: 4-Hydroxybutyrate, −: n.d.

−10 100 –

– – – – – –

4 –

177 170

P(3HB) Ultrahigh-molecular weight P(3HB) P(3HB-co-10% 3HV) P(3HB-co-20% 3HV) P(3HB-co-10% 3HHx) P(3HB-co-17% 3HHx) P(4HB) P(4% 3HB-co-3% 3HV-co-93% 4HB) silkworm (Bombyx mori) silks (fiber and film) spider dragline (Nephila edulis) silks (fiber) Collagen (mammalian tendon)

Glass transition temp, Tg (°C)

Melting point, Tm (°C)

Sample

Table 20.1 Thermal and mechanical properties of biologically derived polymers

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Advanced wound repair therapies Poly(hydroxyalkanoate) (PHA)

3HB OCHCH2CO CH3

n

3HV OCHCH2CO

m

3HH OCHCH2CO

l

3HO OCHCH2CO

CH2

CH2

CH2

CH3

CH2

CH2

CH3

CH2

o

4HB OCH2CH2CH2CO

p

CH2 CH3

20.1 Chemical structures of monomer units of poly(hydroxyalkanoate) (PHA).

Poly[(R)-3-hydroxybutyrate] (P(3HB)) was the first PHA isolated from Bacillus megaterium in the 1920s and later identified as a microbial reserve polyester (Lemoignei, 1926). PHAs are attractive as biomaterials, such as scaffolds for tissue engineering, because of their biocompatibility, processability and wide range of mechanical properties related to the chemistry of the secondary monomer units. The identification of hydroxyalkanoate units other than 3HB as constituents of microbial reserve polyesters has had a major impact on research and commercial interests in these microbial polyesters, since the incorporation of different hydroxyalkanoate units into P(3HB) can change the mechanical, thermal and biological properties (Gordeyev and Nekrasov, 1999; Nakamura et al., 1992; Numata et al., 2004; 2005). In 1988, PHA with 4-hydroxybutyrate units (4HB), the secondary units of PHAs which has higher in-vivo biodegradability in comparison to the other PHAs, was synthesized via bacterial pathways of Ralstonia eutropha (Doi et al., 1988). PHAs containing 4HB units have been particularly attractive as biomaterials for tissue engineering and implantation. Additionally, oligomers and monomers of PHA, which are produced after hydrolysis degradation of PHA materials, have therapeutic and nutritional effects, including for seizure control, metabolic disease control, reduction of protein catabolism, appetite suppression, parenteral nutrition, increasing cardiac efficiency, treatment of diabetes and insulin resistant states, and treatment of effects of neurodegenerative disorders and epilepsy (Martin et al., 1999; Williams and Martin, 2000). PHAs have been investigated as biomaterials for implants such as bone plates, temporary stents, patches and screws (Boeree et al., 1993; Malm et al., 1992; Peng et al., 1996). The first in-vivo study of PHA materials was reported in 1987 and indicated poor in-vivo biodegradation of P(3HB) and an effect of hydration on the ultimate tensile properties of the materials (Miller and Williams, 1987). In contrast, poly[4-hydroxybutyrate] (P(4HB))

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and P(3HB-co-4HB) are hydrolyzed by lipases, in the human body, because of the change in chemistry which helps lipases access the main chain in comparison to the other PHAs (Fig. 20.1). P(4HB) homopolymer has therefore been studied as a strong flexible bioabsorbable material for biomedical applications such as cardiovascular, wound healing, orthopedic, drug delivery and tissue engineering (Martin and Williams, 2003). P(4HB) scaffolds for viable ovine blood vessels suitable for implantation into systemic circulation showed useful function for tissue engineering blood vessels (Opitz et al., 2004). P(4HB)-based trileaflet heart valve scaffolds with 100–240 μm porous structures also exhibited high cell viability and cell growth to form connective tissue between the inside and the outside of porous scaffolds (Sodian et al., 2000a; 2000b; 2002). Poly[(R)-3-hydroxybutyrate-co-(R)-3hydroxyhexanoate] (P(3HB-co-3HHx)), one of the PHAs, was also reported to show lower cytotoxicity and higher biocompatibility in comparison to P(3HB) and poly(l-lactide) (PLLA) (Zhao et al., 2003).

20.3

Silk-derived scaffolds

Silk proteins are produced by a variety of insects, scorpions, and spiders, and form fibrous materials in nature, such as spider orb webs and silkworm cocoons (Altman et al., 2003; Wong Po Foo and Kaplan, 2002). Several features of silk-based materials, such as mechanical properties, solubility and biodegradability, can be controlled by manipulating the secondary structure (Huemmerich et al., 2004; Li et al., 2003). Silk proteins have therefore been explored as scaffolds for cell culture and tissue engineering (Wang et al., 2006). The properties and amino acid sequences of silk proteins are listed in Tables 20.1 and 20.2. Silkworm silk has been used as biomedical sutures for decades, and in textile production for clothes for centuries. Silkworms are easier to domesticate and obtain silk fibroins in comparison to the other silks such as spider silks. The silk fibroins from the cocoon of silkworm Bombyx mori, which is the most studied silkworm silk, contains at least two major fibroin components, a light chain (~25 kDa) and a heavy chain (~392 kDa). The core sequence in the heavy chain includes alanine-glycine repeats with serine or tyrosine. In silkworm cocoons, these two types of silk fibroins are encased in a sericin coat, glue-like proteins that form the composite fibers of the cocoon case. The degradation products of silk fibroin proteins with betasheet structures from the action of proteases, such as alpha-chymotrypsin, has recently been reported, and no cytotoxicity was observed to neurons in-vitro (Numata et al., 2010a). Removal of sericin from silkworm silk is necessary to prepare non-allergic and non-cytotoxic silk-based materials. Recently, methods to extract and regenerate silk fibroin have been developed, and several silk-based biomaterials, such as silk porous scaffolds, silk

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[Gly-Pro-Hyp]n [Val-Pro-Gly-Val-Gly]n MFKLLGLTLLMAMVVLGRPEPPVNSYLPPSDSYGA PGQSGPGGRPSDSYGAPGGGNGGRPSDSYGAPGQG QGQGQGQGGYAGKPSDTYGAPGGGNGNGGRPSSSY GAPGGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGG GGNGNGGRPSSSYGAPGQGQGNGNGGRSSSSYGAP GGGNGGRPSDTYGAPGGGNGGRPSDTYGAPGGGNN GGRPSSSYGAPGGGNGGRPSDTYGAPGGGNGNGS GGRPSSSYGAPGQGQGGFGGRPSDSYGAPGQNQKPS DSYGAPGSGNGNGGRPSSSYGAPGSGPGGRPSDSY GPPASGSGAGGAGGSGPGGADYDND IVEYEADQQG YRPQIRYEGDANDGSGPSGPGGPGGQNLGADGYS SGRPGNGNGNGNGGYSGGRPGGQDLGPSGYSGGRPG GQDLGAGGYSNGKPGGQDLGPGGYSGGRPGGQDL GRDGYSGGRPGGQDLGASGYSNGRPGGNGNGGSDGG RVIIGGRVIGGQDGGDQGYSGGRPGGQDLGRDGYSS GRPGGRPGGNGQDSQDGQGYSSGRPGQGGRNGFG PGGQNGDNDG SGYRY

(Beckwitt and Arcidiacono, 1994; Bini et al., 2006; Hinman and Lewis, 1992). (Orgel et al., 2006). (Indik et al., 1987). (Adams et al., 2000; Hoskins et al., 2007).

[GRGGLGGQGAGAAAAAGGAGQGGYGGLGSQG]n

Collagen Elastin Resilin, Pro-Resilin

(Mita et al., 1994).

[Gly-Ala-Gly-Ala-Gly-Ser]n

Silk (silkworm, hydrophobic region) Silk (spider)

Reference

Amino acid sequence

Protein

Table 20.2 Proteins as biologically derived polymers introduced in this chapter

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Keratin [Homo sapiens]

MTTCSRQFTSSSSMKGSCGIGGGIGAGSSRISSVLAGGSC RAPNTYGGGLSVSSSRFSSGGAYGLGGGYGGGFSSSSSS FGSGFGGGYGGGLGAGLGGGFGGGFAGGDGLLVGSEK VTMQNLNDRLASYLDKVRALEEANADLEVKIRDWYQR QRPAEIKDYSPYFKTIEDLRNKILTATVDNANVLLQIDNA RLAADDFRTKYETELNLRMSVEADINGLRRVLDELTLA RADLEMQIESLKEELAYLKKNHEEEMNALRGQVGGDV NVEMDAAPGVDLSRILNEMRDQYEKMAEKNRKDAEEW FFTKTEELNREVATNSELVQSGKSEISELRRTMQNLEIEL QSQLSMKASLENSLEETKGRYCMQLAQIQEMIGSVEE QLAQLRCEMEQQNQEYKILLDVKTRLEQEIATYRRLL EGEDAHLSSSQFSSGSQSSRDVTSSSRQIRTKVMDVHD GKVVSTHEQVLRTKN

(Marchuk et al., 1985).

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films, hydrogels, coatings and electrospun nanofibers, have been processed from silk solutions (Karageorgiou and Kaplan, 2005; Makaya et al., 2009; Nazarov et al., 2004; Numata and Kaplan, 2010; Sofia et al., 2001; Tamada, 2005). Cloning and expression of native and synthetic silks has been achieved in a variety of host systems using synthetic oligonucleotide versions of consensus repeats or variants of these repeats garnered from sequence data from native genes (Arcidiacono et al., 1998; Hayashi and Lewis, 1998; Hinman and Lewis, 1992). Silk proteins modified by genetic engineering can also be designed to display new features alongside native properties (Bini et al., 2006; Greish et al., 2009; Huang et al., 2007; Megeed et al., 2004; Nagarsekar et al., 2002; Numata et al., 2010b; 2009; Rabotyagova et al., 2009; Szela et al., 2000; Wong Po Foo et al., 2006; Yanagisawa et al., 2007). Silkworm silk from B. mori silkworm (silk-like repeats of GAGAGS) and elastin repeats (GVGVP) have been combined to generate a family of protein copolymers, silk-elastin-like proteins (SELP), constructed by recombinant DNA techniques and utilized as gene and drug delivery systems by forming hydrogels to release adenovirus containing reporter genes (Greish et al., 2009; Megeed et al., 2004). Enhanced gene expression was reported in target cells, up to 10 fold, when compared to viral injection without the SELP, demonstrating utility for head and neck solid tumors. To increase the cell-adhesive ability of silk fibroin for practical use as biomaterials, partial collagen and fibronectin sequences were inserted into dragline silk from Nephila clavipes and silk fibroin from B. mori silkworm (Bini et al., 2006; Yanagisawa et al., 2007). Silk-based 3D scaffolds are attractive biomaterials for bone tissue regeneration because of their biocompatibility and mechanical properties (Altman et al., 2003; Santin et al., 1999; Wang et al., 2008). Two kinds of three-dimensional (3D) porous scaffolds have been reported: one prepared from regenerated silk fibroin using an all-aqueous process, and the other using a process involving hexafluoroisopropanol (HFIP) organic solvent. The scaffolds prepared from aqueous process degrade completely between two and six months in vivo, while those prepared from HFIP persist beyond one year. The degradation of aqueous-derived scaffolds appears to be more homogeneous than that of the HFIP-derived scaffolds, because of widespread cellular invasion throughout the scaffold. In the case of the HFIPderived scaffolds, a higher original silk fibroin concentration (e.g. 17%) and smaller pore size (e.g. 100–200 micron) lead to lower levels of tissue ingrowth and slower degradation (Wang et al., 2008). The 3D silk fibroin scaffolds loaded with bone morphogenetic protein-2 (BMP-2) were successfully developed for sustained release of BMP-2 in order to induce human bone marrow stromal cells to undergo osteogenic differentiation when the seeded scaffolds were cultured in vitro and in vivo

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with osteogenic stimulants for four weeks (Karageorgiou et al., 2006). Recently, adenosine release in the brain of rats via silk-based scaffolds has been studied for refractory epilepsy treatments, and demonstrated therapeutic ability, including the sustained release of adenosine over a period of two weeks via slow degradation of silk, biocompatibility, and the delivery of predetermined dose of adenosine (Li T et al., 2009; Wilz et al., 2008). Silk fibroin scaffolds containing insulin-like growth factor I (IGF-I) were prepared for controlled release in the context of cartilage repair (Uebersax et al., 2008). Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was observed, starting after two weeks and more strongly after three weeks. Tropical tasar silkworm Antheraea mylitta silk-based 3D matrices were also evaluated for in vitro drug release and for the study of cell-surface interactions (Mandal et al., 2009; Mandal and Kundu, 2009). The silk-based matrices contained two different model compounds, bovine serum albumin (66 kDa) and FITC-insulin (3.9 kDa), to characterize release profiles (Mandal et al., 2009). Additionally, silk-based micromolded matrices supported significant enhancement in cell attachment, spreading, mitochondrial activity, and proliferation with feline fibroblasts in comparison to polystyrene plates as controls (Mandal and Kundu, 2009). These studies indicate the potential use of slow degrading silk fibroin 3D scaffolds and tubes loaded with bioactive molecules for drug-releasing biomaterials.

20.4

Collagen-derived scaffolds

Collagen is the most abundant protein in mammals and the main structural component of the extracellular matrix, with (Gly-X-Y)n repeating units longer than 1400 amino acid residues and with three residues per one helical turn structure (Lee et al., 2001). The most common tripeptide unit of collagen is (Gly-Pro-Hyp)n. Collagen is also one of the most studied biologically derived polymer scaffolds because of its biocompatibility, cell adhesion, growth and differentiation promoting properties (Freyman et al., 2001; Weinberg and Bell, 1986). Collagen along with glycosaminoglycans are important factors for cell attachment, proliferation and differentiation (Pieper et al., 1999). Collagen-glycosaminoglycan (CG) scaffolds for skin regeneration were inactive when the mean pore size was below 20 μm or above 120 μm (Yannas et al., 1989). The relationships between cell attachment and viability in scaffolds and the structure of scaffolds has been studied (Yannas et al., 1989). CG scaffolds with a constant composition and solid volume fraction (0.005), but with four different pore sizes corresponding to four levels of specific surface area, were manufactured by lyophilization (O’Brien et al., 2005). The fraction of viable cells attached to the CG scaffold decreased with increasing mean pore size and decreasing surface

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area of the scaffold. The correlation between scaffold specific surface area and cell attachment indicates that cell attachment and viability are influenced by scaffold specific surface area over the range of 95.9 to 150.5 μm of pore sizes for MC3T3 cells (O’Brien et al., 2004; 2005). The CG scaffolds supported osteogenesis of rat mesenchymal stem cells, mouse osteogenic cell line MC3T3 and human osteoblast cell line hFOB, as well as the attachment and proliferation of fibroblasts, chondrocytes and neurons (Byrne et al., 2008; Freyman et al., 2001; Jaasma and O’Brien, 2008; Jaasma et al., 2008; Keogh et al., 2010; McMahon et al., 2008). The disadvantage of collagen-based biomaterials is the rapid degradation rate and lower hydrostability, which results in the rapid loss of mechanical properties in vivo (Angele et al., 2004). Several methods to stabilize collagen scaffolds with biologically derived polymers have been reported. Elastin and glycosaminoglycan enhance the stability of collagen scaffolds when mixed for composite materials (Daamen et al., 2003). Crosslinking can also be used to stabilize collagen by forming molecular networks (Glowacki and Mizuno, 2008). Porous scaffolds of collagen crosslinked with hyaluronate or calcium phosphate demonstrated good biocompatibility and potential as osteochondral implants (Liu et al., 1999; Yaylaoglu et al., 1999). To form scaffolds with adequate mechanical properties, cross-linking elastin-like polymer with collagen has been developed with enzymatically resistant covalent bonds between collagens and elastins to increase mechanical strength in a dose-dependent manner without significantly affecting the porosity or thermal properties of the original scaffolds (Garcia et al., 2009).

20.5

Elastin-derived scaffolds

Elastin, which is mainly composed of Gly, Val, Ala and Pro with a molecular weight of approximately 66 kDa, is present in connective, vascular, and load-bearing tissues, and has highly elastic mechanical properties. Elastin is attractive as a biomaterial for scaffolds for tissue engineering. However, elastin is insoluble and difficult to process, as well as less available in terms of quantities, therefore, there are few reports about elastin as biomaterials scaffolds in comparison to the other protein-based scaffolds. An elastin tissue scaffold derived from bovine nuchal ligament was reported and characterized for mechanical properties (Kirkpatrick et al., 2003). The elastic modulus was 1.2 × 106 ± 1 × 105 Nm−2 (parallel to fiber orientation) (Kirkpatrick et al., 2003). The elasticity of elastin is useful to improve collagen-based tissue-engineered blood vessels, which do not have sufficient mechanical properties for bypass grafts. Hybrid tissue-engineered blood vessels of type I collagen and elastin with either human dermal fibroblasts (HDFs) or rat smooth muscle cells (RASMs) exhibited increased tensile strength (11-fold in HDFs; 7.5-fold in RASMs) and linear stiffness

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moduli (4-fold in HDFs; 1.8-fold in RASMs) compared with collagen control constructs with no exogenous elastin scaffold. These data indicate that the elastin hybrid constructs show useful elastic solid mechanical properties (Berglund et al., 2004). The low ultimate tensile strength of elastin has limited its use in biomaterials. Scaffolds consisting of a purified elastin tubular conduits were strengthened with fibrin bonded layers of acellular small intestinal submucosa for potential use as small diameter vascular grafts (Hinds et al., 2006). The addition of acellular small intestinal submucosa increased the ultimate tensile strength of the elastin conduits nine-fold. Burst pressures for the elastin composite vascular scaffold (1396 ± 309 mmHg) were significantly higher than pure elastin conduits (162 ± 36 mmHg) and comparable to native saphenous veins. The average suture pullout strength of the elastin composite vascular scaffolds is 14.6 ± 3.7 N, significantly higher than the pure elastin conduit (0.40 ± 0.10 N), but comparable to native porcine carotid arteries (13.9 ± 4.3 N) (Hinds et al., 2006). In-vivo cellular repopulation of a tissue-derived tubular elastin scaffold was also reported. Elastin tubes filled with agarose gel containing basic fibroblast growth factor for sustained release of the growth factor showed significantly more cell infiltration at 28 days compared to those without growth factor (Kurane et al., 2007). Elastin scaffolds formed from cross-linked elastin-like polypeptide hydrogels were investigated to identify relationships between scaffold formulation parameters (crosslink density, molecular weight, and concentration) and properties including mechanical, matrix accumulation, metabolite use and production, and histological appearance (Nettles et al., 2010). Crosslink density was the strongest predictor of most outcomes related to neuron functions, followed by elastin-like polymer concentration (Nettles et al., 2010). Recombinant elastin and tropoelastin have recently been reviewed (Almine et al., 2010; Mithieux and Weiss, 2005; Wise and Weiss, 2009). Elastin fibers are predominantly composed of the secreted monomer tropoelastin. The important role of cell interactions with recombinant human tropoelastin includes integrin alpha(V)beta(3) as the major fibroblast cell surface receptor for human tropoelastin (Bax et al., 2009). The C-terminal GRKRK motif of tropoelastin can bind to cells in a divalent cationdependent manner (Bax et al., 2009). Assemblies of the elastin generated by recombinant DNA means, permitted the construction of elastic sponges via chemical cross-linking with bis(sulfosuccinimidyl) suberate. These matrices exhibited a Young’s Modulus from 220 to 280 kPa with linearity of extension to at least 150% (Wu et al., 1999). Synthetic elastin is extensible by 200–370% (Mithieux et al., 2004). The constructs behaved as hydrogels and displayed stimuli-responsive characteristics towards temperature and salt concentrations. Further, the elastin scaffolds have shown in-vitro growth

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and proliferation of cells and have been well tolerated in-vivo (Mithieux et al., 2004; Tu et al., 2010). Recombinant human tropoelastin and α-elastin as biopolymeric materials have been used to fabricate tissue engineered scaffolds by electrospinning and with different cross-linking methods. Cell culture studies confirmed that the electrospun protein scaffolds supported the attachment and growth of human embryonic palatal mesenchymal (HEPM) cells (Li M et al., 2005; Nivison-Smith et al., 2010).

20.6

Resilin-derived scaffolds

Resilin, an entropic elastomer (rubber)-like protein found within structures where energy storage and long-range elasticity are needed, shows an elongation to break of 300–400%, low solubility, and thermal stability up to 140°C (Tatham and Shewry, 2002). The resilience of resilin is approximately 92% due to the covalent cross-links between tyrosine residues (Andersen, 1964; Gosline et al., 2002; Lyons et al., 2007). Cloning and expression of the first exon of Drosophila CG15920 gene, which was identified as encoding a resilin-like protein, showed that this recombinant protein can be cast into a rubber-like biomaterial by rapid photochemical crosslinking (Elvin et al., 2005). Artificial elastomeric proteins that mimic the molecular architecture of titin have been characterized through the combination of well-characterized protein domains GB1 and resilin (Cao and Li, 2007; Lv et al., 2010). The elastomeric proteins containing resilin can be photochemically crosslinked and cast into solid biomaterials. These biomaterials behave as rubber-like materials showing high resilience at low strain, and represent a new muscle-mimetic biomaterial. The mechanical properties of these biomaterials can be fine-tuned by adjusting the composition of the elastomeric proteins, providing the opportunity to develop biomaterials that mimic different types of muscles for application in tissue engineering scaffolds. (Lv et al., 2010).

20.7

Keratin-derived scaffolds

Keratin is the major structural fibrous protein to form hair, wool, feathers, nails, and horns of many kinds of animals, and has a high concentration of cysteine, 7 to 20% of the total amino acid residues, that form inter- and intra-molecular disulfide bonds (Dowling et al., 1986). α-Keratin with helical structures declines and β-keratin appears upon stretching elastin, which affects mechanical, thermal and chemical properties (Pauling and Corey, 1951; 1953). A stable aqueous solution of reduced keratins can be prepared by extracting the proteins from wool with a mixture of urea, mercaptanol, sodium dodecyl sulfate (SDS) as a surfactant, and water at 40–60°C (Yamauchi et al., 1996). A clear film from the keratin solution with glycerol

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can be prepared, is insoluble in water and organic solvents including dimethyl sulfoxide, and is degradable in-vitro and in-vivo. (Yamauchi et al., 1996). Comparative culture assay on keratins, collagen, and glass revealed that the keratins are more adhesive for cells and more supportive of cell proliferation than collagen and glass. (Yamauchi et al., 1998). Keratin sponge scaffolds, with a homogeneous porous microstructure with pore sizes of 100 μm, have been developed by lyophilization of an aqueous wool keratin solution after controlled freezing (Tachibana et al., 2002). Rapid cell growth of mouse fibroblast cells (L929) on the sponge (doubling time 29 hr) for at least 7 days, as well as maximum cell number of 7.4 million, or approximately 37 times higher than on the cell culture dishes, was reported. These data indicate that wool keratin sponges are useful scaffolds for long-term and high-density cell cultivation (Tachibana et al., 2002). Keratin sponge scaffolds chemically modified with CaP-precipitation, carboxyl groups, hydroxyapatite particles, SH groups, lysozymes, and bone morphogenetic proteins (BMP)-2 were also reported (Kurimoto et al., 2003; Tachibana et al., 2005; 2006). Additions of chitosan or glycerol into keratin films provide strong and flexible film as shown in Table 20.1. The composite as well as keratin and chitosan films demonstrate high fibroblast attachment and proliferation for mammalian cell culture (Tanabe et al., 2002). Films were compression molded at 120°C, were insoluble and slightly swelled in water, and demonstrated maximum strength of 27.8 ± 2.9 MPa and Young’s modulus of 1218 ± 80 MPa (Katoh et al., 2004a). A compression-molding/particulate-leaching method was reported for the fabrication of keratin sponges with controlled pore size (<100, 100–300 and 300–500 μm) and porosity (more than 90%) (Katoh et al., 2004b). In-vivo degradation of human hair keratin (HHK) scaffolds was due to extracellular enzymatic degradation by ubiquitin systems, generating particles. Subsequently the satellite cells grown on the scaffolds were activated to proliferate and eventually fused into generated muscle fibers (Qiao et al., 2002). Chicken calamus keratin conduits as a tissue-engineered scaffold material and its degradation products demonstrated low toxicity based on skin sensitization in rats, intracutaneous stimulation in rabbits, acute systemic toxicity in mice, and cytotoxicity to L929 cells (Dong et al., 2007).

20.8

Polysaccharide-derived scaffolds

20.8.1 Chitosan Polysaccharides, which are biosynthesized in woods, plants, algae, bacteria and fungi, consist of a variety of polymers with glycosidic linkages (Fig. 20.2). Polysaccharides have useful properties for biologically derived scaffolds such as non-toxicity, water solubility, stability to variations in pH and

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Advanced wound repair therapies Polysaccharide -Monomeric UnitsCH2OH

CH2OH

OH

O

O

O O

CH2OH

OH

O

OH

O

NHCOCH3

NH2

OH Cellulose

Chitosan

Chitin

COOH

CH2OH O O

O

OH

OH

OH

O

CH3CONH

Hyaluronan

20.2 Chemical structures of monomer units of cellulose, chitosan, chitin and hyaluronan.

can be chemically modified to achieve various functions; disadvantages are low mechanical properties, and poor thermal and chemical stability (Sutherland, 1996). Chitosan is osteocompatible and osteoconductive, enhancing bone formation in-vitro and in-vivo (Muzzarelli et al., 1994). Chitosan scaffolds with porous structures support the differentiation and proliferation of osteoblasts; hence chitosan sponges are effective scaffolding biomaterials for bone formation (Seol et al., 2004). Chitosan has been hybridized with a variety of materials, such as alginate, hydroxyl apatite, calcium phosphate, poly(methylmethacrylate), PLLA, glycerol phosphate, collagen and hyaluronan, for practical applications due to the weak mechanical properties of pure chitosan (Cho et al., 2008; Dang et al., 2006; Funakoshi et al., 2005; Li Z et al., 2005; Madhumathi et al., 2009; Majima et al., 2007; Wang et al., 2010; Zhang et al., 2006; 2008; Zhang and Zhang, 2002). Water-soluble chitosan having double bonds was synthesized by sequential grafting of methacrylic acid (19%) and lactic acid (10.33%) via reactions between amino groups and carboxyl groups with carbodiimide. The water-soluble chitosan gels by thermal treatment at body temperature under the initiation of a redox system using N,N′,N′-tetramethylethylenediamine with gelation times of 6 to 20 minutes (Hong et al., 2006). Various mixed hydrogels of β-chitin and chitosan as a sponge scaffold for rabbit chondrocyte cultures have been

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characterized by histochemical and immunohistochemical methods, and the cartilage-like layer in the chondrocyte-sponge composites were similar to hyaline cartilage. Only the pure β-chitin sponge showed features similar to the cartilage in rabbits based on immunohistochemical staining for type II collagen (Suzuki et al., 2008).

20.8.2 Chitin Chitin, a biopolymer of N-acetylglucosamine with some glucosamine, is the main component of the cell walls of fungi, the exoskeletons of arthropods such as crustaceans and insects, the radulas of mollusks and the beaks of cephalopods. Relatively high biocompatibility of chitin scaffolds with MSCs has been shown in vitro with immunohistochemistry and fluorescence confocal microscopy (Li et al., 2004). Bioresorbable β-chitin sponges as scaffolds for 3D culture of chondrocytes have been developed. Cell layers at the surface of the β-chitin sponge were filled with chondrocytes and abundant extracellular matrix. β-Chitin sponges can therefore be considered biocompatible and useful scaffolds for 3D chondrocyte culture (Abe et al., 2004). Chitin has been combined with several polymers and bioactive molecules to improve mechanical properties and cellular adhesion. Composites of β-chitin with octacalcium phosphate (OCP) or hydroxylapatite prepared by precipitation of the mineral into chitin scaffolds have been investigated and oriented precipitation of OCP crystals was formed inside the chitin layers. Mechanical factors are a main cause for the orientation of OCP crystals with the a-axis almost normal to the chitin fibers, possibly because the compartmentalized space in the chitin governs the orientation of the crystals (Falini et al., 2001; 2002). Porous hydroxyapatite-chitin materials with 25%, 50% and 75% (w/w) hydroxyapatite prepared by freeze-drying have shown non-cytotoxicity, in vivo degradability and supported the proliferation of MSCs-induced osteoblasts, demonstrating the potential of hydroxyapatite-chitin matrices as suitable substrates for tissue engineered bone substitutes (Ge et al., 2004). Hydroxyapatite/collagen/poly(l-lactide) (PLLA) scaffolds reinforced by chitin fibers for bone-tissue engineering had lower cytotoxicity against MSCs as well as approximately 4-fold higher compressive strength than scaffolds without chitin fibers (Li et al., 2006). Porous 3D PLLA scaffolds reinforced by the chitin fibers (30%) demonstrated higher attachment, proliferation, differentiation, and mineralization with osteoblasts than PLLA alone (Li X et al., 2009).

20.8.3 Cellulose Cellulose, a polysaccharide composed of d-glucose via β-1,4 linkages, is the most abundant and renewable biopolymer, and has been studied for

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biomedical applications including scaffolds. Bacterial cellulose has been studied for blood vessel engineering and wound healing (Czaja et al., 2006; Klemm et al., 2001). Bacterial cellulose secreted from Gluconacetobacter xylinus (Acetobacter xylinum) has been explored as a scaffold to support chondrocyte proliferation with providing significant advantages in terms of chondrocyte growth over tissue culture plates (Svensson et al., 2005). Nonwoven cellulose II fabrics have also been used as scaffolds for in vitro cartilage tissue engineering. The scaffolds coated with a calcium phosphate layer show significantly improved cell adherence compared to untreated cellulose fabrics, with excellent proliferation of the adhered chondrocytes. Additionally, calcium was leached out from the precipitated layer, which can lead to a microenvironment that triggers the development of cartilage similar to cartilage repair in the vicinity of subchondral bone (Muller et al., 2006; Pulkkinen et al., 2006). A method to prepare 3D cellulose nanofibril network scaffolds with controlled microporosity has been developed with paraffin wax and starch particles of various sizes in a growing culture of A. xylinum, resulting in bacterial cellulose scaffolds with different morphologies and interconnectivities (Backdahl et al., 2008). Smooth muscle cells (SMCs) attach and proliferate on and into the scaffolds (Backdahl et al., 2008). Further, cellulose hybrid materials such as nanocomposites with hydroxyapatite, gelatin, montmorillonite, cellulose acetate and sodium silicate gel, have been investigated to improve mechanical properties, biocompatibility and cellular adhesion (Fang et al., 2009; Fukuhara et al., 2010; Haroun et al., 2009; Kim et al., 2009; Ponader et al., 2008; Xing et al., 2010).

20.8.4 Hyaluronan Hyaluronan, also called hyaluronic acid or hyaluronate, is composed of repeating units of glucuronic acid and N-acetylglucosamine, and is the main non-protein glycosaminoglycan component of the extracellular matrix (Fraser and Laurent, 1989; Laurent et al., 1986). Hyaluronan also plays an important role in cell proliferation, cell-migration, matrix assembly and tissue development, and may be involved in the progression of some malignant tumors. Many researchers have investigated hyaluronan and composite scaffolds because of the biocompatibility and cellular adhesion of hyaluronan, and some are in clinical practice (Alini et al., 2003; Chang et al., 2006; 2003; Filova et al., 2008; Ko et al., 2009). Hyaluronan scaffolds provide a useful environment where cells down regulate the expression of some catabolic factors as well as reduce the expression of molecules involved in cartilage degenerative diseases (Cavallo et al., 2010; Grigolo et al., 2005). Hyaluronan scaffolds induce the expression of CXCR4 receptor, ChemokineCXCL13, Matrix Metalloproteinase 3, and Intercellular Adhesion Molecule-1 (CD54) and down regulate the expression of Chemokine

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CXCL12, CXCR5 receptor, Matrix Metalloproteinase 13, Tissue Inhibitor of Metalloproteinase-1, and CD44 Antigen, demonstrating a 3D hyaluronan scaffold acts as a signaling molecule for hMSCs (Lisignoli et al., 2006; 2005). Hyaluronan-based scaffolds with bone marrow stromal cells (BMSC) provide successful cell proliferation and differentiation support for repair of bone tissue (Lisignoli et al., 2003; Loken et al., 2008). For tissue-engineered cartilage repair, hyaluronan scaffolds are of interest due to a role for hyaluronan in cartilage development and function. In situ photocrosslinkable hyaluronan was reported as a scaffold for articular cartilage repair. The mechanical and physical properties of the crosslinked photocrosslinkable hyaluronan hydrogels are similar to those of other hydrogels, with compressive and dynamic shear moduli of 0.6 and 0.3 kPa, respectively, and diffusion coefficients of 600–8000 μm2/s depending on molecular weight. (Nettles et al., 2004). The mechanisms of the interaction of a hyaluronanbased scaffold with rat mesenchymal stem cells (rMSCs) and endothelial progenitor cells (EPCs) have been studied to evaluate the potential clinical applications, suggesting that the cells can migrate and adhere inside hyaluronan-based scaffolds, maintain their pre-endothelial phenotype, and express angiogenic factors (Pasquinelli et al., 2008; 2009).

20.9

Conclusions and future trends

Biologically derived scaffolds of 10 different biopolymers were summarized. In the natural environment and in the human body there are many biological polymers which can be considered for scaffolds but have not been characterized for such applications to date. New biopolymers remain to be characterized and relationships clarified between structure and function for biomedical applications. In addition, recombinant DNA techniques have become important in the design of biopolymers for biomedical applications. Recombinant proteins self-assemble into structures at the nanometer and micrometer scales, which may enhance new functions of biomaterials. Furthermore, there are various biomaterials with different mechanical, thermal and biological properties, and there remains a need for comprehensive approach to correlate chemistries and other biopolymer features with structures, functions and diseases, as well as the most suitable biomaterial, tissue engineering and regenerative medicine applications.

20.10 References Aaron, B. B., Gosline, J. M. (1981), “Elastin as a random-network elastomer – a mechanical and optical analysis of single elastin fibers.” Biopolymers, 20, 1247–1260.

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Numata, K., Hamasaki, J., Subramanian, B., Kaplan, D. L. (2010b), “Gene delivery mediated by recombinant silk proteins containing cationic and cell binding motifs.” J Control Release, 146, 136–143. O’Brien, F. J., Harley, B. A., Yannas, I. V., Gibson, L. (2004), “Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds.” Biomaterials, 25, 1077–1086. O’Brien, F. J., Harley, B. A., Yannas, I. V., Gibson, L. J. (2005), “The effect of pore size on cell adhesion in collagen-GAG scaffolds.” Biomaterials, 26, 433–441. Opitz, F., Schenke-Layland, K., Cohnert, T. U., Starcher, B., Halbhuber, K. J., Martin, D. P., Stock, U. A. (2004), “Tissue engineering of aortic tissue: dire consequence of suboptimal elastic fiber synthesis in vivo.” Cardiovasc Res, 63, 719–730. Orgel, J. P., Irving, T. C., Miller, A., Wess, T. J. (2006), “Microfibrillar structure of type I collagen in situ.” Proc Natl Acad Sci U S A, 103, 9001–9005. Pasquinelli, G., Orrico, C., Foroni, L., Bonafe, F., Carboni, M., Guarnieri, C., Raimondo, S., Penna, C., Geuna, S., Pagliaro, P., Freyrie, A., Stella, A., Caldarera, C. M., Muscari, C. (2008), “Mesenchymal stem cell interaction with a non-woven hyaluronan-based scaffold suitable for tissue repair.” J Anat, 213, 520–530. Pasquinelli, G., Vinci, M. C., Gamberini, C., Orrico, C., Foroni, L., Guarnieri, C., Parenti, A., Gargiulo, M., Ledda, F., Caldarera, C. M., Muscari, C. (2009), “Architectural organization and functional features of early endothelial progenitor cells cultured in a hyaluronan-based polymer scaffold.” Tissue Eng Part A, 15, 2751–2762. Pauling, L., Corey, R. B. (1951), “Configuration of polypeptide chains.” Nature, 168, 550–551. Pauling, L., Corey, R. B. (1953), “Compound helical configurations of polypeptide chains: structure of proteins of the alpha-keratin type.” Nature, 171, 59–61. Peng, T., Gibula, P., Yao, K. D., Goosen, M. F. (1996), “Role of polymers in improving the results of stenting in coronary arteries.” Biomaterials, 17, 685–694. Pieper, J. S., Oosterhof, A., Dijkstra, P. J., Veerkamp, J. H., van Kuppevelt, T. H. (1999), “Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate.” Biomaterials, 20, 847–858. Pollock, C. M., Shadwick, R. E. (1994), “Relationship between body mass and biomechanical properties of limb tendons in adult mammals.” Am J Physiol, 266, R1016–1021. Ponader, S., Brandt, H., Vairaktaris, E., von Wilmowsky, C., Nkenke, E., Schlegel, K. A., Neukam, F. W., Holst, S., Muller, F. A., Greil, P. (2008), “In vitro response of hFOB cells to pamidronate modified sodium silicate coated cellulose scaffolds.” Colloids Surf B Biointerfaces, 64, 275–283. Pulkkinen, H., Tiitu, V., Lammentausta, E., Laasanen, M. S., Hamalainen, E. R., Kiviranta, I., Lammi, M. J. (2006), “Cellulose sponge as a scaffold for cartilage tissue engineering.” Biomed Mater Eng, 16, S29–35. Qiao, D. F., Lu, Y. M., Fu, W. Y., Piao, Y. J. (2002), “Degradation of human hair keratin scaffold implanted for repairing injured skeletal muscles.” Di Yi Jun Yi Da Xue Xue Bao, 22, 902–904. Rabotyagova, O. S., Cebe, P., Kaplan, D. L. (2009), “Self-assembly of genetically engineered spider silk block copolymers.” Biomacromolecules, 10, 229–236. Santin, M., Motta, A., Freddi, G., Cannas, M. (1999), “In vitro evaluation of the inflammatory potential of the silk fibroin.” J Biomed Mater Res, 46, 382–389.

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21 Stem cell therapies for wound repair G. G. G AU G L I T Z, Ludwig Maximilians University, Germany and M. G. J E S C H K E, University of Toronto and Sunnybrook Research Institute, Canada

Abstract: Treatment of non-healing wounds has remained difficult, in spite of better understanding of pathophysiologic principles. Besides advances stemming from breakthroughs in recombinant growth factors and bioengineered skin, early data suggest the use of multipotent stem cells in order to accelerate wound healing. Until recently, research has been mostly focused on bone marrow-derived mesenchymal stem cells; however, other types of stem cells, including adipose-derived stem cells (ADSCs) and those derived from hair follicles gain more and more interest for potential application for the repair and regeneration of various damaged tissues. The aim of this chapter is to discuss the use and the potential of this novel technology. Key words: stem cells, adult, embryonic, wound healing, umbilical cord.

21.1

Introduction

Appropriate wound care is critical, and various treatment modalities have been used to improve the wound bed. Besides well-known components of wound care including debridement of the necrotic tissue, removal of edema fluid, decreasing the bacterial burden, and providing the right balance of moisture to the wound bed, more advanced therapeutic approaches, such as topically applied growth factors and cell-based therapies have been developed in recent years. Presently, several technologies are under active development to aid cutaneous wound repair and are discussed elsewhere in this book. However, it is becoming increasingly clear that more radical steps need to be taken to enhance the treatment of chronic and burn wounds in a direction that allows greater effectiveness. Stem cells, defined based on the findings of Ernest A. McCulloch and James E. Till (1, 2), are characterized by: 1) a prolonged self renewal capacity and 2) the ability to differentiate into mature stages and different tissue types by asymmetric replication (3, 4). These properties have led to the expectation that human embryonic stem cells can be useful in understanding disease mechanisms, the development of effective drugs, and the treatment of patients with various diseases and injuries (5, 6). Despite their 552 © Woodhead Publishing Limited, 2011

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unique potential, the use of embryonic stem cells has remained controversial. Ethical concerns regarding the use of human embryos for the purpose of obtaining their cells have been raised by both the scientific community and public media. Even though there are new studies suggesting that embryonic stem cells may be obtained without destroying the embryo (7), it remains unclear whether this approach would eliminate the ethical concerns. To circumvent these issues, Yamanaka et al. (8) produced pluripotent somatic cells by direct reprogramming, and generated pluripotent stem cells from adult human dermal fibroblasts and other human somatic cells, which were comparable to the differentiation potential of human embryonic stem cells (9). In addition, differentiated adult tissues have been shown to harbor pluripotent stem cells with unexpected plasticity (5). Thus, stem cells derived from adult sources may have the same clinical and experimental potential as embryonic stem cells. The opportunity to repair and regenerate tissues injured by disease and trauma is opening the way to new optimistic treatments that need to be carefully evaluated in early clinical trials. These developments are building on the successes of bone marrow hematopoietic stem cell (HSCs) transplants that have more than 30 years of patient applications in blood diseases and cancer. This chapter focuses on presently discussed sources of stem cells, their possible mechanism of action and their clinical applicability particularly for the healing of acute and chronic cutaneous wounds.

21.1.1 Embryonic stem (ES) cells ES cells are pluripotent stem cells that are harvested from the inner cell mass of the pre-implantation blastocyst (3–5 day-old embryo), and have been obtained from mice, non-human primates, and humans (10, 11). ES cells can be maintained in culture as undifferentiated cell lines or induced to differentiate into many different lineages, including blood cells, neural cells, adipocytes, muscle cells, and chondrocytes, among others (5, 12). The ability of researchers to efficiently manipulate ES cells to differentiate into specifically directed cells will provide means of an unlimited supply of cells that may be used, not only for the growth of implantable tissues, but also for testing new drugs to cure diseases, and in the identification of potentially problematic genes (11, 13–15). Thus, ES cells have had and will have an enormous impact on biology and medicine. However, in spite of their tremendous potential, ES cells are the subject of considerable controversy. Opponents of ES cell use most often question the morality of using cells obtained from destroyed embryos, an act they consider equivalent to destroying human life (16). They fear the possibility that the use of human ES cells could lead to the almost industrial production of human embryos for the sole purpose of obtaining their cells. Current research has been directed towards developing methods that will permit the

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use of ES cells while eliminating the ethical concerns surrounding their use. Methods to produce human ES cells without destroying embryos were proposed in the President’s Council on Bioethics report in 2004 (17). Two of these methods were further investigated in individual proof of concept studies (7, 18) which gleaned promising results. Questions, however, still point to the ethical use of these methods for many involved in these debates (19). The use of stem cells derived from adult tissues, and not those derived from embryonic tissues, avoids many of the ethical concerns that may arise from the use of stem cells in research applications.

21.1.2 Adult stem (AS) cells AS cells, also referred to as somatic stem cells or mesenchymal stem cells (MSC), are those mature, adult cells that are undifferentiated and found in a specific tissue or organ (20). These cells are self-renewing, and are able to differentiate into major specialized cell types that serve to maintain the integrity of and repair the tissues in which they are found (10, 20). In addition to the capability of self-renewal throughout the entire lifetime of an organism, AS cells are characterized by being clonogenic (a single adult stem cell should be able to generate a line of genetically identical cells) and

Immediately applicable?

Immune response by a host (rejection)

Yes

N/A-temporal coverage (need to remove)

Basically yes

Results varied, but often rejected

Autograft

No

No

Stem cell-based new substitute(s)

Yes

No (+regenerative activity)

Synthetic dressing

Cultured cell base (fibroblast/keratinocyte)

21.1 Summary of current skin substitutes available for wound coverage.

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being able to differentiate into cells with a mature phenotype regarding morphology, specific cell surface markers, and characteristic behavior (21). Unlike ES cells, which are defined by their origin from the ICM of blastocysts, AS cells do not have such a definitive basis and are likely characterized by being set aside during fetal development and restrained from differentiation. AS stem cells are often determined by labeling and tracking in vivo after re-transplantation or, when grown in vitro, they can be manipulated by adding growth factors or introducing genes to determine what differentiated cell types they will yield. It is often difficult to distinguish adult, tissue specific stem cells from progenitor cells, which are found in fetal and adult tissues and are partly differentiated to give rise to certain cell types of a tissue (21). Besides the bone marrow (BM), other adult tissues such as peripheral blood, brain, spinal cord, skeletal muscle, epithelia of the skin and digestive system, and pancreas contain stem cells. However, because of the ease with which they are obtained, human mesenchymal stem cells (hMSCs) derived from bone marrow have received considerable attention. Human umbilical cord blood has also been explored as an alternative source of stem cells to repopulate the bone marrow in the treatment of diseases in children and adults (22). In addition, adipose tissue has been identified as a source of multipotent cells that have the capacity to differentiate to cells of adipogenic, chondrogenic, myogenic, and osteogenic lineages when cultured with the appropriate lineage specific stimuli (20, 23). However, research on adult stem cells has been slow, largely because great difficulty has been encountered in maintaining adult non-mesenchymal stem cells in culture. There are challenges involved in maintaining and expanding long-term cultures of adult stem cells in large numbers. Isolation has also proven to be quite problematic as these cells are present in extremely low numbers in the adult tissue. While current use of adult stem cells is still limited, there is great potential in future utilization of such cells for the use of tissue specific regenerative therapies. The advantage of adult stem cells is that they can be used in autologous therapies, thus avoiding any immune rejection complications. Another area of current study is the production of pluripotent somatic cells by direct reprogramming. Reprogramming is a technique that involves de-differentiation of adult somatic cells to produce patient-specific pluripotent stem cells without the use of embryos. Cells generated by reprogramming would be genetically identical to the somatic cells and would not be rejected by the donor. This method also avoids the technical limitations of nuclear transfer into oocytes. Takahashi and Yamanaka were the first to discover that mouse embryonic fibroblasts (MEFs) and adult mouse fibroblasts can be reprogrammed into an induced pluripotent state (IPS) (24). In recent studies the authors demonstrated that reprogramming can be done with human cells (9).

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21.2

Frequently utilized sources of adult stem cells

21.2.1 Bone marrow-derived stem cells Bone marrow contains hematopoietic stem cells and it is also the most recognized source of mesenchymal stem cells. Stem cells obtained from bone marrow are found in the stroma of the marrow. These cells are multipotent, and are therefore able to differentiate into lineages of cells such as adipocytes, osteocytes, myocytes, tenocytes, and neural cells (20, 25, 26). The ability of bone marrow-derived stem cells to contribute to a number of different tissues has been demonstrated in vitro and in vivo in animal models and, more recently, in human transplantation chimeras (27). These cells are typically obtained from bone marrow aspirates from marrow transplant donors. When cultured in vitro, bone marrow stem cells exhibit a fibroblast-like morphology. Marrow stromal cells have been studied and, to date, certain cell surface markers have been identified that are useful in cell selection and determination of preparation of marrow stem cell populations (28). In addition to their ability to differentiate into multiple cell lineages, the use of marrow stem cells is advantageous because they offer a source of cells that is isolated and expanded in vitro with relative ease. The number of cells may be significantly increased by subculturing a small sample of donor tissue (14, 28). Bone marrow-derived cells, used to help heal chronic wounds, have been of particular interest. Fathke et al. (29) showed in the chimeric mouse model that distant bone marrow-derived cells may contribute to the reconstitution of the dermal fibroblast population in cutaneous wounds. Bone marrow stroma cells were further found to synthesize higher amounts of collagen, FGF, and VEGF, when compared to native dermal fibroblasts, indicating a potential use for accelerating wound healing (30). Ichioka et al. (31) investigated the effect of a bone marrow-impregnated collagen matrix on wound healing in a microcirculatory mouse model and observed significant increases in angiogenesis. Furthermore, patients with chronic leg ulcera treated with this method demonstrated successful wound closure. Falanga et al. (32) successfully utilized a fibrin polymer spray to apply cultured autologous mesenchymal stem cells obtained from bone marrow aspirates to wounds. The technique accelerated the rate of healing of acute and nonhealing cutaneous wounds in both humans and mice. This approach may represent a feasible method for introducing cells into wounds. There are, however, some difficulties related to these techniques. Stem cells must be cultured in sufficient numbers for topical application and grow in the wound for a therapeutic response. Another limitation in severe burn trauma is that bone marrow suppression has been observed either as a result of silver sulphadiazine toxicity (33) or sepsis (34, 35). Ultimately, the number of bone marrow mesenchymal stem cells significantly decrease with age (36).

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21.2.2 Umbilical cord-derived stem cells Human umbilical cord blood is a rich source of hemopoietic stem cells for clinical application (37, 38). McGuckin et al. (39) proposed umbilical cord blood to be one of the largest untouched sources of stem cells with characteristic features such as naive immune status. However, the presence of mesenchymal stem cells in umbilical cord blood remains controversial. Studies by Erices et al. (40) showed that mesenchymal stem cells together with hematopoietic precursors are circulating in the blood of preterm fetuses, while Mareschi et al. (41) failed to isolate these cells in preterm umbilical cord blood. In contrast, Romanov et al. (42) found that cord vasculature contains a large amount of mesenchymal stem cell-like elements. These elements form colonies of fibroblastoid cells that were successfully expanded in culture. They suggested that the umbilical cord stroma could thus be utilized as an alternative source of mesenchymal stem cells for experimental and clinical investigation. A study by Kamolz et al. (43) indicated that stem cells from umbilical cord blood are able to differentiate into epithelial cells under in vitro conditions and suggested their use as a starting material for isolation and expansion of cells in large skin defects. It should be pointed out that umbilical cord blood-derived stem cells are usually obtained from allogenic sources, potentially leading to immunological rejection upon transplant or transfusion. Phan and colleagues recently found the amniotic membrane of the umbilical cord to be an extremely rich source of stem cells for burns resurfacing (unpublished data). The isolated cells, termed “cord lining stem cells”, can be divided into subpopulations of epithelial cells (cord lining epithelial cells) (44) and mesenchymal cells (cord lining mesenchymal cells) (Fig. 21.2). They express stem cell markers, such as Oct-4 and Nanog, and have been shown to form healthy colonies in culture. Our group has recently shown that the amniotic membrane is a source of mesenchymal stem cells that can differentiate into bone, cartilage, and fat (Fig. 21.3, 44). Both have been successfully utilized to treat partial thickness and full thickness burns as well as chronic diabetic wounds in an ongoing clinical case series (unpublished data). Growing these cells on specific scaffolds creates an accessible way of applying these cells to the wound. Immunological rejection of these cells has not been observed in ongoing trials.

21.2.3 Adipose-derived stem cells (ADSCs) ADSCs represent another available source of pluripotent cells which have characteristics similar to BM-MSCs (45) and can differentiate into various lineages (23, 46, 47). ADSCs can be obtained from the processing of either

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(a) Sources of stem cells Placental amniotic membrane

Cord lining membrane

Cord lining membrane

(b) Mesenchymal stem cells

Epithelial stem cells

21.2 (a) shows the tissues used for SC isolation. Freshly isolated tissues were cut and cultured as described in the text, and approximately 10 to 14 days after starting the culture, we observed significant numbers of cells migrating from the explants. (b) shows representative phase contrast images of the two types of cells isolated from the umbilical cord and amnion. The majority of cells are mesenchymal (fibroblastic, left), but we also observed small populations of epithelial-like cells (right). With permission from Kita et al. (44).

liposuctioned or excised fat. Adipose tissue contains 100 to 1000 times more pluripotent cells on a percubic centimeter basis than bone marrow (48, 49). ADSCs have surface markers and gene profiling similar to BM-MSCs (45). Given their convenient isolation compared with BM-MSCs and extensive

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21.3 Characteristics of mesenchymal stem cells. (a) Expression of CD73 and CD105 analyzed by flow cytometry. (b) Differentiation of mesenchymal stem cells into osteogenic, adipogenic, and chondrogenic lineages. Undifferentiated cells or differentiated cells were stained with Alizarin Red S (osteogenic), Oil-Red O (adipogenic), or Safranin O (chondrogenic). Cell pellet after chondrogenic differentiation is shown circled. Scale bars = 100 μm (adipogenic and chondrogenic) and 200 μm (chondrogenic), respectively. (c) CFU-F assay. Left: no cells, middle and right, dish #1 and 2. With permission from Kita et al. (44).

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proliferative capacities ex vivo, ADSCs hold great promise for use in wound repair and regeneration. Kim et al. recently investigated the effect of ADSCs on human dermal fibroblasts (HDF), which play a well known key role in skin biology such as wound healing, scar and photoaging. Co-culture of ADSCs and HDFs increased collagen and fibronectin synthesis and proliferation and migration activity of HDFs. ADSCs produced various growth factors such as PDGF, insulin-like growth factor (IGF) and KGF in addition to the previously reported growth factors, such as bFGF, TGF-β, HGF, and VEGF. In an animal model, ADSCs significantly reduced the wound size and accelerated the re-epithelialization from the edge, suggesting that ADSCs may be used for the treatment of photoaging and wound healing. Theoretically, the abundance of isolated stem and therapeutically active cells from adipose eliminates the requirement of cell expansion in tissue culture facilities as is required with bone marrow derived cells and makes these cells readily available to the clinician, at the bedside. Human liposuction aspirates was used by Huang et al. (50) to culture adipose-derived mesodermal stem cells and differentiate them into chondrogenic cells. A study by Kim et al. (45) indicated that adipose-derived stem cells promoted human dermal fibroblast proliferation by direct cell-to-cell contact and by secretory induced paracrine activation which significantly accelerated the re-epithelialization of cutaneous wounds. Beanes et al. (51) further observed that human connective tissue stem cells from fat lose their stem cell potential with age.

21.2.4 Cutaneous stem cells In mammals, the epidermis is a multilayered epithelium that is composed of hair follicles (HFs), sebaceous glands and interfollicular epidermis. The interfollicular epidermis, defined as the portion of the epidermis located between the orifices of hair follicles, regenerates throughout adult life in order to replace terminally differentiated cells that are continuously shed from the surface of the skin and also to renew the hair follicle (52). The regeneration of the epidermis and the hair follicle is sustained by many different types of epidermal stem cell, which also participate in the repair of the skin after injuries. In addition to their self-renewing capacity and multipotency, these cells are quiescent with a low tendency to divide, but upon injury are characterized by an extensive and sustained self-renewal capacity (52). Recent advances in methods in isolating cutaneous stem cells have lead to a greater understanding of the potential use for these cells. Hair follicles with their ability for self-renewal represent currently the most promising source of multipotent stem cells (53). Using transplantation of the murine bulge region, Oshima et al. (54) demonstrated that bulge cells could repopulate the epidermis, sebaceous glands, and the epithelial layers

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of the hair follicle. Roh et al. (53) showed that stem cells extracted from the human bulge region can be induced to exhibit hair follicle differentiation and form epidermal and sebaceous cells in vitro as well as form an epidermis and sebaceous cells in vitro, and this supports the multi-potential capacity of human cutaneous stem cells. In addition, cells from the hair follicle are currently used clinically to generate epithelium used for chronic wound coverage. The above evidence has overwhelmingly demonstrated that the cells located in the hair follicle bulge region represent multi-potent, tissuespecific epithelial stem cells. Most authors agree that these stem cells will become an important source for therapeutic applications, which is based on their accessibility and the easy access to skin.

21.3

Clinical applications of stem cells to wound healing

As excellently summarized in a recent review by Lau and colleagues(55), encouraging results from stem cell-based treatment strategies in postinfarction myocardial repair (56) have led to application of similar strategies in order to treat skin wounds, where positive effects on all phases of wound healing have been reported with various types of MSC (45, 57–62). In addition, the implantation of HFs in IntegraTM skin equivalent templates for transplantation onto a human patient’s burn wound accelerated reepithelialization, minimized skin graft failure by reconstituting the epithelial stem cell pool, and was felt to produce cosmetically satisfactory results (62). Other than transplantation, recruiting endogenous stem cells to the site of injury presents an alternative for the treatment of cutaneous wounds. For example, intradermal injection of cutaneous T-cell attracting chemokines and chemokine (C-C motif) ligand-17 and ligand-21 resulted in a significant acceleration of skin regeneration via recruiting BM-derived keratinocyte precursors and BM-MSC, respectively (63, 64). Increased MSC migratory rate has been shown to be induced by HGF (65), basic fibroblast growth factor (bFGF) (66, 67) and CXCL5 (67) in vitro. Thus, HGF, bFGF and CXCL5 may be exploited as BM-MSC chemoattractants. To enable a sustained, long-term and localized delivery of these diffusible factors, various forms of ECM-like material have been developed and examined as carriers (65, 68). However, application of stem cells seems advantageous over administering single biological diffusible factors because stem cells can interact with their environment and release multiple wound healing factors. As shown by Krause et al. enriched populations of hematopoietic stem cells homing to bone marrow within 48 hours could incorporate and function in a variety of tissues, including stomach, kidney, lung, and skin (26). In a human study of chronic non-healing wounds, the group of Badiavas et al. showed that directly applied bone marrow-derived cells can lead to wound

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closure and possible rebuilding of tissues (58). The lack of an efficient delivery system for these autologous bone marrow-derived cultured cells this study was encountered in a recent study the same group where hMSC were successfully applied to non-healing and acute wounds, using a specialized fibrin spray system (69). However, despite the above mentioned improvement in wound healing using various stem cell lines, several issues need to be considered before administering stem cells to wound patients. For example, functionality of stem cells decreases with age, thus older patients may not present the perfect population as donors (70–72). Using a mouse model, Schatteman et al. demonstrated that BM-MSC from old mice inhibit rather than promote wound healing when applied to wounds in diabetic mice (72). Also, the risk of immunological rejection upon transplant or transfusion must be considered if using stem cells from allogenic sources. The mode of stem cells delivery is another frequently discussed current issue. Ideally, stem cells should retain their stemness until delivered and should maintain localized at the wound site in order to enhance engraftment. For such purposes, an acellular dermal matrix was employed by Altman and colleagues as a carrier for delivering ADSC to the wounds locally (60). This method successfully prevented systemic distribution of the ADCS and may thus be superior to simply injecting stem cells in the wound site (60). Other concerns of BMMSC-based wound healing therapy include the increased risk keloid scaring since BM-MSC release VEGF which has been linked to excessive scar formation (61).

21.4

Conclusions

Cutaneous wound healing requires a well-orchestrated integration of the complex biological and molecular events of cell migration and proliferation, as well as extracellular matrix deposition, angiogenesis, and remodeling. Stem cells, due to their ability to differentiate into various tissue types by asymmetric replication thus represent a promising tissue repair strategy. A variety of sources, such as bone marrow, peripheral blood, umbilical cord blood, adipose tissue and skin/ hair follicles, have been utilized to isolate stem cells to modulate the healing response of acute and chronic wounds. Recent data have demonstrated the feasibility of autologous ASC therapy in cutaneous repair and regeneration. However, further research in order to solve the many questions on experimental and clinical application of stem cells in wound healing is necessary.

21.5

Acknowledgement

The authors would like thank Dr. Katsu Kita, PhD for his support and help.

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References

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24. Takahashi K, Yamanaka S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676. 25. Nakagawa H, Akita S, Fukui M, Fujii T, Akino K. (2005) Human mesenchymal stem cells successfully improve skin-substitute wound healing. Br J Dermatol 153: 29–36. 26. Krause DS, Theise ND, Collector MI, et al. (2001) Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 105: 369–377. 27. Hematti P, Sloand EM, Carvallo CA, et al. (2002) Absence of donor-derived keratinocyte stem cells in skin tissues cultured from patients after mobilized peripheral blood hematopoietic stem cell transplantation. Exp Hematol 30: 943–949. 28. Barry FP, Murphy JM. (2004) Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 36: 568–584. 29. Fathke C, Wilson L, Hutter J, et al. (2004) Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair. Stem Cells 22: 812–822. 30. Han SK, Yoon TH, Lee DG, Lee MA, Kim WK. (2005) Potential of human bone marrow stromal cells to accelerate wound healing in vitro. Ann Plast Surg 55: 414–419. 31. Ichioka S, Kouraba S, Sekiya N, Ohura N, Nakatsuka T. (2005) Bone marrowimpregnated collagen matrix for wound healing: experimental evaluation in a microcirculatory model of angiogenesis, and clinical experience. Br J Plast Surg 58: 1124–1130. 32. Falanga V, Iwamoto S, Chartier M, et al. (2007) Autologous bone marrowderived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng 13: 1299–1312. 33. Gamelli RL, Paxton TP, O’Reilly M. (1993) Bone marrow toxicity by silver sulfadiazine. Surg Gynecol Obstet 177: 115–120. 34. Gamelli RL, He LK, Liu H. (1995) Recombinant human granulocyte colonystimulating factor treatment improves macrophage suppression of granulocyte and macrophage growth after burn and burn wound infection. J Trauma 39: 1141–1146; discussion 1146–1147. 35. Shoup M, Weisenberger JM, Wang JL, Pyle JM, Gamelli RL, Shankar R. (1998) Mechanisms of neutropenia involving myeloid maturation arrest in burn sepsis. Ann Surg 228: 112–122. 36. Rao MS, Mattson MP. (2001) Stem cells and aging: expanding the possibilities. Mech Ageing Dev 122: 713–734. 37. Huss R. (2000) Isolation of primary and immortalized CD34-hematopoietic and mesenchymal stem cells from various sources. Stem Cells 18: 1–9. 38. Hows JM. (2001) Status of umbilical cord blood transplantation in the year 2001. J Clin Pathol 54: 428–434. 39. McGuckin CP, Forraz N, Baradez MO, et al. (2005) Production of stem cells with embryonic characteristics from human umbilical cord blood. Cell Prolif 38: 245–255. 40. Erices A, Conget P, Minguell JJ. (2000) Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 109: 235–242. 41. Mareschi K, Biasin E, Piacibello W, Aglietta M, Madon E, Fagioli F. (2001) Isolation of human mesenchymal stem cells: bone marrow versus umbilical cord blood. Haematologica 86: 1099–1100.

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42. Romanov YA, Svintsitskaya VA, Smirnov VN. (2003) Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells 21: 105–110. 43. Kamolz LP, Kolbus A, Wick N, et al. (2006) Cultured human epithelium: human umbilical cord blood stem cells differentiate into keratinocytes under in vitro conditions. Burns 32: 16–19. 44. Kita K, Gauglitz GG, Phan TT, Herndon DN, Jeschke MG. (2010) Isolation and characterization of mesenchymal stem cells from the sub-amniotic human umbilical cord lining membrane. Stem Cells Dev 19(4): 491–520. 45. Kim WS, Park BS, Sung JH, et al. (2007) Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts. J Dermatol Sci 48: 15–24. 46. Zuk PA, Zhu M, Mizuno H, et al. (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7: 211– 228. 47. Kim CG, Lee JJ, Jung DY, et al. (2006) Profiling of differentially expressed genes in human stem cells by cDNA microarray. Mol Cells 21: 343–355. 48. Strem BM, Hicok KC, Zhu M, et al. (2005) Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med 54: 132–141. 49. Aust L, Devlin B, Foster SJ, et al. (2004) Yield of human adipose-derived adult stem cells from liposuction aspirates. Cytotherapy 6: 7–14. 50. Huang JI, Hedrick MH, Lorenz P. (2000) Chondrogenesis of human adipoderived mesodermal stem cells. Surg Forum 583–585. 51. Beanes SR, Zhu M, Lorenz HP. (2000) Aging decreases the differentiation potential of human stem cells Plastic Surgery Research Council 45th Annual Meeting, Seattle, WA. 52. Tiede S, Kloepper JE, Bodo E, Tiwari S, Kruse C, Paus R. (2007) Hair follicle stem cells: walking the maze. Eur J Cell Biol 86: 355–376. 53. Roh C, Lyle S. (2006) Cutaneous stem cells and wound healing. Pediatr Res 59: 100R–103R. 54. Oshima H, Rochat A, Kedzia C, Kobayashi K, Barrandon Y. (2001) Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 104: 233–245. 55. Lau K, Paus R, Tiede S, Day P, Bayat A. (2009) Exploring the role of stem cells in cutaneous wound healing. Exp Dermatol 18: 921–933. 56. Amado LC, Saliaris AP, Schuleri KH, et al. (2005) Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci U S A 102: 11474–11479. 57. Francois S, Mouiseddine M, Mathieu N, et al. (2007) Human mesenchymal stem cells favour healing of the cutaneous radiation syndrome in a xenogenic transplant model. Ann Hematol 86: 1–8. 58. Badiavas EV, Falanga V. (2003) Treatment of chronic wounds with bone marrow-derived cells. Arch Dermatol 139: 510–516. 59. Falanga V, Saap LJ, Ozonoff A. (2006) Wound bed score and its correlation with healing of chronic wounds. Dermatol Ther 19: 383–390. 60. Altman AM, Matthias N, Yan Y, et al. (2008) Dermal matrix as a carrier for in vivo delivery of human adipose-derived stem cells. Biomaterials 29: 1431–1442.

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61. Wu Y, Chen L, Scott PG, Tredget EE. (2007) Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells 25: 2648–2659. 62. Navsaria HA, Ojeh NO, Moiemen N, Griffiths MA, Frame JD. (2004) Reepithelialization of a full-thickness burn from stem cells of hair follicles micrografted into a tissue-engineered dermal template (Integra). Plast Reconstr Surg 113: 978–981. 63. Inokuma D, Abe R, Fujita Y, et al. (2006) CTACK/CCL27 accelerates skin regeneration via accumulation of bone marrow-derived keratinocytes. Stem Cells 24: 2810–2816. 64. Sasaki M, Abe R, Fujita Y, Ando S, Inokuma D, Shimizu H. (2008) Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol 180: 2581–2587. 65. Zhao J, Zhang N, Prestwich GD, Wen X. (2008) Recruitment of endogenous stem cells for tissue repair. Macromol Biosci 8: 836–842. 66. Schmidt A, Ladage D, Schinkothe T, et al. (2006) Basic fibroblast growth factor controls migration in human mesenchymal stem cells. Stem Cells 24: 1750–1758. 67. Nedeau AE, Bauer RJ, Gallagher K, Chen H, Liu ZJ, Velazquez OC. (2008) A CXCL5- and bFGF-dependent effect of PDGF-B-activated fibroblasts in promoting trafficking and differentiation of bone marrow-derived mesenchymal stem cells. Exp Cell Res 314: 2176–2186. 68. Liu Y, Cai S, Shu XZ, Shelby J, Prestwich GD. (2007) Release of basic fibroblast growth factor from a crosslinked glycosaminoglycan hydrogel promotes wound healing. Wound Repair Regen 15: 245–251. 69. Falanga V. (2005) Wound healing and its impairment in the diabetic foot. Lancet 366: 1736–1743. 70. Chambers SM, Goodell MA. (2007) Hematopoietic stem cell aging: wrinkles in stem cell potential. Stem Cell Rev 3: 201–211. 71. Van Zant G, Liang Y. (2003) The role of stem cells in aging. Exp Hematol 31: 659–672. 72. Schatteman GC, Ma N. (2006) Old bone marrow cells inhibit skin wound vascularization. Stem Cells 24: 717–721.

21.7 ADSCs HSCs ES cells AS cells MSC BM MEFs IPS FGF

Appendix: list of abbreviations Adipose-derived stem cells Hematopoietic stem cell Embryonic stem cells Adult stem cells Mesenchymal stem cells Bone marrow Mouse embryonic fibroblasts Induced pluripotent state Fibroblast Growth Factor

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Vascular Endothelial Growth Factor Human dermal fibroblasts Platelet Derived Growth Factor Insulin-like Growth Factor Keratinocyte Growth Factor Transforming Growth Factor Hair Follicles C-X-C motif chemokine 5

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22 Electrical stimulation for wound healing K. BA L A K AT O U N I S, Oxford Brookes University, UK

Abstract: Electrical stimulation has been applied on wounds and its effects have been studied for several decades. Based on the presence of the current of injury, and in order to investigate the importance of maintaining the endogenous current for wound healing enhancement, a significant amount of research has been undertaken. Many types and forms of stimulation have been tried and tested. This chapter attempts to present progress in research regarding electric stimulation in wound healing. Key words: electrical stimulation, wound healing, current of injury, galvanotaxis, low intensity current, microcurrents, high voltage pulsed current, HVPC.

22.1

Introduction

Electrical stimulation is widely used as a physical agent in medicine and rehabilitation (Prentice, 2005). The use of electric current in tissue healing has been studied and practiced due to the discovery of a current that is produced during injury. The current produced by the wound itself appeared to be decreasing in intensity over time (Jaffe & Vanable, 1984). This decrease was linked to a plateau in tissue healing. Therefore, external application of electrical current has been provided in order to support and promote the repair and regeneration phase. Electrical stimulation has also been shown to influence cell migration proliferation and various other mechanisms. Electrical stimulation may be highly variable in form and parameters. Various forms of currents such as direct currents (DC), pulsed direct currents, high- or low-voltage pulsed currents (HVPC, LVPC), alternative currents (AC), and low-intensity currents (LIC) are available (Kloth, 2005). Direct current (Fig. 22.1 (a)) is the continuous, unidirectional flow of charged ions. Negatively charged ions move toward the positive pole (anode) and positively charged ions move toward the negative pole (cathode). Direction of current is determined by the polarity chosen. Continuous DC has no pulses or waveform (American Physical Therapy Association, 2001). Pulsed direct current (Fig. 22.1 (b)) is the continuous bidirectional flow of charged ions in which a change in direction of flow occurs at a given time period. 571 © Woodhead Publishing Limited, 2011

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Alternating current (Fig. 22.1 (c)) is the brief unidirectional or bidirectional flow of charged ions in which each pulse is separated by a longer off period of lack of current flow. Pulsed current is described by its waveform, amplitude, duration, and frequency. Pulsed current can have two waveforms, monophasic or biphasic. A monophasic pulse represents a unidirectional flow of ions, where intensity of current changes. The biphasic pulsed current waveform presents with bidirectional flow. The biphasic waveform may be asymmetric or symmetric (American Physical Therapy Association, 2001). High voltage pulsed current (HVPC) usually presents very short-duration and twin triangular pulses (American Physical Therapy Association, 2001). It has been shown that no pH changes occur in human skin following 30 minutes of HVPC stimulation, which underlines the lack of risk of burns (Newton & Karselis, 1983).

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Current of injury

One of the first to report the presence of current of injury was Dubois-Reymond, in 1843. The current of injury is created by the wound and an electric field of an intensity less than 1 mA is produced. It extends to a periwound radius of 2–3 mm and the gradient gradually diminishes from 140 mV/mm to 0 mV/mm (McGinnis & Vanable, 1986; Jaffe & Vanable, 1984). It has been reported that the transportation of Na+ into the cell, maintains a difference in voltage, of 20–40 mV (Barker et al., 1982; Illingworth and Barker, 1980). It has been reported that the current of injury will eventually decrease if the wound is left open, but can be preserved when a moist occlusive dressing is applied (Griffin et al., 1991; Jaffe & Vanable, 1984). As the wound heals, the intensity of the current of injury is also reduced (Jaffe & Vanable, 1984). By considering that healing appears to be promoted through the use of occlusive dressings (Alvarez et al., 1983a; Winter, 1972), which retain the current of injury within the wound environment, it is postulated that the current of injury plays a significant role in wound healing repair and regeneration (Ojingwa & Iserrof, 2003). Therefore, it can be claimed that LIC may resemble the natural electric field/current created following injury, thus enhancing a complex biological mechanism of wound healing (Kloth & McCullogh, 1996). One of those mechanisms is galvanotaxis. Galvanotaxis is a term describing the attraction of positively/negatively charged cells towards the opposite pole of an electric field (either positive or negative) (Nishimura et al., 1996; Sheridan et al., 1996). Macrophage cells migrate towards the anode, whereas neutrophils migrate to either pole. Directional migration is provoked in the following: epithelial cells, endothelial cells, fibroblasts and neutrophils (Fukushima, 1953; Monguio, 1933). Galvanotaxis is an important phenomenon, since it may be a regulatory in vivo cell movement mechanism. Factors influencing migration might be several biological cues such as chemical (Tai et al., 2009). Galvanotaxis is mediated by polarized activation of various signaling pathways such as PI3 kinases/pten, integrins (Zhao, 2009). It has also been reported that leukocytes migrate towards the cathode when inflammation or infection is present. Fibroblasts and human keratinocytes appear to migrate towards the cathode (Canaday & Lee, 1991; Bourguignon & Bourguignon, 1987; Bourguignon et al., 1986; Ericksson & Nutticelli, 1984). The effect of positive polarity on accelerating reepithelialization is in line with the concept of galvanotaxis, since epidermal cell migration is directed towards the anode (Greenberg et al., 2000; Mertz et al., 1993). The biological processes underlying galvanotaxis are under investigation. One of the main proposed theories is lateral electrophoresis resulting in changes in the plasma membrane and protein redistribution (Ojingwa & Iserrof, 2003).

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The physiologic effects of electrical stimulation will be presented in the following section. Emphasis will be placed on studies investigating the effect of electrical stimulation on changes in angiogenesis, tissue oxygenation, DNA synthesis and receptor levels. Then the effectiveness of types of current directly on wound healing will be presented and discussed.

22.3

Physiological effects of electrical stimulation

22.3.1 Wound angiogenesis It has been reported that an increase in capillary density in venous leg ulcers takes place through the application of ES, following several months of absence of significant healing (mean increase of 43.5%) (Junger et al., 1997). In another trial, monophasic pulsed current was applied at 140 μs pulse duration, 630 μA, at 128 pulses per second pps and then to 315 μA at 64 pps when significant healing had occurred. Initially the negative electrode was placed on the wound and polarity was reversed at 7–14 days, for another 3–10 days. Capillary density improved from 8.05 capillaries/mm2 to 11.55 capillaries/mm2 (P < .039). In a clinical study, where the same electrical stimulation device was used, placing the anode on the wound resulted in faster reepithelialization than when placing the cathode (2 days earlier). Creation of new vascular tissue was observed. The trial took place in burn wounds of pigs. Other studies (Mertz et al., 1993) report that initiating stimulation by placing the cathode, instead of the anode, on the wound and then reversing the polarity, resulted in increased reepithelialization up to 20%, than when solely positive or negative polarity was applied (Greenberg et al., 2000).

22.3.2 Improvement of tissue oxygenation It is postulated that ES increases oxygen tension for a limited time period. The increase in oxygen concentration results in the oxidation of bacteria by neutrophils (Gagnier et al., 1988). It has been shown in studies (Gagnier et al., 1988), that local transcutaneous partial pressure of oxygen (PtcO2), increases when administering negative monophasic pair spiked waveform or symmetric biphasic square waveform in patients with spinal cord injury SCI. The improvement of tissue oxygenation using ES is a significant fact for SCI related ulcers. In spinal cord injured patients it has been suggested that the number of adrenergic receptors is reduced below the injury level. Vascular alterations are supported to take place and thus present with reduced bloodflow thus tissue oxygenation, and PtCO2. Dodgen et al. (1987), delivered monophasic, paired spike current through the cathode placed on the gastrosoleus muscle on the wounds of diabetic individuals. Asymmetric biphasic waveform was also used so as not to

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induce muscle contraction. Transcutaneous oxygen levels (PtcO2) were measured with oximetry before and after the session. Diabetic individuals showed significant increases in PtcO2 30 min after stimulation. Generally, results from the studies support the hypothesis that ES increases local perfusion primarily and secondly increases cutaneous oxygen saturation (Kloth, 2005). A paradox in wound healing has been noted. Even if perfusion is enhanced, wound healing may still fail. Factors affecting wound healing may also include re-injury affecting perfusion or the presence of hibernating tissue (Ennis et al., 2008). Future research is expected to clarify this paradox.

22.3.3 Other effects DNA and collagen synthesis have been shown to increase under the effect of a continuous electrostatic field on fibroblast cells (100 V/cm) in a period of 14 days. HVPC was also used, and protein synthesis was accelerated by 160%. Regarding the parameters used for stimulation, voltages exceeding 250 V appear to inhibit protein synthesis. The addition of insulin to electrically stimulated cells resulted in an increase of Ca++ uptake and protein synthesis (Falanga et al., 1987; Bassett & Hermann, 1968). Receptor levels of Transforming growth factor-b (TGF-b), that appear to participate in collagen synthesis, have been shown to be upregulated at 100 V and 100 pps. ATP and aminoacid uptake were found to be increased in other studies (Cheng et al., 1982). Conclusively, acceleration of wound healing using ES has been shown to occur in vitro through increase in Ca release, upregulation of insulin, TGF-b and protein synthesis.

22.4

Antibacterial effects of electrical stimulation

The preparation of wound bed is supported by four basic elements: tissue management, inflammation and infection control, moisture balance and epithelial regeneration (Falanga, 2004; Schultz et al., 2003). Perhaps the most important element is preventing infection, by which the necessary environment for the inflammation to subside, and wound healing to take place, is guaranteed. Antiseptics commonly used are slow release forms of silver or iodine (Drosou et al., 2003). Such medications do not inhibit healing or produce allergic reactions (Apelqvist & Ragnarson, 1996; Ug & Ceylan, 2003). Electrical stimulation has been shown to promote bacteriostatic and bactericidal effects. Microamperage direct current using platinum electrodes produced bacteriostatic effects, while alternating current produced only minor effects over Escherichia coli in vitro (Rowley, 1972). Microampere

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DC was also shown to yield antibacterial effects on Pseudomonas or proteus organisms on human wounds (Wolcott et al., 1969). Antibacterial effects of microampere direct current were revealed on Pseudomonas aeruginosa, at 1 mA 69 and Staphylococcus aureus (Barranco et al., 1974). Specifically silver anode electrodes at 0.4 and 4.0 μA, inhibited S aureus (Ong et al., 1994; Alvarez et al., 1983b). Electric direct current of 100 μA using silver wire electrodes and the anode, developed a bactericidal influence on gram positive bacteria and gram negative bacilli (Laatsch et al., 1995; Ong et al., 1994). HVPC at various intensity levels (50–800 mA) and 100 pps for 30 min had no antibacterial effects, while continuous DC produced bacteriostatic effects at 1, 5, 10 mA for S aureus (Guffey & Asmussen, 1989). HVPC has been supported to have an effect on pH in trials, while other studies show no effect (Kincaid & Lavoie, 1989; Newton & Karselis, 1983). The antibacterial effects of LVPC have been presented using the dermapulse system, an electrotherapy device providing low intensity current, on gram negative and gram positive pathogens. All organisms were significantly reduced with the application of the dermapulse system (Daeschlein et al., 2007).

22.5

The effect of high voltage pulsed current (HVPC) on wound healing

There is a substantial amount of research studies on the influence of HVPC on wound healing. The use of HVPC for a period of 14 weeks has been shown to promote ischemic wound healing, increase arteriolar vasodilation and dermal capillary formation (Goldman et al., 2004). Another study showed that HVPC (150 V, 100 pps, 100 μs pulse duration) with cathode on wound, administered for 45 minutes, 3 times per week for 4 weeks, resulted in wound surface area reduction to 50%. The negative electrode was placed on the wound (with saline moist gauze) (Houghton et al., 2003). In another trial where HVPC was applied (n = 15), 12 of 15 neuropathic diabetic foot ulcers healed. Treatment was administered for a mean period of 2.6 months, for one hour, 3 days per week. The anode was placed on the wound site (Alon et al., 1986). Another trial that resulted in significant wound healing of venous leg ulcers, delivered HVPC to 33 individuals for 50 min 6 days per week for approximately 7 weeks, through saline moist gauze. Other treatments administered to other groups were topically applied medication and the Unna boot. Initially, for up to 3 weeks, the treatment electrode was of negative polarity until pus and slough were removed and subsequently polarity was reversed. Compression bandaging was also used on all wounds. Wound

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size was significantly decreased (P < 0.001). Degree of granulation tissue development and rate of pus clearance was enhanced with HVPC compared to the other treatments (Franek et al., 2000). The efficacy of HVPC on wound healing in SCI patients has also been investigated. When HVPC is provided for 1 hour per day for 20 days (100 pps, 200 V) wound size significantly decreased (Griffin et al., 1991). A retrospective trial underlined the beneficial effect of HVPC on ischemic wounds (100 V, 100 pps, 1 hour per day). Over one year, 90% of wounds had been healed. Still the time frame that ES was provided was very long – a fact which does not exclude the possibility of wound healing occurring due to natural healing unrelated to ES (Goldman et al., 2003).

22.6

The effect of low intensity direct currents (LIDC) on wound healing

Microcurrents appear to accelerate wound healing and also appear to present with an axtioxidative effect (Lee et al., 2009). The effect of LIPDC has been investigated (Junger et al., 1997), on 15 venous leg ulcers non responding to treatment. An intensity of 630 μA, frequency of 128 pulses per second and pulse duration of 140 μs were selected with the cathode on the wound for one to two weeks. Polarity was reversed after 3–10 days. Intensity was reduced to 315 μA (64 pulses per second) when healing had reached a significant level. Treatment was provided daily for 30 minutes. Skin perfusion increased from 13.5 to 24.7 (40 mm Hg being an intact skin value) and capillary density increased to 43.5% (P < .039). Mean wound size reduced up to 63% (P < 0.01). Microampere direct current has been shown to enhance wound healing. A trial involving 15 individuals with venous leg ulcers reported mean healing rate of 14.4% and subsequent mean volume reduction of 85%. The trial provided treatment for a total of 6 weeks and 6 hours per day. Parameters used were 0.2–0.8 mA and polarity was reversed (Lee et al., 2009). Another clinical trial including 3 individuals with venous leg ulcers yielded positive results (Assimakopoulos, 1968). In a double-blind study (Wood et al., 1993), 74 patients with stages II and III chronic decubitus ulcers received treatment comprising electrical stimulation of 300–600 μA. Following 8 weeks post-treatment in the ES group, 58% of wounds had healed, compared to only 1% in the control group. The rest of the ulcers in the control group on the contrary worsened. A statistically significant enhanced healing was observed (P < 0.0001). Apart from venous leg ulcers, low intensity currents have been applied in research on ischemic wounds. Wound healing was studied in 83 patients

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with ischemic wounds (Wolcott et al., 1969). Treatment was very extensive in terms of ES application, three sessions per day, 2 hours each were delivered. Intensity reached 200–800 μA with the negative electrode being placed on the wound. On day 3, polarity was reversed, given that no infection had appeared. In the event of presence of infection, reversal was cancelled to when infection had subsided. Reversal was delayed a further 3 days. Polarity reversal took place due to its reported antibacterial effects when utilized in similar current parameters. (Rowley et al., 1974) – 45% of the wounds healed in a mean of 9.6 weeks, and the remaining wounds reached healing of a mean of 64.7% for a time frame up to 7.2 weeks (mean). In another study, electric stimulation was applied to 6 patients with bilateral ischemic skin ulcers. The parameters of LIDC were the same as in Wolcott et al. (1969), and, polarity was reversed one time. Comparison was made between the two sides that received standard, or standard treatment and ES treatment (Gault & Gatens, 1976). Healing rate of the standard treatment side was 14.7% compared to 30% in the other side. Results showed a notable acceleration of healing using ES. A larger number of patients were treated with ES. One hundred patients received LIDC treatment on ischemic wounds. Mean healing rate reached 28.4% at seven days. Assimakopoulos (1968) studied three patients with venous leg ulcers. By providing ES of an intensity of 100 μA, healing was completed in 6 weeks. LIDC was studied on 30 patients with various ulcers located either over the sacrum or the lower limb below the knee. The treatment group received electrical stimulation of 200–800 μA for 2 hours, twice per day, with an interval of over 2–4 hours, 5 days per week, for 5 weeks plus standard treatment. The negative electrode was positioned on the wound. Reversal of polarity was applied, similarly as in Wolcott et al. (1968). Treatment was continued until complete healing was reached. Results revealed statistically significant enhancement of wound healing of 1.5 to 2.5 times greater in the ES group (Carley & Wannappel, 1985). A systematic review on the effect of low intensity currents on wound healing has presented protocols of application of low intensity currents (Balakatounis & Angoules, 2008).

22.7

Other types of electrical stimulation applied to wounds

22.7.1 Combination with the application of heat The application of heat along with ES has been shown to produce a carryover effect in blood flow. The application of ES has been combined with heat to

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investigate their effect on wound healing. When applied on non-healing chronic ulcers together for 30 minutes over one month (30 Hz, 250 microsec, 20 mA) wound size decreased to 55.5% of its initial size (Suh et al., 2009). The effect of heat along with ES has also been reported by Petrofsky et al. (2007) who applied ES of 20 mA for 30 minutes for 3 times per week for 4 weeks at either 32 or 37°C. Blood flow almost doubled, implying an indirect acceleration of wound healing (Petrofsky et al., 2007). Furthermore, biphasic ES of 20 mA for 30 minutes, 3 times per week for 4 weeks at 32°C resulted in increase in healing up to 70% and increased blood flow. However, blood flow did not increase in the center of the wound (Lawson & Petrofsky, 2007).

22.7.2 Other types of electrical current applied for wound healing Low frequency transcutaneous nerve stimulation, an alternative application of ES, has been applied to chronic venous leg ulcers. Treatment lasted 5 minutes for 2 times per day for 12 weeks. Blood flow was significantly improved but beneficial effects may be due to compression bandaging that was provided at the same period (Ogrin et al., 2009). Biphasic asymmetric pulsed current (80 pps, 1 ms pulse duration, amplitude to tingling for 20 min per day) was applied to 64 patients with neuropathic diabetic foot ulcers. Treatment consisted of standard care and 20 minutes twice per day for 12 weeks. In each week polarity was reversed. A percentage of 42% of wounds healed with ES compared to 15% with standard care (Lundberg et al., 1992). Biphasic symmetrical pulsed current has been shown to complete healing of chronic ischemic lower leg ulcerations (6 of 24 presented with beginning or advanced distal gangrene) over one year. Pain free walking distance was also increased from 87.5 m to 421.25 m. ES was administered for 20 minutes daily. Pulse frequency ranged from 1–2 pps, amplitude 15–10 mA (Debreceni et al., 1995). A trial involving 114 diabetic wounds received either symmetrical or biphasic assymetrical PC and produced a 60% improved healing rate compared to standard treatment (Baker et al., 1997). Another trial found that ES with local wound care using a Dacron-mesh silver nylon stocking worn 8 hours by night and weight off-loading, wound healing was enhanced. ES parameters were 50 V, 80 pps, 100 μs pulse duration (Peters et al., 2001). Povidone has cytotoxic effects and has been shown in a study to have a negative influence in wound healing when combined with ES (Katelaris et al., 1987).

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22.8

Discussion

Standard care is commonly provided in clinical studies. Treatment protocols of ES last only 30–60 minutes daily and are administered 5–7 days per week. Thus, in the remaining time every day, standard treatment cannot be withheld, due to ethical reasons. Most clinical studies compare the effect of ES with simultaneous provision of standard care to control groups receiving standard care. Several other studies compare wound healing before and after treatment. An overall view of research on electrical currents showed that HVPC has been linked with positive results on wound healing. Treatment periods from 3 weeks and everyday to every other day for 45–60 minutes have been found to enhance or promote wound healing. HVPC has been successfully applied and studied on ischemic ulcers, pressure ulcers, neuropathic ulcers. Regarding LIDC, current research indicates that LIDC of 200–800 microA is effective in promoting and accelerating wound healing. It is emphasized that in no study was blood or serous exudate observed, showing that the intensity range of 200 to 800 μA is suitable for low-intensity electrical stimulation, presenting with no side-effects. On LIPDCs, studies showed that an intensity of 630 μA may stimulate healing of ulcers that did not respond with standard compression. Currents of 300 to 600 μA, for stages II and III pressure ulcers applied daily for at least 30 minutes for 4 to 8 weeks were supported to be effective in wound healing. Reversal of polarity in LIPDC has been proposed in the absence of infection. When healing is not progressing, reversal may again take place. Other forms of ES have also been studied, such as low frequency transcutaneous nerve stimulation, biphasic asymmetric or symmetric pulsed current yielding encouraging results on the role of ES for wound healing. The combination of ES with the application of heat revealed a significant increase in wound healing. Still, the extent of the contribution of healing has not been separately studied. A comparison of the effects of the different types of current on wound healing has not been attempted. The large variability of treatment characteristic parameters, wound types and other population characteristics, render comparison unfeasible. The majority of studies though have applied HVPC and LIC on wounds. Furthermore an overview of the effectiveness of low intensity currents has been undertaken recently, presenting specific protocols of application of LIC on wounds (Balakatounis & Angoules, 2008). Recent research highlights the importance of the characteristics of electrodes. It is evident that in electrotherapy appropriate electrode design has to be selected in order to ensure proper spreading of current. Research on appropriate electrode selection is necessary due to the fact that tissue

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during injury/inflammation is not homogenous, therefore there are variable areas of low or high tissue resistance (Petrofsky et al., 2007). New devices have been developed, enabling electrode size and shape selection according to wound size and shape. Weber et al. (2009), have presented a device delivering HVPC of 60–120 Hz and pulse duration of 90–110 mus, or direct current, in which up to 49 electrodes in array may be used. In that way electrodes are more appropriate to deliver current to the wound and thus facilitate wound healing (Weber et al., 2009). In another research study, three-channel ES is proposed to be preferred to two-channel, since it has been found to provide better current dispersion and tissue penetration (Suh et al., 2009). Regarding electrode selection, research is necessary in order to clarify the considerations and facts relating to electrode selection in the healing of wounds. There are also clinical implications to be taken into consideration. Wound healing is a challenge for the medical team and the clinic. Wounds, apart from being a medical condition on their own, affect patient recuperation and rehabilitation in several ways. Application of wound healing treatments may necessitate dedicating treatment time (McCulloch, 1998), and possibly may take time from other activities that may also be important for the patient recuperation. In that way other issues may be overlooked and other medical concerns, e.g. deep vein thrombosis, may receive less attention and therefore delay treatment, progress, or even discharge. Pain is a common symptom and depending on the location of the wound, prolonged presence of an ulcer may worsen the quality of life of the patient or discourage movement, such as when a pressure ulcer is dealt with. The attention needed to avoid infection often discourages patient transfers or leaving the room/ ward so as to minimize risk of infection through contact with additional surfaces that may not be sterilized. Accelerating wound healing may lead directly to faster rehabilitation of the patient. In turn this may be translated into reducing hospitalization time, improvement of provided services, and cost effectiveness. Advances in stem cell research have received much attention in recent years and research has focused on the effect of ES on stem cells. Direct current electric fields appear to elongate and align perpendicularly cultured human adipose tissue derived stem cells (hASCs). This finding may direct future researchers to investigate the effect of current on stem cells and relate findings to wound healing and conduct relevant studies, thus expanding the knowledge base in the specific field which is generally promising (Tandon et al., 2009). Further research is required to specify which parameters and which types of current can be applied successfully in which types of wounds. Furthermore, the time and frequency of treatment need to be investigated in order to promote evidence-based treatment protocols. Special attention needs to

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be drawn to the significance of applying the electrodes correctly in a clean wound site, and providing secure proper delivery of current. At a cellular level, research may continue to discover the effects of electric fields and current on various types of cells such as epithelial cells, leukocytes, and create a larger pool of evidence to support electrical stimulation as a treatment option for wound healing.

22.9

Conclusion

There is wealth of evidence to support the effectiveness of various forms and types of current in treating wounds. Overall, it may be proposed that electrical current applied for wound healing may be pulsed or continuous, of high voltage or low intensity, and is proposed to be applied for over 30 minutes at least for 5–7 times per week. The negative electrode is usually applied on the wound site. The combination of ES with heat also appears promising. The rationale supporting the effect of electrical stimulation on wound healing is mainly galvanotaxis, cell migration and cell modulation through which regeneration and repair are accelerated.

22.10 References Alon G, Azaria M, Stein H. (1986). Diabetic ulcer healing using high voltage TENS [Abstract]. Phys Ther.; 66:775. Alvarez O, Mertz P, Eaglstein W. (1983a). The effect of occlusive dressings on collagen synthesis and reepithelialization in superficial wounds. J Surg Res.; 35:142–8. Alvarez O, Mertz P, Smerbeck R, et al. (1983b). The healing of superficial skin wounds is stimulated by external electrical current. J Invest Dermatol.; 81(2):144–8. American Physical Therapy Association (2001). Electrotherapeutic terminology in physical therapy. Alexandria (VA): APTA. Apelqvist J, Ragnarson T. (1996). Cavity foot wounds in diabetic patients: a comparative study of cadexomer iodine ointment and standard treatment. An economic analysis alongside a clinical trial. Acta Derm Venereol.; 76:231–5. Assimacopoulos D. (1968). Low intensity negative electric current in treatment of ulcers of leg due to chronic venous insufficiency: preliminary report of three cases. Am J Surg.; 115:683–7. Baker L, Chambers R, DeMuth S, et al. (1997). Effects of electrical stimulation on wound healing in patients with diabetic ulcers. Diabetes Care.; 20(3):405–12. Barker A, Jaffee L, Vanable J Jr. (1982). The glabrous epidermis of cavies contains a powerful battery. Am J Physiol.; 242:R258–66. Balakatounis KC, Angoules AG. (2008). Low-intensity electrical stimulation in wound healing: review of the efficacy of externally applied currents resembling the current of injury. Eplasty.; 16(8):e28.

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Barranco S, Spadero J, Berger T, et al. (1974). In vitro effect of weak direct current on Staphylococcus aureus. Clin Orthop.; 100:250–5.91. Bassett C, Herrmann I. (1968). The effect of electrostatic fields on macromolecular synthesis by fibroblasts in vitro [Abstract]. J Cell Biol.; 39:9. Bourguignon G, Bourguignon L. (1987). Electric stimulation of protein and DNA synthesis in human fibroblasts. FASEB J.; 1(5):398–402. Bourguignon G, Bergouignan M, Khorshed A, et al. (1986). Effect of high voltage pulsed galvanic stimulation on human fibroblasts in cell culture. J Cell Biol.; 103:344a. Canaday D, Lee R. (1991). Scientific basis for clinical application of electric fields in soft tissue repair. In: Brighton C, Pollack S, editors. Electromagnetics in biology and medicine. San Francisco: San Francisco Press. Carley PJ, Wainapel SF. (1985). Electrotherapy for acceleration of wound healing: low intensity direct cur rent. Arch Phys Med Rehabil.; 66(7):443–6. Cheng N, Van Hoof H, Bockx E, et al. (1982). The effects of electric currents on ATP generation, protein synthesis, and membrane transport in rat skin. Clin Orthop.; 171:264–72. Daeschlein G, Assadian O, Kloth LC, Meinl C, Ney F, Kramer A. (2007). Antibacterial activity of positive and negative polarity low-voltage pulsed current (LVPC) on six typical Gram-positive and Gram-negative bacterial pathogens of chronic wounds. Wound Repair Regen.; 15(3):399–403. Debreceni L, Gyulai M, Debreceni A, et al. (1995). Results of transcutaneous electrical stimulation (TES) in cure of lower extremity arterial disease. Angiology.; 46: 613–8. Dodgen P, Johnson B, Baker L, et al. (1987). The effects of electrical stimulation on cutaneous oxygen supply in diabetic older adults. Phys Ther.; 67(5):793. Drosou A, Falabella A, Kirsner R. (2003). Antiseptics on wounds: an area of controversy. Wounds.; 15(5):149–66. Ennis WJ, Meneses P, Borhani M. (2008). Push-pull theory: using mechanotransduction to achieve tissue perfusion and wound healing in complex cases. Gynecol Oncol.; 111(2 Suppl):S81–6. Erickson C, Nuccitelli R. (1984). Embryonic fibroblast motility and orientation can be influenced by physiological electric fields. J Cell Biol.; 98:296–307. Falanga V. (2004). Wound bed preparation: science applied to practice. In: European Wound Management Association (EWMA), editor. Position document: wound bed preparation in practice. London: MEP Ltd;. p. 2–5. Falanga V, Bourguignon G, Bourguignon L. (1987). Electrical stimulation increases the expression of fibroblast receptors for transforming growth factor-beta. J Invest Dermatol.; 88:488–92. Franek A, Polak A, Kucharzewski M. (2000). Modern application of high voltage stimulation for enhanced healing of venous crural ulceration. Med Eng Phys.; 22:647–55. Fukushima K, Senda N, Inui H, et al. (1953). Studies of galvanotaxis of leukocytes. Med J Osaka Univ.; 4(2–3):195–208. Gagnier K, Manix N, Baker L, et al. (1988). The effects of electrical stimulation on cutaneous oxygen supply in paraplegics. Phys Ther.; 68(5):835–9. Gault WR, Gatens PF Jr. (1976). Use of low intensity direct current in management of ischemic skin ulcers. Phys Ther.; 56(3):265–9.

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Goldman R, Brewley B, Zhou L, Golden M. (2003). Electrotherapy reverses inframalleolar ischemia: a retrospective, observational study. Adv Skin Wound Care.; 16(2):79–89. Goldman R, Rosen M, Brewley B, Golden M. (2004). Electrotherapy promotes healing and microcirculation of infrapopliteal ischemic wounds: a prospective pilot study. Adv Skin Wound Care.; 17(6):284–94. Greenberg J, Hanly A, Davis S, et al. (2000). The effect of electrical stimulation (RPES) on wound healing and angiogenesis in second degree burns. Proceedings of the 13th Annual Symposium on Advanced Wound Care; 1–4; Dallas, TX. Griffin J, Tooms R, Mendlus R, et al. (1991). Efficacy of high voltage pulsed current for healing of pressure ulcers in patients with spinal cord injury. Phys Ther.; 71(6):433–42. Guffey J, Asmussen M. (1989). In vitro bactericidal effects of high voltage pulsed current versus direct current against Staphylococcus aureus. J Clin Electrophysiol.; 1:5–9. Houghton P, Kincaid C, Lovell M, et al. (2003). Effect of electrical stimulation on chronic leg ulcer size and appearance. Phys Ther.; 83(1):17–28. Illingworth C, Barker A. (1980). Measurement of electrical currents emerging during the regeneration of amputated finger tips in children. Clin Phys Physiol Meas.; 1:87–9. Jaffe L, Vanable J. (1984). Electrical fields and wound healing. Clin Dermatol.; 2(3):34–44. Junger M, Zuder D, Steins A, et al. (1997). Treatment of venous ulcers with low frequency pulsed current (Dermapulse): effects on cutaneous microcirculation. Der Hautartz.; 18:879–903. Katelaris P, Fletcher J, Little J, et al. (1987). Electrical stimulation in the treatment of chronic venous ulceration. Aust N Z J Surg.; 57:605–7. Kincaid C, Lavoie K. (1989). Inhibition of bacterial growth in vitro following stimulation with high voltage, monophasic pulsed current. Phys Ther.; 69(8):651–5. Kloth LC. (2005). Electrical stimulation for wound healing: a review of evidence from in vitro studies, animal experiments, and clinical trials. Int J Low Extrem Wounds.; 4(1):23–44. Kloth LC, McCulloch JM. (1996). Promotion of wound healing with electrical stimulation. Adv Wound Care.; 9(5):42–5. Laatsch L, Ong P, Kloth L. (1995). In vitro effects of two silver electrodes on select wound pathogens. J Clin Electrophysiol.; 7(1):10–5. Lawson D, Petrofsky JS. (2007). A randomized control study on the effect of biphasic electrical stimulation in a warm room on skin blood flow and healing rates in chronic wounds of patients with and without diabetes. Med Sci Monit.; Jun.; 13(6):258–63. Lee BY, Al-Waili N, Stubbs D, Wendell K, Butler G, Al-Waili T, Al-Waili (2009). Ultra-low microcurrent in the management of diabetes mellitus, hypertension and chronic wounds: report of twelve cases and discussion of mechanism of action. Int J Med Sci.; Dec 6;7(1):29–35. Lundeberg T, Eriksson S, Malm M. (1992). Electrical nerve stimulation improves healing of diabetic ulcers. Ann Plast Surg.; 29(4):328–31. McCulloch JM. (1998). The role of physiotherapy in managing patients with wounds. J Wound Care.; 7(5):241–4.

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McGinnis M, Vanable J Jr. (1986). Voltage gradients in newt limb stumps. Prog Clin Biol Res.; 210:231–8. Mertz P, Davis S, Cazzaniga A, et al. (1993). Electrical stimulation: acceleration of soft tissue repair by varying the polarity. Wounds.; 5(3):153–9. Monguio J. (1933). Über die polare Wirkung des galvanischen Stromes auf Leukozyten. Z Biol.; 93:553–9. Newton R, Karselis T. (1983). Skin pH following high voltage pulsed galvanic stimulation. Phys Ther.; 63(10):1593–6. Nishimura K, Isseroff R, Nuccitelli R. (1996). Human keratinocytes migrate to the negative pole in direct current electric fields comparable to those measured in mammalian wounds. J Cell Sci.; 109:199–207. Ogrin R, Darzins P, Khalil Z. (2009) The use of sensory nerve stimulation and compression bandaging to improve sensory nerve function and healing of chronic venous leg ulcers. Curr Aging Sci.; 2(1):72–80. Ojingwa JC, Isseroff RR. (2003). Electrical stimulation of wound healing. J Invest Dermatol.; 121(1):1–12. Ong P, Laatsch L, Kloth L. (1994). Antibacterial effects of a silver electrode carrying microamperage direct current in vitro. J Clin Electrophysiol.; 6(1):14–8. Peters E, Lavery L, Armstrong D, et al. (2001). Electric stimulation as an adjunct to heal diabetic foot ulcers: a randomized clinical trial. Arch Phys Med Rehabil.; 82:721–4. Petrofsky JS, Lawson D, Suh HJ, Rossi C, Zapata K, Broadwell E, Littleton L. (2007). The influence of local versus global heat on the healing of chronic wounds in patients with diabetes. Diabetes Technol Ther.; 9(6):535–44. Prentice WE. (2005). Therapeutic Modalities in Rehabilitation. 3rd ed. New York: McGraw Hill. Rowley B. (1972) Electrical current effects on E. coli growth rates. Proc Soc Exp Biol Med.; 139:929–34. Rowley B, McKenna J, Chase G, et al. (1974). The influence of electrical current on an infecting microorganism in wounds. Ann N Y Acad Sci.; 238:543–51. Schultz G, Sibbald G, Falanga V, et al. (2003). Wound bed preparation: a systemic approach to wound management. Wound Repair Regen.; 11(2 Suppl):S1– 28. Sheridan D, Isseroff R, Nuccitelli R. (1996). Imposition of a physiologic DC electric fields alter the migratory response of human keratinocytes on extracellular matrix molecules. J Invest Dermatol.; (4):642–6. Suh H, Petrofsky JS, Lo T, Lawson D, Yu T, Pfeifer TM, Morawski T. (2009). The combined effect of a three-channel electrode delivery system with local heat on the healing of chronic wounds. Diabetes Technol Ther.; 11(10):681–8. Tai G, Reid B, Cao L, Zhao M. (2009). Electrotaxis and wound healing: experimental methods to study electric fields as a directional signal for cell migration. Methods Mol Biol.; 571:77–97. Tandon N, Goh B, Marsano A, Chao PH, Montouri-Sorrentino C, Gimble J,VunjakNovakovic G. (2009). Alignment and elongation of human adipose-derived stem cells in response to direct-current electrical stimulation. Conf Proc IEEE Eng Med Biol Soc.; 1:6517–21. Ug A, Ceylan O. (2003). Occurrence of resistance to antibiotics, metals and plasmids in clinical strains of Staphyloccus spp. Arch Med Res.; 34(2):130–6.

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Weber SA, Vonhoff PA, Owens FJ, Byrne J, McAdams ET. (2009). Development of a multi-electrode electrical stimulation device to improve chronic wound healing. Conf Proc IEEE Eng Med Biol Soc.; 1:2145–8. Winter G. (1972). Epidermal regeneration studies in the domestic pig. In: Maibach H, Rovee D, eds. Epidermal Wound Healing. Chicago, IL: Year Book Medical Publishers; 71–112. Wolcott L, Wheeler P, Hardwicke H, et al. (1969). Accelerated healing of skin ulcers by electrotherapy: preliminary clinical results. South Med J.; 62:795–801. Wood JM, Evans PE, III, Schallreuter KU, et al. (1993). Multicenter study on the use of pulsed low-intensity direct current for healing chronic stage II and stage III decubitus ulcers. Arch Dermatol.; 129(8):999–1009. Zhao M. (2009). Electrical fields in wound healing – An overriding signal that directs cell migration. Semin Cell Dev Biol.; 20(6):674–82.

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23 Negative pressure wound therapy M. S. M I L L E R, The Wound Healing Centers of Indiana, USA

Abstract: This chapter discusses negative pressure wound therapy (NPWT) as an effective modality for treating challenging wounds. The chapter reviews the historical development of NPWT and the scientific basis behind its use, including its known effects on wounds. The chapter explores the future trends of NPWT. Key words: negative pressure wound therapy, NPWT, negative pressure, chronic wound care, subatmospheric, non-healing wounds, Boyle’s law.

23.1

Introduction

Of the numerous available options for promoting healing of open wounds, the use of negative pressure has unquestionably been the most controversial. Despite the paucity of reproducible scientific evidence documenting efficacy, there is an almost panacea mentality surrounding its use. The controversies surrounding negative pressure are multiple and all too often generate a fanatical allegiance to one or more techniques or theories, all too often when the support is grounded in emotion rather than factual information. Whereas wound dressing choices are mostly dependent on the wound parameters, the choice of negative pressure systems tends to be based on economic factors and more, passion. The literature runs the gamut from what wounds and conditions respond best, to what pump systems provide the ideal environment for healing. In this chapter, the goal will not be to identify the best choice in a given situation or even stratify the many options. I leave it to the reader to glean from the available literature, their own experiences and their economic factors to make such a choice regarding which specific negative pressure entity is best suited for a given situation. Recognizing that the use of negative pressure as a medical treatment is rooted in antiquity, this chapter is dedicated to providing the opportunity for the reader to look at the many components of negative pressure science and practices as a starting point and then decide which colors of this spectrum best suit their vision. As a point of clarification, it is recognized that the myriad geographical areas, companies and practitioners have different terms for this modality. My choice to use the phrase “negative pressure” is based solely on the prevalent use of this term in the literature which identifies the generic science without commercialization. 587 © Woodhead Publishing Limited, 2011

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23.2

History of negative pressure wound therapy

The history of the use of negative pressure has been primarily influenced by the perceived needs of the types of injuries and illnesses for a given culture. When people lived only an average of 30 years (CDC 1999), the wounds that occurred were primarily from injuries, burns, or animal bites and more often than not, infected. Now with the increased longevity of mankind, the wounds have become much different in their character. The earliest uses of negative pressure appear to have been more on intact skin than used to treat wounds. The technique of “cupping” has been seen in many cultures with the earliest record of cupping in the Ebers Papyrus dated about 1550 BC. This document is considered by some to contain the most important medical records of ancient Egypt. “Cupping” was know as cucurbitula, which is derived from Latin “curcurbita” meaning a gourd, or made in the shape of a gourd. There were several ways to create suction in the skin/cup interface. One was to heat the air in the cup before application to the skin; as the air cooled, a suction developed. The other is to have a hole in the cup through which suction may be applied. Once suction was established, the hole was plugged, usually with wax. There is also evidence of cupping dating back to 1000 BC in China. The ancient Greeks, including Hippocrates (ca 400 BC), used the technique for internal disease and structural problems. Historically, cupping devices have been made from many different materials such as animal horns, bamboo, wood, metals (copper and bronze), pottery, or glass. Bailey (English Dictionary) defines a cupping glass as “a sort of glass vial applied to the fleshy part of the body to draw out corrupt blood and windy matter.” Traditional Chinese cupping is said to realign and balance the flow of one’s vital energy or life force known as chi along meridians, which are influence points for internal organs. In “wet” cupping, the skin is scraped or punctured to create a route for toxins to escape. Cupping massage is a technique where the patient’s back is rubbed with a special salve that promotes blood flow and makes the skin conductive. The therapist then pulls a large massage cupping glass with a very rounded edge across the back with slow, even movements. For cupping to be effective, it can only be used on softer tissue that will allow a seal to be created around the edge of the cup. Cupping draws blood to the surface area of the body where the cups are applied thus resulting in localized blood-flow stimulation. The cup may commonly leave a circular ring or bruising. As understanding of the physiology of how the process of tissue healing progressed, Professor August Bier of Germany described a new concept in the early 1890s called “hyperemic treatment”. The goal of this treatment

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was to artificially increase the quantity of blood (inflammation) in a given diseased part of the body. Bier used a suction apparatus applied to the intact skin for the induction of this hyperemic response which he felt would cause a rush of blood into the area, not only on the skin surface but also the deeper tissue layers. Bier’s aim was to “increase this beneficent inflammatory hyperemia resulting from the fight of the living body against invasion.” He believed, “the blood must continue to circulate; there must never be a stasis of the blood” (p. 19). In 1908, Drs Willy Meyer and Victor Schmieden published a comprehensive manual of hyperemic treatment based on the work of Bier. This appears to be one of the first documented uses of an apparatus applying a subatmospheric pressure to skin wounds. They claimed that If we place such a glass over a diseased area which presents a sinus in its middle, the pus and with it bacteria are aspirated from the depth, slowly and painlessly; often necrotic tissue, or even sequestra of small size, are brought to the surface. This suction effect is particularly valuable, for the granulations lining the fistulous tract or the abscess cavity are in this manner also brought under hyperemia, and the current of secretion, thus directed outwardly, bathes and cleanses them gently but thoroughly.

They used specially designed “suction glasses” which were applied to the skin for six times per day for five minutes with intervals of three minutes between the applications in order to give the edema and hyperemia an opportunity to disappear. Subsequently, Professor Bier’s assistant, Dr E. Klapp, patented the first suction pump for wound care known as the “Klapp Cup” which was used for “local inflammations and abscesses.” Victor T. Junod (1809–1881), a French physician, invented the “Junod’s boot” which was an airtight case into which the lower leg was inserted and the air was then exhausted. It was used to divert a portion of the blood temporarily from the general circulation (Fig. 23.1). With these unique devices providing a novel way to promote healing, more new innovations regarding this technology were developed. In 1952 a German patent was granted illustrating the use of negative pressure applied to an open wound using absorbent tampons made of natural sponge, rubber sponge, foam rubber, cellulose sponge, gauze, cotton, or the like. The patent describes the avoidance of damages associated with excess fluid accumulation, as well as reduction in frequent and uncomfortable dressing changes. In 1971 Atmos Fritzsching & Co, of Germany, was granted a patent for the use of a vacuum pump, catheters or cannula, for the intermittent suction of fluids. The device provided for automatic control of the drainage process within adjustable ranges of negative pressure. During pre-settable pause

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(a)

(b)

23.1 (a) Representative drawing of Junod’s Boot, (b) Representative drawing of modification of Junod’s Boot for the upper extremity (Lawrence Brown Orleans, Indiana, USA).

intervals, lower negative pressure was established and maintained to avoid blockage of the catheter, and backflow of fluids. In 1976 Dr Paul Svedman of Sweden developed a device which used a foam based wound contact layer to provide a conduit from the suction source to the wound bed. From 1986 to 1991 five papers from the Russian medical literature demonstrated the effectiveness of NPWT. These were published as “The Kremlin Papers” by Blue Sky Medical. Included in these were several papers that identified unique properties of negative pressure that had not been previously considered or recognized. Below is a description of some of these papers. In 1986, Davydov and colleagues found that vacuum therapy in 229 subjects with purulent lactation mastitis reduced the bacterial load and septic

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complications, reduced time to healing, normalized the immune process, reduced scar tissue formation, and reduced hospital stays. Their findings suggested that when combined with surgical debridement, NPWT resulted in improved immune response, improvements in microbial control, less scarring, and shorter healing times. Interestingly, they applied the negative pressure for 20 minutes initial treatment, followed by twice daily application for 2.5 to 3.0 hours for 5–6 days. In 1986, Kostiuchenok et al. found that NPWT in combination with surgical debridement in 221 subjects with various purulent wounds reduced the bacterial burden and resulted in improved wound healing. He used short average treatment times lasting less than 10 minutes on average at negative 100mmHg. In 1987 Usopov and Yepifanov used a rabbit surgical wound model to compare NPWT to a passive drainage control group. They found that negative pressures of −75 to −80 mmHg increased drainage with minimal or no hemorrhage. They then treated 1616 humans with various wounds. Those with “sterile wounds” were treated with continuous NPWT at −70 to −80 mmHg for 2–3 days while those with “purulent wounds” were treated with debridement and irrigation and continuous NPWT at −30 mmHg to −40 mmHg for 6–8 days. They found that pressures lower than −80 mmHg in both study components provided active drainage without the likelihood of hemorrhage. In 1988 Davydov et al. performed bacteriologic and cytologic study of purulent wounds utilizing tissue biopsies in wounds of 226 patients who received NPWT at −1 to −1.5 atmospheres (1 hour per day for 6 days) following surgical debridement. Their control group of 212 patients received surgical debridement only. Their findings were that compared to debridement alone, the NPWT group had lower bacterial counts and shorter periods of purulence and inflammation. In 1989 Katherine Jeter RN, and then surgery resident Mark Chariker MD and others, published the results of the treatment of seven patients with incisional and enterocutaneous fistulae, using a unique combination of products to deliver negative pressure to the wound bed. They devised a closed suction wound draining system that incorporated the use of moist gauze dressing, a Jackson-Pratt Mini Snyder flat drain, and a transparent adhesive film dressing to seal the wound, which was connected to a suction source. The use of the system reduced dressing changes, adding to patient comfort and ease of nursing care. Substantial cost savings were noted from use of gauze versus other conventional dressings, and from staff time savings. Interestingly, however, Chariker reported that this method decreased rather than increased the degree of granulation tissue leading to erroneous assumptions of this technique of negative pressure to remove fluid without any promotion of wound healing.

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In 1991 Davydov et al. performed a retrospective review of 744 subjects, examining various treatment modalities for treating purulent wounds following aggressive surgical debridement. He identified 406 subjects having received NPWT; 338 subjects received surgical debridement alone. The authors noted “a pronounced immunocorrecting effect” associated with vacuum therapy specifically that the “vacuum subjects” experienced fewer infectious complications and needed fewer repeat surgeries than the group that received debridement alone. Interestingly, in looking for other possible modalities that might have a similar beneficial effect, they reported that other modalities examined (fluid pulse jets, enzymatic debridement, laser and ultrasound necrolysis, and forced suction), were considered not “universal enough to affect all elements of the biological system of healing during the inflammatory phase.” In 1993 W. Fleischmann of the University of Ulm, Germany, published the results of NPWT treatment in 15 patients with open fractures. Polyvinyl alcohol foam was used as the wound contact layer, which was then covered by transparent polyurethane dressing. Drainage tubes were connected to a suction device. No bone infections occurred in any of the patients. The authors noted, “Efficient cleaning and conditioning of the wound, with marked proliferation of granulation tissue.” In 1997 Morykwas and Argenta, of Wake Forest University, WinstonSalem, North Carolina, published three articles describing a “new method of wound control and treatment.” They also used “subatmospheric pressure” that was applied through a closed system to open wounds for a period of 48 hours using an open cell polyurethane sponge as the wound contact layer, which was then sealed with an adhesive drape. Studies in a pig model, and in patients with chronic and difficult wounds demonstrated the promotion of wound healing. The authors determined that “the application of controlled subatmospheric pressure creates an environment that promotes wound healing.” The subsequent product developed from these findings was named VAC for vacuum assisted closure and marketed by Kinetic Concepts, Inc. better known as KCI. For the next 7 years, this was the sole commercially marketed entity using negative pressure for wound healing. In 2004, a new version received approval for the Center for Medicare and Medicaid Services reimbursement code for Negative Pressure Wound Therapy (Blue Sky Medical – Versatile 1 Negative Pressure Pump System) and became the second consideration for use in this manner. The initial product introduced was called the Kremlin Kit and was based on the Russian literature in which a topical suction device was applied to open wounds. Subsequently, with the discovery of the Chariker-Jeter article, the use of a gauze/drain combination became available in contrast to the original polyurethane foam version. Subsequently, a lawsuit was filed by KCI regarding patent infringements against Blue Sky Medical. This suit was decided in August of 2006 in favor

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of Blue Sky Medical in which it was found that the Blue Sky device and dressings did not infringe on the KCI patents. This decision opened up the door to the numerous incarnations of negative pressure currently on the market and in development.

23.3

The science of negative pressure

The term “negative pressure” does not imply the absence of pressure within a given container but rather pressure that is less than atmospheric pressure (760 mmHg/1 atmosphere). The Ideal Gas Laws of physics, specifically Boyle’s Laws as well as Poiseuille’s Rule, are the two key links connecting science to clinical application. It is imperative that all clinicians be aware of these tenets as without them, the ability to define the appropriateness of therapy and all its implications becomes based on marketing, hype and emotion, not science. Boyles Law specifically explains that for a given gas, there is a relationship between its temperature (T), the volume that that a specific amount of that gas will occupy (V) and the pressure that that volume gas will exert in a closed environment (P). In his experiments Boyle observed that when temperature remained constant, the volume of a gas is inversely proportional to the pressure exerted by the gas. Holding any of these three variables constant (temperature, pressure or volume), allows for a direct relationship between the other two. PV = kT where k is a constant, or Pressure * Volume = Constant * Temperature The value of the constant k depends on the units used for the other quantities but once the units are fixed, k is also fixed. When any one of the quantities V, P, or T is changed, one or two of the others must change so that the equation above still holds. One fact that must be recognized is that in any given negative pressure system, there is not a perfect and complete absence of any molecules (i.e. a vacuum). Regardless of the efficiency of the pump, a perfect vacuum is not attainable (nor desired) and so the presence of any gas molecules in our closed systems assures the pertinence of the Ideal Gas Laws. For example, at a given constant temperature, increasing the pressure in a specific, volumedelineated vessel would result in decreased volume in that vessel. Conversely, reducing the pressure would result in an increased volume in that same closed environment. Recognizing that for any given gas, this law changes only as these parameters change, allows for an elegantly simple understanding of how the concept of negative pressure exists. If one then recognizes that the generation of the pressure (and hence the volume) within a closed, sealed negative pressure dressing is set by an outside controlling entity (the pump),

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then the pressures themselves are subject only to the settings of the pump and its ability to maintain them. Since the environment is closed, the pressure generated by the pump should be identical to that within the entire negative pressure dressing and hence, at the surface of the wound. The caveat is that the presence of another substance in the system (fluid, another type of gas, etc.) potentially adds another variable to the equation by destroying the uniformity of the viscosity and hence can dramatically alter the pressure within the system, regardless of what the pump is generating and what is actually present at the wound surface. This particular conundrum is best defined by Poiseuille’s Law. This is defined as: the calculation of the rate at which an incompressible viscous fluid flows through a cylindrical pipe. The general principles of this law are that small changes in the internal diameter of a lumen can make a big difference in the rate of flow and more pertinent to our situation, as viscosity of the “fluid” within that lumen increases, the flow rate will decrease unless the pump compensates. This explains why variations in pressures are noted between those measured at the wound interface versus the pressure settings of the pump when there is drainage in the tubing connecting the wound to the pump. The addition of a fluid within the tubing changes the viscosity of the contents of the tubing as a whole. While it is assumed that the pump should be placed at the same height as the patient (wound) to minimize variations between the pressures at the wound surface and those generated by the pump, in fact, the Ideal Gas Laws do not address this as a variable if the tubing contains only gas. Once again, if the entire system is filled solely with gas and the pressure is set at the pump, then the entire closed system will be at the same pressure. However, if fluid is present in the tubing, the change in viscosity will alter the dynamics of the system thus placing a greater demand on the pump to overcome the effects of gravity on the fluid if the same pressure is to be maintained. In short, changes in the viscosity of the system and/or the effects of gravity should have a minimal effect on the desired pressure to be applied to the wound unless the pump itself is unable to compensate for them. Clearly an understanding of Boyle’s and Poiseuille’s laws is integral to understanding the fundamentals of how negative pressure is generated, transmitted and applied to the surface of wounds. It is important to recognize that the porosity of the currently available wound contact layers (foam, gauze, etc.) preclude their being considered as having “viscosity” and therefore, the concept of their altering, modifying or improving on the suction is not scientifically correct.

23.4

The pathophysiologic mechanisms of action of negative pressure

It is important to appreciate the simplicity in which a negative pressure system operates. Once the marketing, hype, and misconceptions are elimi-

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Suction conduit to the wound contact layer Transparent film adherent to skin

Suction not yet applied to the wound contact layer. It remains in its natural, non-compressed state.

Wound Bed

23.2 Negative pressure setup in the relaxed state. Fluid is removed through the suction conduit With suction applied, the wound contact layer flattens, exerting pressure on the wound itself as well as transmitting suction to the wound.

23.3 Negative pressure setup in the contracted state with suction applied.

nated, the most basic fundamentals of a negative pressure system become quite universal. In Fig. 23.2, the wound base is covered with a wound contact layer into which (or contacted by) a suction conduit (usually some form of flexible tubing or connecting device) is inserted, which itself is connected to a suction pump (not shown). A transparent film encompasses the wound, the suction conduit as well as several centimeters of the adjacent skin to assure a sealed chamber. This represents the wound prior to initiation of suction. In Fig. 23.3, suction has been applied through the conduit. In accordance with Boyle’s Law and Poiseuille’s Rule, the negative pressure has uniformly been distributed throughout the wound contact layer, which is now compressed against the wound bed. This has caused a positive pressure to be applied against the wound bed or to be more accurate, causes coaptation of the dressing to the wound, the wound to the dressing and the wound to

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itself as the negative pressure causes a reduction in the wound size as an effect of active therapy. While the visible changes on the NPWT dressing with respect to the wound may appear similar in acute and chronic wounds, the effects of NPWT differ when used on chronic versus acute wounds such as in burns, trauma or immediately postoperatively. In wounds with acute inflammation, the suction removes edema and the compression of the dressing against the wound bed reduces the accumulation of additional edema fluid, by pushing against the blood vessels and lymphatic vessels, thereby opposing the hydrostatic and oncotic forces that otherwise favor leaks. The inflammatory process that is initiated with acute injury creates the biophysical pressures that tend to push fluids out of the bloodstream into the tissues (into the extracellular space or leaking from an open wound), and the counteracting pressures that tend to “pull” those fluids back into the tissues. The Landis–Starling Equation is used to explain the movement of fluids from the blood vessels into the extracellular spaces and vice versa based on numerous factors that are represented by these hydrostatic and oncotic forces.

The Landis–Starling Equation, which describes what is known as the “Starling Pressures” Hydrostatic pressure gradient

Colloid osmotic pressure gradient

Jv = Kf [Pc − Pif] − σ [πp − πif] Kf is the proportionality constant, and Jv is the net fluid movement between compartments. The movement of fluid out of the vessels is defined as positive, and inward force/movement is defined as negative. (J If Jv is positive, fluid will tend to leave the capillary (filtration). If negative, fluid will tend to enter the capillary (absorption). This equation has a number of important physiologic implications, especially when pathologic processes grossly alter one or more of the variables. The movement of fluid depends on six variables: Capillary hydrostatic pressure (Pc); Interstitial hydrostatic pressure (Pi); Capillary oncotic pressure (πc); Interstitial oncotic pressure (πi); Filtration coefficient (Kf); Reflection coefficient (σ)

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While a detailed explanation of this equation is beyond the scope of this chapter, a simple overview is appropriate to identify the basics of what happens when negative pressure is applied to an acute wound. The amount of liquid that will leak out of a blood vessel is proportional to the hydrostatic pressure (the outward, leaking force of the “head” of hydraulic pressure) minus the oncotic pressure of all the substances (plasma proteins, for example) in the blood vessel that tend to retain water. When negative pressure is applied to the wound, the compressive force of the dressing pushing against the wound (and arguably the wound pushing against the dressing) applies a strong physical force of compression against the blood vessels in the area of a wound, and that compression acts on the Starling forces. At their most basic, the forces: (1) prevent fluids from leaking out of the blood vessels into the tissues and (2) push fluids already leaked into the tissues back into the blood vessels. NPWT produces a manifold effect on the development of inflammatory reactions in a wound, regulating them and directing the wound process toward primary healing. The overall effect is to result in removal of inflammatory exudates from the wound-removal of inflammation and suppuration. One of the byproducts of the acute inflammatory response is edema, which contains lactic acid, a by-product of the partial (incomplete) metabolism of glucose (sugar). The removal of this lactic acid reduces the ongoing inflammatory process and sequelae potentially resulting in a reduction of the untoward symptoms associated with injury. Additional benefits of this modality include: decreased pain in the area, decreased edema, and overall, decreased induration. Mechanical cleansing of the wound surface as a result of negative pressure therapy involves the removal of pus, necroses, microbes, and foreign bodies. This occurs through promotion of autolytic debridement which is a natural process of cellular and tissue degradation occurring in enclosed, moist wounds. In addition, Coles (1956) showed there is change in the blood circulation in the treated area producing a localized hyperemia due to improved microvascular blood flow. This results in increased lymphocyte presence in the area of the wound, and subsequent rapid activation of phagocytosis due to increases in acid phosphomonoesterase activity. Normalization of pH dynamics in the wound as a result of normalization of the oxidation-reduction processes in the wound tissues is due to improved microcirculation. This establishes a natural antibacterial environment by changing the acid reaction of the wound toward neutralization or weak alkalescence, which is detrimental to microorganisms. The application of negative pressure to the wound surface creates suction and compression, which deforms the underlying cells. This in turn changes the expression of immediate-early genes (IEGs), which in turn leads to increases in cellular proliferation and protein synthesis. Additionally, the

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cellular effects include dynamic neutrophilic reactions and a transition from degenerative to restorative phase. Negative pressure tends to strongly prevent the denaturation of collagen fibrils in the zone of stasis of a burn, and other acute tissue injuries. In simplest terms, the appropriate use of negative pressure wound therapy in both acute and chronic wounds potentially results in the rapid closure of virtually all types of burns. It must be recognized that while these effects are independent of the wound contact layer used, the fullest manifestation of the aforementioned must always be juxtaposed against numerous factors including the phase of wound healing when applied, the duration of the therapy, periodicity of therapy, and pressures used among a plethora of others. The fullest effect of this therapy continues to be discovered but in simplest terms can be summarized now for convenience: • • • • • • • •

• • • •

Increases blood flow in those vessels that are already present. Promotes growth of new blood vessels (angioneogensis), providing a permanent supply of fresh blood. Promotes formation of granulation tissue. Brings oxygen and nutrients into the wound, which promote healing. Removes edema so well that it can even shrink tissues that have become grossly swollen with lymphedema. Removes lactic acid and subsequent reduction of pain. Maintains a moist wound bed. Creates compression (as well as suction), which pushes back on the blood vessels and thereby decreases the amount of edema and/or serum leaking out of the vessels and into the wound. Kills bacteria, decontaminating the wound. Protects collagen from being denatured. Brings fresh supplies of growth factors into the wound. Brings tissues together (coaptation).

23.5

The search for the perfect negative pressure technology

Even as this chapter was being developed, the author identified new additions to the commercially available negative pressure products and technologies. Negative pressure devices based on mechanical rather than electrical power were introduced, a cannisterless system in which the dressings rather than the pump retain and contain the drainage, and dressings based on the concepts of Bier which provide negative pressure to the wound in a non-contact, encompassing fashion (Miller Technique) rather than directly in contact with the wound surface.

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The question is not whether the current incarnations of negative pressure provide a desired effect and outcome. Rather, it is the realization that evolution is necessary for survival, be it of living organisms or technology. A therapy that currently meets the needs of a given population will likely in the future, need to adapt or become replaced by more advanced, better adapted incarnations. The identifying of future trends for a given technology is both extraordinarily difficult and yet immoderately simple. It is not so much as attempting to be a negative pressure Nostradamus but rather looking in simplest terms at the problems, misconceptions, and holes in the explanatory theories and clinical practices of the current technology and devices and then identifying potential solutions. The potential development of these devices is left then up to the imagination, intelligence and drive of potential inventors who support the clinical practice of wound healing. One of the most basic pathophysiologic concepts is that of activation and relaxation. All living things from the microscopic to the macroscopic level must have a period of quiescence to balance out that of activation, the chance to replenish, rejuvenate and restore the homeostasis. Human physiology in all aspects is based on these two opposite but equal aspects. The respiratory activation and relaxation of the diaphragm, the relaxed refilling and contractile ejection of the cardiac contents, the peristaltic ebb and flow of food down the gastrointestinal tract and urine down the ureters; one cannot identify a physiologic process in which activity is not matched with an approximately equal period of inactivity. The autonomic nervous system is itself based on this concept. Imagine if you will, an attempt to inhale to a maximum without exhalation or attempting to hold a given muscle group in perpetual flexion. There are built in physiologic safeguards which assure that for every action, there are equal and opposite reactions to maintain homeostasis but more to allow for maximal effect of a given physiologic process. Even the most robust bodybuilder must take time off after their workout to allow for their body to reap the benefits resulting in muscular hypertrophy. In contrast, the current theories of negative pressure act in complete opposition to this normal physiology. The application of negative pressure to a given wound for periods of up to 72 hours with the only period of respite being the time needed to change the dressing is the penultimate of illogic. The fact that healing even occurs in the face of such discordance with the most basic of physiologic principles leads to the obvious conclusion that maximal affect would occur if periods of complete relaxation, replenishment and rejuvenation were allowed to occur. Indeed, there are examples in the negative pressure literature in which this theory is clearly practiced. Davydov et al. 1986 applied negative pressure for 20 minutes initially and then 2.5 to 3 hours per day for a periods of 5–6 days. In 1988 Davydov

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reported applying negative pressure to wounds for periods of 1 hour per day for 6 days. Kostiuchenok in 1986 used negative pressure in treatment times less than 10 minutes. Early US case reports of non-continuous therapies were published introducing the concept of periodic therapy (6–8 hours per day). It is acknowledged that the majority of negative pressure treatments take place in situations and locations that are at best infrequently, and at worst rarely, monitored by trained personnel in regards to the technology and more the machines and dressings. What this creates is a disconnect between what is actually taking place at the bedside and what is being reported to the clinician. How can an accurate decision be made regarding what parameters need to be set or changed on the machine and more, what actions need to take place. There are multiple scenarios in which a pump system fails or becomes problematic and while the ultimate solution is for the clinician to go to the bedside and assess the situation, their proximity makes what could be a simple solution, one of decisions made in absentia. While the current machines have alarms, alerts, readouts and recordings for events, both good and bad, there is still a disconnect regarding the ability of the clinician to identify a problem (or lack thereof) and institute a solution in “real time”. We can envision an elegant solution for which the currently available technology exists. It would be easy to construct a negative pressure system in which a symbiotic relationship between the dressing and the pump itself would provide real time information on multiple parameters including the chemical profile of the wound, information pertinent to the wound itself, wound drainage, the status of the dressing, functional parameters of the pump and an overall assessment of the numerous factors related to the wound and its treatment. The addition for the off site clinician to have the numerical and visual data needed to make clinical assessments and changes would be a welcome change from the current second hand interpretation based on all too frequently misinterpreted or misunderstood findings. The late night phone call from a harried nurse or exasperated family member could be resolved by simply looking at the downloaded information on a nearby computer screen. Further, this data would be an integral part of the medical record. Smart dressings and smart pumps appear to be a potential consideration for the future. Current practice of wound care mandates that the dressings are changed at standardized intervals. The accepted regimen for the currently available wound contact dressing negative pressure systems falls between every other and every third day. The rationale for this does not appear to have a scientific basis, but rather appears to be based on the concerns that negative pressure dressings do not differ appreciably from “regular” wound dressings. The buildup of materials beneath dressings including increased bioburden, buildup of exudates, and the concept of a dirty wound that needs

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to be “cleaned to promote healing”, as well as the obsessive need to view the wound to assure that all is well have led to a self-perpetuating, unsupported medical practice. There does not appear to be any definitive documentation or evidence which provides a rationale for a specific interval for dressing changes that maximizes healing. This failure of critical thinking has led to a mentality of dressing change paranoia based solely on emotionality and presumption. In their article, “Stretching” Negative Pressure Wound Therapy: Can Dressing Change Interval be Extended in Patients with Open Abdomens?, Stawicki and Grossman (2006) presented data suggesting that in certain conditions, the interval of NPWT dressing changes could be extended to 7 days and provide several citations to support this. However, they too fall prey to the prevalent “standards” instead of science when they further stated “While it is appropriate to change NPWT dressings frequently (i.e., every 12 to 24 hours) in the setting of severe and moderate wound contamination, it seems that dressing change regimens in the setting of minimally contaminated and clean wounds may be spaced farther apart than the currently recommended 24- to 72-hour interval.” While it should be noted that this statement had no supporting citations, nonetheless, the authors are to be credited with considering prolongation of the dressing change interval. Of course, the obvious questions are to define “minimally” vs. “moderate” vs. “severe” wound contamination but more, recognizing that negative pressure removes contamination regardless of the quantity, even recognizing that the larger the amount, the “longer” it may take to remove, does the amount of contamination really present a consideration into the question of what interval the dressing needs to be changed? The actual defining point for long-term use of the same negative pressure dressing is mitigated by the dressings themselves. Using a biodegradable wound dressing would be problematic, since its beneficial effect on the system would be reduced as the structure and function concurrently degrade. For certain current dressings, the potential for adverse effects due to ingrowth of the granulation tissue into the wound dressing, mandates that the dressing be removed before potentially catastrophic effects (bleeding, pain, avulsion of needed tissue, fragmentation and subsequent retention, etc.) occur. Another concern is the ability to monitor the wound during active therapy. The most obvious solution therefore is to consider a dressing that does not degrade, does not allow for the problems related to in-growth, and allows for “real-time” visualization of the wound. Other considerations need to include the ease of application, ease of removal, ability to maintain a seal under various conditions (patient movement, patient position, connecting tubing traction), alterations in the skins condition (sweating, friability, etc.), and in keeping with our basic tenets, the ability to self-clean.

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Clearly, there are an infinite number of revisions, additions and deletions in the currently available negative pressure technologies and products with the potential to improve (and detract from) the plethora of wound healing parameters. It is imperative to recognize that no two patients are alike and more, no two wounds are alike. The current “one size fits all” mentality that has been pervasive in the marketing and institution of negative pressure therapy is, at best, inappropriate and, at worst, illogical and without scientific basis. The most basic tenet must be that the more adaptive the therapy is to the specific patient and wound characteristics, the greater the likelihood of a faster, more cost-effective, more patient-friendly wound healing outcome.

23.6

Conclusions

From the earliest incarnations of these negative pressure wound therapies, the goal has clearly been to create technologies and devices that had the widest applicability. Despite this, NPWT has become the wound care embodiment of the popular phrase; “one size fits all.” The focus has clearly been away from the patient-centered concept but rather towards mass utilization through identification of those wounds and conditions that were in some way, potentially amenable to NPWT and then, making sure that the wound ultimately fit the therapy. If negative pressure wound therapy is to achieve its highest potential, the focus must be on its ability to adapt to the infinite number of wound variables that create non-healing wounds. It is not so much as looking only for square pegs to put in a square hole or changing the peg to fit the hole but rather to have holes that change to meet the needs for every, and any, given peg. With the most basic understanding that every patient and more, every wound is different, then critical thinking mandates that flexibility and adaptability of therapeutic intervention is the most fundamental way to treat and thus, heal wounds. The perpetual attempt to bluntly force or modify unique pegs so they fit into standard holes can only result in frustration, financial waste, and the highest likelihood of violating the most basic of medical tenets “primum non nocere” (First do no harm). The ultimate solution is to have a technology that has mass applicability with adaptive specificity.

23.7

Acknowledgement

The assistance of Eric Newgent DO, MS of Baraboo, Wisconsin, USA is acknowledged for his research skills and insights into the organization of the chapter to assure it provides a concise, even-handed presentation on the topic.

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References and further reading

Ahmadi A (2008), ‘The efficacy of wet-cupping in the treatment of tension and migraine headache’, Am J Chin Med, 36, 37–44. American Heritage Stedman’s Medical Dictionary, Houghton Miffin Company, Available at: http://dictionary.reference.com/browse/Poiseuille’s law Accessed October 5, 2010. Argenta L C (1997), ‘Vacuum-assisted closure: a new method for wound control and treatment: animal studies and basic foundation’, Annals of Plastic Surgery, 38, 563–76. Argenta L C, Morykwas M J (1997), ‘Nonsurgical modalities to enhance healing and care of soft tissue wounds’, J South Orthop Assoc., 6, 279–88. Avery C (2000), ‘Negative pressure wound dressing of the radial forearm donor site’, Int J Oral Maxillofac Surg, 29, 198–200. Bagautdinov N A (1986), ‘Variant of external vacuum aspiration in the treatment of purulent diseases of the soft tissues’, Current Problems in Modern Clinical Surgery: Interdepartmental Collection, edited by V. Ye Volkov et al. (Chuvashia StateUniversity, Cheboksary, USSR), 94–96. Baharestani M (2009), ‘V.A.C. Therapy in the management of pediatric wounds: clinical review and experience.’ Int Wound J, 6 Suppl, 1–26. Bailey N (1742), English Dictionary, London, printed for R. Warf, A. Ward, J. and P. Knapton, and T. Longman. Barker D E (2000), ‘Vacuum pack technique of temporary abdominal closure: A 7-year experience with 112 patients’, The Journal of Trauma–Injury, Infection and Critical Care, 48, 201–7. Bartels C G (2001), ‘The vacuum sealing technique. A new procedure to cover soft tissue defects after resection of leiomyosarcoma’, Hautarzt, 52, 653–657. Best Practice Statement: Gauze-based Negative Pressure Wound Therapy. Wounds UK, Aberdeen, 2008. Bryan S (2009), ‘Case study: negative pressure wound therapy in an abdominal wound.’ Br J Nurs, 18, S15–6, S18, S20–1. CDC (1999), ‘Ten great Public Health Achievements – United States 1900–1999’, MMWR Morb Mortal Wkly Rep 48, 241–3. Capobianco CM (2009), ‘An overview of negative pressure wound therapy for the lower extremity’, Clin Podiatr Med Surg., 26, 619–31. Carson S N (2004), ‘Healing skin grafts over chronic wounds with vacuum assisted closure and silver dressings’, Available at: http://www.silverlon.com/studies/ HSG001.pdf. Chariker M E (1989), ‘Effective management of incisional and cutaneous fistulae with closed suction wound drainage’, Contemporary Surgery, 34, 59–63. Coles, D R, Greenfield A D M (1956), ‘The reactions of the blood vessels of the hand during increases in transmural pressure’, J. Physiol, 131, 277–289. Contractor D (2008), ‘Negative pressure wound therapy with reticulated open cell foam in children: an overview.’, J Orthop Trauma, 22(10 Suppl), S167–76. Davydov Y A (1986), ‘Vacuum therapy in the treatment of purulent lactation mastitis’, Vestnik Khirugii, 137, 66–70. Davydov, Y A (1980), ‘Concepts for the clinical-biological management of the wound process in the treatment of purulent wounds by means of vacuum therapy’, Vestnik Khirugii, Jul. 7, 132–136, and 8 page English translation thereof.

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Davydov Y A (1988), ‘The bacteriological and cytological assessment of vacuum therapy of purulent wound’, Vestnik Khirugii, 10, 48–52. Davydov, YA (1991), ‘Concepts for clinical biological management of the wound process in the treatment of purulent wounds using vacuum therapy’, Vestnik Khirugii, 2:132–135. Ennis W J (2008), ‘Push–pull theory: Using mechanotransduction to achieve tissue perfusion and wound healing in complex cases’, Gynecologic Oncology, 111, Supplement 1, S81–S86. Fleck C A (2004), ‘When negative is positive: A review of negative pressure wound therapy’, Extended Care Product News, 92, 20–5. Fleischmann W (1993), ‘Vacuum sealing as treatment of soft tissue damage in open fractures’, Unfallchirurg, 96, 488–92. Gray M (2004), ‘Is negative pressure wound therapy effective for the management of chronic wounds?’, J Wound Ostomy Continence Nurs, 31, 101–5. Herndon, (2002), Total Burn Care, 2nd Edition, New York, W.B. Saunders Publishers. Ingber D (1991), ‘Extracellular matrix and cell shape: potential control points of inhibition and angiogenesis’, J Cell Biochem, 47, 236–41. Jing C (2000), ‘An experimental study on systemic inflammatory response syndrome induced by subeschar tissue fluid’, Burns, 26, 149–55. Johnson T (1634), The Works of that Famous Chirurgion Ambrose Parey, London, printed by T. Cotes and R. Young. Kostiuchenok (1986), ‘Vacuum treatment in the surgical management of suppurative wounds’, Vestnik Khirugii 137:18–21. Meyer, W. Schmieden, V (1908), Biers Hyperemic Treatment In Surgery, Medicine, And The Specialties: A Manual of Its Practical Application, W.B. Saunders Company. Miller M S (2005), ‘Using negative pressure for wound therapy’, Podiatry Management, 24, 121–8. Miller M S (2006), ‘Treating wound dehiscence with an alternative system of delivering topical negative pressure’, Journal of Wound Care, 15, 321–4. Miller M S (2006), ‘Treating a non-healing wound with negative pressure wound therapy’, Advances in Skin & Wound Care, 19, 202–5. Miller M S (2006), ‘Negative pressure wound therapy: An option for hard to heal wounds’, Nursing Homes/Long Term Care Management, 55, 56–9. Miller M S (2008), ‘Negative wound pressure therapy – alternatives to VAC’, Journal of Wound Technology, 1, 15–20. Miller M S, Bybordi F (2009) ‘Negative pressure Darwinism: survival of the fittest paradigm’, Wounds, 7, 192–7. Miller M S, Lowery C (2005), ‘Negative pressure wound therapy – a rose by any other name’, Ostomy Wound Management, 51, 44–9. Philbeck T E Jr. (1999), ‘The clinical and cost effectiveness of externally applied negative pressure wound therapy in the treatment of wounds in home healthcare Medicare patient’, Ostomy Wound Manage, 45, 41–50. Pollak A N (2008), ‘Use of negative pressure wound therapy with reticulated open cell foam for lower extremity trauma’, J Orthop Trauma., 22(10 Suppl), S142–5. Senchenkov A (2006), ‘Application of vacuum-assisted closure dressing in penile skin graft reconstruction’, Urology, 67, 416–19.

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Stawicki S P, Grossman (2006), ‘Comparative studies of combination therapy Silverlon®/VACTM vs. VACTM alone’, Available from: http://www.silverlon.com/studies/ vac_dressing_combination.html. Stawicki S P, Grossman M (2007), ‘ “Stretching” negative pressure wound therapy: can dressing change interval be extended in patients with open abdomens?’, Ostomy Wound Management, 53, 26–9. Steenvoorde P (2006), ‘Negative pressure wound therapy to treat peri-prosthetic methicillin-resistant Staphylococcus aureus infection after incisional herniorrhaphy. A case study and literature review.’, Ostomy Wound Manage, 52, 52–4. Sumpio BE (2008), ‘Role of negative pressure wound therapy in treating peripheral vascular graft infections’, Vascular, 16, 194–200. Svedman, P (1979), ‘A dressing allowing continuous treatment of a biosurface’, IRCS Medical Science: Biomedical technology, Clinical Medicine, Surgery and Transplantation, 7, 221. Svedman, P. (1983), ‘Irrigation treatment of leg ulcers’, Lancet, 322, 532–34 Svedman, P. et al. (1986), ‘A dressing system providing fluid supply and suction drainage used for continuous of intermittent irrigation’, Annals of Plastic Surgery, 17, 125–33. Torbrand C (2008), ‘Sympathetic and sensory nerve activation during negative pressure therapy of sternotomy wounds’, Interact Cardiovasc Thorac Surg., 7, 1067–70. Tse, (2006), American Negative Pressure Wound Therapy, Beijing, PRC, Scientific and Technical Documents Publishing House. Available from: http://www.csttechnology.com/woundcare.html Ubbink D T (2009), ‘Negative pressure therapy for surgical wounds’, Ned Tijdschr Geneeskd. 153, A365. Vestnik Khirurgii (1991), The Kremlin papers: A Collection of Published Studies Complementing the Research and Innovation of Wound Care 1986–1991. Blue Sky Medical. Vikatmaa P (2008), ‘Negative pressure wound therapy: a systematic review on effectiveness and safety’, Eur J Vasc Endovasc Surg., 36, 438–48. Yusupov Yu (1987), ‘Active Wound Drainage’, Vestnik Khirurgi, 138, 7.

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24 Debridement methods of non-viable tissue in wounds D. J. L E A P E R, Imperial College London, UK and Cardiff University, UK, S. M E AU M E, Hôpital Charles Foix, France, J. A P E L Q V I S T, University Hospital of Skåne, Sweden, L. T E O T, Hopital LaPeyronie, France and F. G O T T R U P, Copenhagen Wound Healing Centre, Denmark

Abstract: Debridement is a key element of wound care, particularly wounds healing by secondary intention. These wounds are often chronic with one or more underlying aetiologies, prone to contamination and infection, and delayed healing related to excessive exudate and necrotic material. By removing adherent dead or foreign material, with its associated extra bioburden, the risk of critical colonisation and infection are minimised, thereby reducing the need for antimicrobial therapy. Recognition of the need and extent of debridement should be part of any wound treatment plan. There are several methods of debridement ranging from the crude use of wet-to-dry dressings to more sophisticated use of negative pressure therapy. Whichever method is used needs involvement of the whole multidisciplinary team to enable optimal benefit. Key words: debridement, chronic wounds, pressure ulcer, diabetic foot ulcer, venous ulcer, infection, bioburden, colonisation, biofilm, wound dressings, antimicrobials, hydrotherapy, larva therapy.

24.1

Introduction

Debridement of wounds, using a sharp, surgical technique is a skill that is easily acquired but that is often avoided because of inexperience, or a fear of breaking ‘rules’, or even litigation. Debridement is a major function of the surgeon who should be a member of a multidisciplinary team. Without this support, front line wound care practitioners, who can clearly see the need for debridement, may have to turn to one of many other methods or technologies because someone with the skill or authority is not available. These methods can give as complete a result and be quite sophisticated, such as high pressure irrigating devices, but are slower and may need several sessions (the penalty for increased safety!). The military use of wet-to-dry dressings is unacceptable because of its related pain and the use of chemical debridement, particularly the use of hypochlorite disinfectant solutions, has 606 © Woodhead Publishing Limited, 2011

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been banned by many. Many other methods exist as adjuncts and involve enzymatic preparations; autolytic hydrogels and hydrocolloids; and reintroduction of time-honoured methods, such as larvatherapy; and newer technologies, such as negative pressure therapy. The removal of bridging necrotic tissue from wounds with its contained bioburden and risk of delayed healing, infection, sepsis, organ dysfunction and risk of death may seem obvious but wound debridement has, surprisingly, been subjected to systematic review and analysis. The inevitable conclusion that ‘more research is needed’ is unlikely to be responded to, based on the reality, cost and impracticality of involving the huge diversity and aetiology of wounds that may need debridement. This disparity of opinion between ‘scientists’ who do not treat wounds, and ‘clinicians’ who do, is likely to worsen.1,2 Nevertheless, debridement is a key part of advanced wound repair therapies and this chapter will address its indications, methods and benefits. In this chapter, the practical clinical and the supporting scientific aspects of debridement have been addressed. Much of the evidence is poor and further clinical studies are needed in what is a key part of modern wound management.

24.2

Background

The term debridement is derived from the 19th-century French word ‘desbrider’, which means to unbridle a horse. It was recognised in military practice that soft tissue injuries could heal if tissues on the surface of a shrapnel wound were excised, rather like removing a horse’s saddle. The exposed healthy tissue beneath was then less likely to become infected and healing by secondary intention was thereby encouraged with a considerable reduction in complications. Debridement is not therefore the removal of debris but a much more active process of removal of dead or necrotic tissue, infected material or foreign material from the surface or depths of a wound. Wounds of all aetiologies may present this need for debridement from an incised wound which has become infected with necrotising fasciitis (Meleney’s synergistic hospital gangrene, Fournier’s gangrene), to a pressure sore which presents with an adherent ‘cap’ of dead tissue. The aim is to lay the foundation for a clean wound bed, a term which originated from plastic surgery where the wound is optimally prepared to take a split thickness skin graft; or in this more general context to prepare the wound bed, together with control of oedema, minimisation of colonisation/infection and an optimal blood supply, to heal an open wound by secondary intention. In 1995 the European Tissue Repair Society (ETRS) led a working party consensus on wound debridement.3 It struggled to agree on some terms, although scab (comprising mainly an adherent coagulum of platelets and fibrin) was considered to be physiological and an eschar (comprising

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24.1 Black toe associated with peripheral vascular disease with a line of demarcation.

adherent dead tissue and foreign material) was pathological. Both scab and eschar impede healing by secondary intention in the optimal moist environment and may need removal but an eschar is more likely to be associated with infection. Gangrene, necrosis, ischaemia, devitalised are further terms which have specific uses. They may be difficult to recognise; dead tissue may present with a line of obvious demarcation, such as the black toe of a patient with peripheral vascular disease or after an embolus; sometimes tissue viability is hard to delineate and need recognition using supra-vital dyes, biopsy, measurement of transcutaneous tissue oxygen tension, Doppler ultrasound or near infra-red spectroscopy as examples. Recognition of burn depth may present similar difficulty prior to tangential excision and grafting. Common to many of these conundrums is the need to remove scab or eschar before any accurate assessment of a wound can be made for a treatment plan. The recognition of dead tissue remains essentially clinical; specific tests have not become usable in daily clinical practice. There are exceptions when the clinical diagnosis is very easy: a demarcated black toe may be left to auto-amputate (Fig. 24.1) and an adherent painless dry pressure sore of the heel in a patient with terminal illness can be managed expectantly (Fig. 24.2).

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24.2 Black heel (dry, painless, no smell or exudate) not needing debridement in a terminally ill patient.

24.3

Complications of non-viable tissue in wounds and the need for debridement

Non-viable tissue, and anaerobic conditions, in particular, encourage infection, and infection is the single most potent factor which delays healing in wounds of all types. This may be overt secondary infection by facultative skin pathogens such as Staphylococcus aureus, which may lead to death of further tissue needing debridement (‘wet gangrene’; as opposed to ‘dry gangrene’ where there is non-infective mummification). Anaerobic conditions, particularly in immunocompromised patients with type I diabetes or who have been on long-term steroids, may encourage the development of gangrene, which may or may not be gas forming. This may be due to a synergistic ‘soup’ of organisms but in military injuries where there is considerable tissue damage with foreign body material, including clothing and soil, gas gangrene may be caused by Clostridium perfringens. In many of these situations radical debridement of tissue, or limb amputation, may be needed to save life together with intensive care and organ support. The larger the bioburden is, within or colonising a wound (acute or chronic), relates directly to infective complications. Organisms can act in synergy, depending on aerobic or anaerobic conditions and is facilitated by the presence of non-viable tissue and contamination. If this is not removed by early, complete and repeated debridement, the risk of local invasive infection (cellulitis, lymphangitis, lymphadenopathy, and bacteraemia) becomes greater. Depending on the virulence and size of the bacterial load and host response, this may have systemic effects mediated through

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systemic inflammatory effects, which may be excessive or inappropriate leading to sepsis, sepsis syndrome and septic shock, multiple organ dysfunction syndrome (MODS) and multiple organ failure (MOF), and death: Systemic Inflammatory Response Syndrome (SIRS) consists of any two of • • • •

>38°C >90 beats/min >20 breaths/min >12 × 109/l

Pyrexia Tachycardia Tachypnoea WBC

(or <36°C) (or PaCO2 <4.2 kPa) (or <4 × 109/l)

Sepsis is SIRS with a documented source of infection (in this case an infected wound). Sepsis-like syndromes, without a documented infection, can be seen in burns, extended injury and after haemorrhage and ischaemia. Severe sepsis (or sepsis syndrome) is sepsis with evidence of MODS. Septic shock is severe sepsis refractory to fluid therapy, requiring inotropes to maintain mean arterial pressure. MODS/MOF includes: • • • • • • • •

acute tubular necrosis (oliguria, rising urea, creatinine) acute respiratory distress syndrome (ARDS with PaO2/FiO2 < 30; PaO2 < 9.3 kPa; increasing respiratory non-compliance) haemodynamic instability (systemic vascular resistance <800 dyne/s/ cm3; lactate >1.2 mmol/l) gastrointestinal complications (stress ulcers, ileus, inflammation) hepatic complications (rising bilirubin, albumin, AST) haematological complications (falling platelets, disseminated intravascular coagulopathy) metabolic complications (including insulin resistance) neurological complications (measured with Glasgow coma score).

The mediators involved in this cause the release of a complex cascade of endothelial, macrophage and white cell, and platelet-mediated pro-inflammatory cytokines, nitric oxide, reactive oxygen species, arachidonic acid derivatives and eicosanoids, kinins and other proteases, which lead to further tissue damage if not controlled. The stimulus to this response is tissue damage and release of bacterial exo and endotoxins. Management of systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), and multiple organ failure (MOF) requires multidisciplinary treatment with wound care, intensive care and organ support with anti-infective strategies using antibiotics and topical antimicrobials.

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24.3 Necrotic pressure sore with infection and sepsis.

24.4 Hernia wound infected with Meleney’s synergistic gangrene of the abdominal wall needing urgent extensive debridement.

These syndromes are also seen in open and chronic wound care if early and complete debridement is not undertaken. It has been long recognised that there is a ‘pre-infective’ condition in open wounds, which more recently has been packaged in a continuum of contamination-colonisation-‘critical colonisation’-local infection-systemic infection (and sepsis). An inadequately debrided stage IV pressure sore (Fig. 24.3) can lead to MOF and death just as rapidly as an infected acute surgical wound can do and needs debridement (Fig. 24.4). Inadequately or incompletely debrided wounds promote bacterial growth as a culture medium and risk colonisation, and infection, with antibiotic resistant organisms.4 Dead tissue or even scab may splint contraction in a healing open wound (Fig. 24.5), even without the

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24.5 Dry gangrene at the wrist splinting healing by contraction, following extravasation of IV drugs.

presence of infection and of course the dry environment associated with exposure may delay epithelialisation. Of course antimicrobials are optimally used in conjunction with debridement. However, antibiotics cannot treat dead tissue, particularly in the diabetic foot, where prolonged treatment risks development of resistance and emergence of unwanted organisms. Whenever antibiotics are used, they should, as far as possible, be reserved for narrow spectrum use based on culture and sensitivities; only when there is spreading or life-threatening infection should broad spectrum antibiotics be used empirically and based on ‘best-guess’ sensitivities. The topical antimicrobials have a major role in debridement and as a barrier to colonisation. Some of the disinfectants, such as the hypochlorites, have been used as very effective debriding agents with success in wound bed preparation prior to split thickness skin grafting. They are, however, toxic to healing tissues and the less toxic antiseptics may serve as well when used as an adjunct to alternative methods of debridement. Military injuries present further challenges. High velocity injuries cause intense tissue damage in addition to the ricocheting and tumbling effect of shrapnel and specialised bullets. With high velocity even a small mass can cause diffuse tissue injury based on the release of kinetic energy (from the formula ½ mv2). Behind the entry into tissues is a sucking, cavitation effect which leaves a large amount of dead tissue and entry of clothing and environmental material such as soil. This gives an anaerobic tissue with foreign body material and a rich source of sores which is ripe for the development of Clostridium perfringens gas gangrene. This is the wound which needs the classic ‘debride-ing’ and removal of all underlying non-viable tissue and

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24.6 Thigh wound after debridement and later successful healing by secondary intention.

contamination. Such a wound must never be closed by primary suture (Fig. 24.6).

24.4

Presence of biofilm

In many circumstances the material removed by debridement, particularly from chronic open wounds, contains biofilm associated with contaminating or colonising organisms. Many practitioners mistake shiny, fibrinous wound exudate as biofilm but there is no diagnostic yet available which supports this assertion; this diagnostic test will come. It is likely that many open wounds have superficial biofilm but this can only be recognised using sophisticated tests such as confocal or scanning electron microscopy, denaturing gel gradient electrophoresis (DGGE) or polymerase chain reaction (PCR) techniques. The logic for including the removal of biofilm with general wound debridement is obvious and devices for atraumatic and techniques for repeated removal of wound surface biofilm, rather like regular brushing of teeth and biofilm, are likely to be devised and implemented when biofilm is known to be present. The persisting presence of biofilm allows colonising bacteria to remain as persister cells, being capable of planktonic growth leading to infection, when conditions are optimal for their growth. Bacteria in surface biofilm, planktonic or persister, can also promote activation of white cells and macrophages in the wound bed with inappropriate inflammatory responses which delay healing. These include excessive release of proteases and persistence of nitric oxide and reactive oxygen species from stimulated macrophages at the wound edge. Whilst in their protective biofilm microenvironment, these bacteria can resist other host defences as

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well as the action of topical antiseptics and systemically administered antibiotics.

24.5

Organisation of debridement

Debridement should be undertaken with a multidisciplinary team, although the techniques involved can be learned by all members of the team. The diabetic foot ulcer is a classic example: debridement can range from the excision of excessive callus, which increases the risk of underlying tissue damage, bleeding and infection; to early debridement of superficial dead tissue and release of pus; through to the specialised early recognition of underlying osteomyelitis and dead bone which needs skilled surgical interventions to reduce the need for extensive amputation. In addition there is the need for investigation of and improvement of arterial inflow when there is associated peripheral vascular disease, skilled dressing care and offloading methods as well as the correct decision to use specific antimicrobials. This conjunction of physician, surgeon, endocrinologist, nurse, podiatrist, microbiologist, and radiologist applies to all wounds needing debridement and reduces the risk of misinterpretation of the ‘unusual wound’. Debridement of a malignant ulcer could lead to disastrous haemorrhage or the risk of dissemination; malignant ulcers tend to have an exophytic edge and may present with ‘clean’ granulation tissue which bleeds readily. A biopsy, which can often be taken with no or a local anaesthetic/eutectic mixture of local anaesthetic (EMLA) cream, ideally from the growing edge, can confirm the diagnosis.

24.6

Timing and types of debridement

Radical and complete debridement allows removal of all necrotic or ischaemic tissue and foreign body material with any excessive host response to colonisation and infection, such as fibrinous material and coagulated exudate. All these undesirable factors may, in addition to delaying healing, promotion of an acute inflammatory response and acting as a bacterial growth medium, act physically to splint wound closure. It is logical therefore that this material is removed at an early stage with the added benefit that a complete assessment can be made of the wound beneath to aid further management and enable successful healing. Where debridement is undertaken depends on available facilities and the types of anaesthesia which can be used. Many methods are available but the scientific measurement of pain is not easy.5 Debridement can be undertaken with patient consent in regular, pre-ordained sessions at the bedside or in a treatment room with less need for anaesthesia, or in an operating theatre with full general anaesthesia to allow completion in one session (Fig. 24.7a–d).

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(b)

(c)

(d)

24.7 Several wounds needing different anaesthetic approaches for debridement: (a) diabetic ulcer (anaesthetic may not be needed because of neuropathy but this should never be assumed without testing!); (b) wound edge necrosis after amputation needing superficial debridement (eutectic mixture of local anaesthetic (EMLA) cream or local anaesthetic soak can be used68); (c) surgical wound infection and superficial dehiscence after debridement prior to and after secondary suture; (d) gangrenous tendon in leg ulcer (needs operating theatre and general/epidural anaesthetic).

The opportunity to undertake randomised clinical trials has been grasped but the Cochrane Collaboration, as expected, assert that more research is needed.6,7 Because of the reality and diversity of clinical practice, let alone the prohibitive costs of randomised, controlled clinical trials, this is not likely to happen.8 There are several methods of debridement: i. Surgical ii. Mechanical (wet-to-dry)

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616 iii. iv. v. vi. vii. viii. ix. x.

Advanced wound repair therapies Chemical (disinfectants such as hypochlorites) Enzymatic Hydrotherapy and irrigation (including ultrasound) High pressure therapy (Versajet, Curapulse, etc.) Autolytic dressings (foams, films, hydrogels and hydrocolloids) Debridement using honey Larvatherapy Negative pressure therapy (NPT)

24.6.1 Surgical debridement This requires a degree of surgical skill with scalpel and scissors and a working knowledge of anatomy. Both can be learnt and some institutions may insist that a practitioner is certified to do this. Instruments used, scalpel, forceps and scissors, must be of adequate quality when undertaken at the bedside or in the patient’s home. There is no place for disposable plastic forceps or dressing scissors, which are not suitable for purpose. Appropriate anaesthesia is the key to success and should be a single, rapid and complete procedure. Anatomical knowledge is needed for areas at risk including superficial vessels (such as the radial artery in Fig. 24.5), nerves and joint spaces. If debridement is undertaken by a novice, they must learn to know when to stop, always have help and know what to do if there is a complication such as excessive haemorrhage. Bleeding is the most difficult as it can obscure vision; the procedure should be ended and bleeding arrested. An assistant can also wipe the patient’s (or the operator’s) brow!

24.6.2 Mechanical debridement (wet-to-dry) This option may be used by less confident practitioners, particularly in military situations. It causes pain and is unsuitable but when a gauze dressing becomes dry and adherent to a wound, its removal gives a ‘complete’ debridement and exposure of healthy bleeding tissues. This is a procedure which is NOT recommended by the authors.

24.6.3 Chemical debridement In the military arena chemicals, which are really disinfectants, have been used extensively to debride wounds. Most of these are the hypochlorite solutions with eponymous names such as EUSOL, Dakin’s and Milton. Because they were given a bad press, because of the experimental evidence of toxicity to healthy tissues, they became excluded from clinical practice.9,10 They are probably very efficient at sterilising a wound surface with their chemical debriding action and are used by some plastic surgeons for wound

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bed preparation prior to split thickness skin grafting. Their prolonged clinical use is probably damaging, bearing in mind there are so many other less toxic methods for debridement, and delays wound healing.

24.6.4 Enzymatic debridement This has been the subject of several trials and systematic reviews11–14 and with the conclusions that more research is needed. The non-specific enzymatic combination of streptokinase-streptodornase introduced several decades ago has been replaced by several preparations of bacterial-derived collagenase for the debridement of burns and chronic skin ulcers. The method is slower than surgical, mechanical or chemical debridement but is usable by a wide range of lesser-skilled practitioners and it appears to be safe and without complications. Many enzymatic preparations are inactivated by antiseptics, such as silver-based antiseptics.

24.6.5 Hydrotherapy and irrigation techniques for debridement Irrigating a wound with a continuous or intermittent flow of fluid delivered under mild pressure, between 8 and 12 pounds per square inch (psi), is sufficient to remove devitalised tissue and bacteria in the wound.15,16 Some whirlpool hydrotherapy systems use highly pressurized saline delivered, via a nozzle, at between 12,800 and 15,000 psi.17 Whirlpool debridement is used for large wounds in which adherent material has to be detached from the wound bed. These devices allow successive slices of tissues to be harvested, which are then immediately extracted from the wound bed using an appropriate aspirating device which prevents contamination of the surrounding normal tissues. Some practitioners, mainly in central Europe and Germany, have described a combined whirlpool device using active antiseptic solutions such as Lavasept or Prontosan. These biguanides are used at a low concentration (0.1%). The facility to irrigate wounds postoperatively, using a modified negative pressure therapy device, has yet to be confirmed for its effectiveness in reducing numbers of micro-organisms and leading to enhanced wound closure.18 The use of ultrasound may facilitate debridement.

24.6.6 High pressure therapy (hydrojets) for debridement Wound cleansing has always been used for wound care and now high pressure hydrojets have been added to the armamentarium. Cleansing can be undertaken using water projected using a hand syringe, to remove devitalised tissues located in undermined areas, but pressure cannot be regulated. The

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pressure is usually low, but the risk of particulate matter splattering and contaminating the surroundings is appreciable. There is potential for more powerful hydrojet devices than simple hand held syringes which give a more controlled water jet. DebritomTM is a high pressure device which has been developed to remove all devitalised areas using a very powerful jet, using a compressor, with a hand piece to concentrate the pressure. This device needs a set of handpieces to be sterilised and re-used, limiting the cost to the machine and the sets of handpieces. Tents have been developed to protect the environment from projection of micro-organisms. This technique is still under evaluation, but an initial series has evaluated the level of pain, which remains moderate, the numbers of stages needed to completely debride the wound, and the adaptability and ambulatory use in the ward.19 VersajetTM was initially developed in Germany but has been used more widely in the USA and Europe. Versajet is a device which has a combination of three effects: debridement, aspiration and removal of slough and devascularised tissue. The handpiece is connected under sterile conditions to the aspiration machine. Based on the Venturi effect, removal of tissues is acheived without any contamination of the surrounding tissues which is a real advantage over the other comparable systems. However, the cost of the machine and of the disposable handpiece is high relative to other systems. However, the final arbiter should be the cost effectiveness of such a device which has been derived after a randomised controlled study. The cost effectiveness of Versajet has been evaluated with a positive input on the global wound management costs.20 The advantages of this technique are the selectivity of debridement and a reduction in blood loss, although this must be established from well-designed studies.

24.6.7 Autolytic debridement using dressings The hydrogels and hydrocolloids may be used to soften up adherent eschar for a few days to greatly facilitate sharp debridement.21 A Cochrane review has assessed the evidence for the effectiveness of types of debridement as a treatment for diabetic foot ulcers.22 Several randomised controlled trials (RCTs), evaluating methods of debridement in the treatment of diabetic foot ulcers, were analysed. The outcome criteria had to include either complete healing or the rate of healing. Five RCTs were identified; three assessed the effectiveness of a hydrogel as a debridement method, one RCT evaluated surgical debridement, and one RCT evaluated larval therapy. Pooling the results of the three hydrogel RCTs suggested that hydrogels are significantly more effective than gauze or standard care in healing diabetic foot ulcers (absolute risk difference 0.23; 95% CI: 0.10, 0.36). Surgical debridement and larval therapy showed no significant benefit in these small trials of the analysis. Other debridement methods such as enzyme prepara-

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tions or polysaccharide beads have not been evaluated in RCTs of patients with DFU. The reviewers’ conclusions were that there is evidence to suggest that hydrogel dressings increase the healing rate of diabetic foot ulcers but more research is needed to evaluate the effects of a range of widely used debridement methods.

24.6.8 Debridement using honey For thousands of years honey has been used in different products but the earliest documented evidence of its medical use for wound care is estimated to be from 2500BC.23,24 A renewed interest has been developing over the last 5–10 years during which time several new products impregnated with honey have been introduced. Honey is a complex natural substance that may contain as many as 600 components.25 It is a highly concentrated sugar product with a low moisture content and low acidity, which is likely to prevent growth of vegetative microbial cells. Additional antimicrobial activity may be based on contained enzymes, such as glucose oxidase, which converts glucose to hydrogen peroxide and gluconic acid.26,27 The antimicrobial effects are active against MRSA from infected or colonised wounds.23,28 While this broad antimicrobial spectrum of activity is well known the mechanism of the debriding action of honey is not. Honey has not been reported to have a direct proteolytic activity,29 so it may be assumed that honey removes attached slough, necrotic tissue and eschar by facilitating autolytic debridement. This is probably achieved through the high sugar content of honey, which causes an osmotic withdrawal of fluid (lymph) from the wound bed, that is replaced from the underlining circulation. However, honey has demonstrated a faster rate of debridement than using hydrogels, which suggests that honey may have a stimulatory action on proteases in the wound bed.29 The osmotic-induced outflow of lymph may also lead to extra oxygenation and an improved supply of nutrients to the growing cells, resulting in improved healing. Differences in the degree of activation of proteolysis between different types of honey may account for the differences in speed of debridement which has been reported. Another mechanism of action could relate to the effect honey has on biofilm production in wounds. Honey has been shown to prevent biofilm formation in vitro at concentrations below minimum inhibitory concentrations (MIC)24 and to interfere with the ability of biofilm-producing bacteria, such as Pseudomonas aeruginosa and Salmonella enteriditis to adhere to cell surfaces. The antimicrobial effect of honey may lead to other clinically associated effects other than debridement, such as reduction of malodour, improved granulation and epithelialisation, and decreasing oedema. Any wound containing slough, necrotic tissue or eschar presents an indication for honey debridement. However, each patient has to be evaluated

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for the most appropriate debridement type; in cases where autolytic debridement is indicated, honey may give a faster type of debridement, particularly if the wound is critically colonised or locally infected. Honey is well accepted by most patients, and few contraindications have been identified.30 Honey has shown less effect in treating arterial leg ulcers and scleroderma and may even cause deterioration. Some patients may experience initial pain and a stinging sensation, but only a few patients experience severe pain and mild analgesics may overcome this. Allergy to honey is rare and anaphylaxis has not been reported.23 ‘Raw’ honeys should probably not be used because they contain yeasts and bacteria31 but they may proffer a probiotic effect. The clinical evidence for the effectiveness of honey debridement is weak. In a trial involving leg ulcers the effect of manuka honey was compared to a hydrogel.32 The debriding effect of honey may be used in different types of acute wounds, such as infected surgical wounds, and also for chronic venous leg, pressure and diabetic foot ulcers. A range of products, such as dressings, are available which contain honey. The amount of honey typically applied depends on the size of the wound and the amount of exudate produced by the wound. Differences in speed of debridement may also relate to differences in how well the honey is kept in contact with the wound bed and to what degree it is diluted by exudate. In practice this means that the frequency of dressing changes that are needed depends on the amount of exudate produced in the wound. Despite the current weak clinical evidence for its use honey may become established as a usable alternative for debridement of wounds. Its advantages are that it is a natural existing and readily available inexpensive product, which has shown no development of resistance, and is well accepted by patients.

24.6.9 Larvatherapy (maggot debridement therapy) The beneficial effects of maggots in the process of wound healing have been known for centuries. Over the last ten years maggot debridement therapy (MDT) has been used in clinical practice, in Europe and the US, for the treatment of various types of severely infected wounds with successful healing results.33,34 It has been suggested that MDT debridement works through mechanical and chemical mechanisms.35,36 Mechanical debridement is effected by the specific mandibles or ‘mouth hooks’ of the maggots and their rough bodies which both scratch the necrotic tissue. Furthermore, maggots produce excretions (ES) which possess proteolytic enzymes that can dissolve the dead and/or infected matrix on the wound bed.36 The longer standing hypothesis was that the mechanical debridement aspect was the responsible mechanisms for the effectiveness of MDT, but current studies do not support this and offer evidence that the chemical mechanisms are the underlying aspects of its success.37 The excretions and secretions of

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maggots contain allantoin, sulfydryl radicals, calcium, cysteine, glutathione, embryonic growth stimulating substance, growth stimulating factors for fibroblasts, carboxypeptidases A and B, leucine aminopeptidase, collagenase and serine proteases (trypsin-like and chymotrypsin-like enzymes, metalloproteinase and aspartyl proteinase).38 A further in vivo study supports the theory that the direct mechanical action of free-range maggots is limited. In this research, larval therapy with free-range maggots and maggots in Biobags® was compared with hydrogel application and showed faster debridement with the maggots.39 Although the larvae in Biobags® needed 28 days to debride and free-range maggots only 14 days, both therapies were very effective in debridement compared to hydrogel, which took 72 days to clean the wounds. Maggot ES can prevent, inhibit and break down biofilms of various bacteria on commonly used prosthetic materials and thus, it may in future research, be shown to provide a new treatment for biofilm-associated infections of orthopaedic biomaterials.40 The maggots used in MDT are Lucilia sericata larvae and cannot cause myasis in humans because their actions are limited to necrotic tissue in the wound and spare healthy tissue. MDT can be used for acute and chronic wound infections. The reported success rates of their use vary from 80 to 90%.41 The clinical indication for improved wound healing by the use of maggots has been shown in diabetic ulcers, ischaemic leg ulcers, osteomyelitis, burn wounds, as postoperative treatment for necrotising fasciitis or for the prevention of (further) amputations.42–45 MDT is not indicated for open wounds in the abdominal cavity, because of the risk of organ damage, and other contra-indications are for pyoderma gangrenosum, septic arthritis, or patients having immunosuppressive therapy.46,47 Caution is also advised in treating wounds near to large arteries and veins. Currently there are two modes of application of MDT. First, freely crawling larvae can be applied to the wound bed covered by a gauze bandage to keep them captured (Fig. 24.8a). In the second mode maggots are enclosed in special Biobags® (Biomonde GmbH, Barsbüttel, Germany) which consist of a sterile Nylon gauze network, with a foam dressing containing a polyvinyl alcohol spacer48 (Figs. 24.8b and 24.8c). The network of the Biobag® is permeable and permits the migration of maggot excretions/secretions (ES) onto the wound. The bag facilitates the application of MDT and also the inspection of the wound bed during the treatment. The effectiveness of the MDT captured in bags or in free-range application seems to be equal,49 but in the case of complicated undermined cavity wounds free larvae may be preferable. A quantity of 5–10 maggots per square centimetre wound surface is advised for 3–4 days consecutively after which the bags containing maggots should be replaced in combination with lavage of the wound.49 Furthermore, it is necessary daily to keep the wound surface wet with physiological saline. However, it

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(b)

(c)

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24.8 (opposite) Use of larvatherapy: (a) Maggots free just after placement in the wound; (b) Maggots enclosed in a Biobag® before placement in the wound; (c) Maggots in a Biobag® after three days in the wound.

has to be borne in mind that excessive flushing can drown the larvae. Additionally, the covering gauze bandage should be changed daily to prevent odour and avoid the dressing to be filled with wound fluid, which could also drown the larvae. Sometimes there are concerns about the resistance expressed by patients to the utilisation of MDT, but in general patient acceptability has been found to be high.50 Information in the form of handouts and posters facilitates acceptance by patients and relatives. There are no severe side effects reported following MDT. Sometimes a tickling feeling of the crawling maggots is noted, but when the captured method is used there are fewer complaints about this sensation. Exceptionally, when treating patients with leg ulcers associated with ischaemia, an increase in pain may be reported when MDT is used.47 The origin of this reported pain during maggot application is not known, since the wound healing effect of maggots is not related to their direct crawling action on the wound surface. No allergic reactions have been reported. Future studies need to investigate the composition of maggot excretion and secretion (ES) and determine the effective substances which are responsible for the debridement, including the remodeling effect on the extracellular matrix components and the reduction of biofilm formation.

24.6.10 Topical negative pressure therapy See Chapter 23 by Miller.

24.7

Scoring the effectiveness of debridement

Scoring systems have been devised to assess whether debridement has been adequately performed. For example, three aspects of debridement were defined in a classification adapted to diabetic foot ulcers:51 presence of callus, undermining of the ulcer edge and necrotic tissue in the wound bed. A score of 0–2 was applied to each of these categories using the following criteria: 0 = debridement needed but not undertaken 1 = debridement needed and undertaken 2 = debridement not needed. A total, ranging from 0 to 6 could therefore be defined, with the highest number being the optimal score. This scoring system, termed as the

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debridement performance index (DPI) by its authors, evaluates both the adequacy of debridement and its quality. The authors validated this scoring system and determined its predictive value for wound closure after 12 weeks of observation by applying it to 143 patients with diabetic foot ulcers who had been treated in a clinical trial involving either standard therapy (n = 65) or the application of a bioengineered skin construct (n = 78). Each diabetic foot ulcer was evaluated using sequential digital photographs and calculation of the DPI score was started at day 0, before initiation of either treatment. Results showed that the lower the baseline debridement performance index the lower the incidence of ultimate wound closure at 12 weeks (p = 0.0276). Patients with a debridement performance index between 3 and 6 were 2.4 times more likely to heal their ulcers than those with a score of 0–2. After controlling for treatment type, the debridement performance index was found to be an independent predictor of wound closure (odds ratio 2.4: 95% confidence intervals 1.0–5.6).

24.8

Debridement in the diabetic foot

It has been claimed that every 30 seconds, a lower limb is amputated because of complications of diabetes and that 20–40% of total expenditure on the management of diabetes can be attributable to the diabetic foot.52 Of all amputations in diabetic patients 85% are preceded by a foot ulcer which subsequently deteriorates to a severe infection or gangrene.53,54 The complexity of diabetic foot ulcers necessitates an in-depth knowledge of the underlying pathophysiology and a multifactorial approach in which aggressive management of infection and ischaemia is of major importance and includes: • • • • • • • • • •

improve circulation remove oedema control pain treat infection improve metabolic control non weight-bearing wound management foot surgery improve general condition programme for support and implementation.

Debridement is an essential component in such a strategy to achieve healing. Key factors to recognize in the choice of debridement technique are infection, ischaemia and the extent of tissue loss/involvement: (i). Peripheral arterial disease (PAD), ischaemia and decreased tissue perfusion. The most important factors related to development of foot

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ulcers are peripheral neuropathy, minor foot trauma, foot deformity and decreased tissue perfusion.52–54 It is essential to distinguish between different types of ulcers, especially with respect to predisposing factors such as neuropathy or neuroischaemia/ischaemia.55–57 In large cohort studies in Europe neuroischaemic or ischaemic ulcers accounted for 50–58% of all diabetic foot ulcers admitted to specialist care and as a consequence PAD and neuropathy are frequently present in the same patient.55–57 An important observation relating to the consequence of PAD is that many diabetic individuals with ischemia do not have severe rest pain or claudication.52–57 For early recognition of the need for revascularisation a diabetic foot ulcer should be considered ischaemic until proven otherwise by extensive clinical examination and non-invasive vascular testing. In principle, no patient should undergo amputation or foot surgery without previous vascular assessment with a view to the possibility of vascular intervention52,54,58,59 not only to avoid amputation but also improve the possibility of healing when there is decreased perfusion. (ii). Diabetic foot infection. An infection in the diabetic foot is a limbthreatening condition and was the immediate cause for amputation in 25–50% of diabetic patients.52,54,60–63 Often there is a combination of ischaemia and infection among events leading to amputation in the diabetic foot. In the EURODIALE study 25–75% of patients at various centres were considered to have a wound infection at time of admission.55,57 In several studies the outcome of deep foot infection has been related to extent of tissue involved, co-morbidity and coexisting peripheral vascular disease.52,64–67 Infection should always at least be considered whenever local signs (pain, swelling, ulceration and exudation) develop even when these signs are less marked than expected. In almost 50% of patients with diabetes and deep foot infections signs such as increased white blood cell count, erythrocyte sedimentation rate, C-reactive protein concentration and body temperature were absent, resulting in delay in diagnosis and optimal treatment.60–63 The absence of these signs might be explained by factors such as impaired immunodefences, decreased peripheral circulation and poor metabolic control. Some patients with diabetic foot infection also have a worsening of their glycaemic control but other blood tests are not exclusive. Continuous extension of soft tissue infection to underlying bone poses both diagnostic and therapeutic challenges.60–63 Radiological diagnosis is often difficult because changes suggesting osteomyelitis usually takes several weeks to become visible on X-ray. The difficulty in distinguishing osteomyelitis from osteoarthropathy is well recognised. To evaluate the presence of and extent of a deep foot infection bone scan, MRI or CT can be

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The choice and timing of debridement techniques in the diabetic foot can be described in terms of initial damage control to save the leg and life of the patient, secondary eradication of devitalised tissue to improve or facilitate wound healing and finally to achieve a foot that can maintain ambulation. In the case of an acute deep foot infection, with or without osteomyelitis, surgical intervention should always be considered because in prospective studies more than 80% of patients needed a surgical procedure in order to achieve healing.60 In the case of a deep soft tissue infection, or abscess, or necrotising teno-vaginitis, immediate surgery is usually mandatory.60–63 Antibiotic treatment for diabetic foot infection should be based on an understanding of anti-infective principles, the most important of which is the severity of infection. This also dictates the mode of antibiotic administration; initial treatment is always empirical and should be associated with debridement and sampling of tissue for culture. In this phase, in the presence of active extensive infection, the wound should be debrided immediately regardless of the need for revascularisation. In the next phase, when the acute infection is under control, the need for revascularisation and evaluation of tissue perfusion should always be considered, together with implementation of the total multidisciplinary strategy.42,60–64 The following need and choice of type of debridement is dependent on the level of decreased perfusion to the foot and the extent of tissue loss, not only to achieve healing of an ulcer, but also to maintain ambulation. A diabetic foot ulcer should always be considered ischaemic until proven otherwise by extensive clinical examination and non-invasive, vascular testing to identify those patients in need of revascularisation to improve perfusion to achieve healing.65 In this situation, without a limb-threatening infection, the blood supply to the wound/extremity should be optimised before surgical debridement to ensure that potentially viable tissue is not unnecessarily removed. This can be achieved by waiting 4–8 weeks after bypass surgery or 3–4 weeks after endovascular surgery.64 The plantar fore-foot ulcer caused by increased mechanical stress, usually localised to the metatarsal heads or plantar region of the first digit and characterised by surrounding callosities, constitutes a special challenge in the management of the neuropathic diabetic foot due to the high probability of re-ulceration and development of infection, particularly osteomyelitis in underlying bone. Off-loading, or redistribution of peak pressure under the plantar area, and mechanical removal of callosities and debris is mandatory to achieve healing.66 The best evidence for treating chronic osteomy-

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elitis in the diabetic foot is limited.63 However, curing chronic osteomyelitis generally requires removal of all involved bone.61–63 There are studies which have shown that curing osteomyelitis, particularly in the digits, can occur without surgical removal.60,63 In patients with diabetes mellitus there is a substantial delay in wound healing, which has been related to several abnormalities, such as decreased concentrations of growth factors, increased protease activity, abnormalities of the extracellular matrix reduced fibroblast function and impaired nutritive function. Several non-surgical debridement techniques have been suggested, not only to remove dead tissue when surgery is not favourable/ advisable, or to change/improve the condition of the wound to increase the probability of healing and shorten wound healing time. Encouraging results have been presented but at the moment there is no consensus regarding indications for and value of these treatments.67

24.9

Conclusions

Debridement is a well-recognised and integral part of both acute and chronic wound care. If necrotic tissue and contaminating materials, including biofilm, are not debrided they can delay healing; promote and prolong inflammation and infection; thereby making an impact on quality of life. The need for debridement should be recognised early and effected by individual practitioners, or by referral through the multidisciplinary team approach, using one or more of the many methods which are available. These methods may involve single stage, complete surgical excision with appropriate analgesia, through enzymatic and autolytic dressings to the several methods of physical and biological removal. Continual assessment of the wound, and the patient as a whole, is needed to achieve optimal results.

24.10 Sources of further information and advice Baharastani M. (Ed.). The clinical relevance of debridement. Springer-Verlag. Berlin, Heidelberg, New York. 1999. Cherry G, Hughes MA (Eds). The second Oxford European wound healing course handbook. Poitif Press, Oxford. 2010. Gray David, Acton Claire, Chadwick Paul, Fumerola Sian, Leaper David, Morris Claire, Stang Duncan, Vowden Kathryn, Vowden Peter, Young Trudie. Consensus guidance for the use of debridement techniques in the UK. Wounds UK 2010; 6. Leaper DJ, Harding KG (Eds). Wounds. Biology and Management. Oxford University Press. 1998. Shukla K, Mani R, Teot L, Pradhan S (Eds). Management of Wound Healing. Japee Brothers Medical Publishers. New Delhi. India. 2007.

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Teot L, Banwell PE, Ziegler UE (Eds). Surgery in Wounds. Springer-Verlag. Berlin, Heidelberg, New York. 2004.

24.11 References 1. Gottrup F, Apelqvist J, Price P. On behalf of the EWMA Patient Outcome Group. Outcomes in controlled and comparative studies on non-healing wounds: recommendations to improve the quality of evidence in wound management. Journal of Wound Care 2010; 19: 237–268 2. Ashby R, Bland JM, Cullum N, Dumville J, Hall J, Kang’ombe A, Madden M, O’Meara S, Soares M, Torgerson D, Watson J, White R. Reflections on the recommendations of the EWMA Patient Outcome Group document. Journal of Wound Care 2010; 283–285 3. Consensus report on wound debridement (European Tissue Repair Society). ETRS Bulletin 1995; 2: 97–136 4. Ellis SL, Finn P, Noone M, Leaper DJ. Eradication of methicillin-resistant Staphylococcus aureus from pressure sores using warming therapy. Surgical Infections 2003; 4: 53–55 5. Claeys A, Gaudy-Marqueste C, Pauly V, Pelletier F, Trucheter F, Boye T, Aubin F, Schmultz JL, Grob JJ, Richard MA. Management of pain associated with debridement of leg ulcers: a randomised, multicentre, pilot study comparing nitrous oxide-oxygen mixture inhalation and lidocaine-prilocaine cream. Journal of the European Academy of Dermatology and Venereology 2010 Jun 21 (Epub ahead of print) 6. Dryburgh N, Smith F, Donaldson J, Mitchell M. Debridement for surgical wounds. Cochrane Database of Systematic Reviews 2008, Issue 3. Art. No.: CD006214. DOI: 10.1002/14651858. CD006214.pub2 7. Edwards J, Stapley S. Debridement of diabetic foot ulcers. Cochrane Database of Systematic Reviews 2010, Issue 1. Art.No.: CD003556. DOI:10.1002/14651858. CD003556.pub2 8. Gottrup F. Debridement: Another evidence problem in wound healing (editorial). Wound Repair and Regeneration 2009; 17: 294–295 9. Leaper DJ, Simpson RA. Antiseptics and healing. Leading article. Journal of Antimicrobial Chemotherapy 1986; 17: 135–137 10. Leaper DJ. Eusol. Editorial. British Medical Journal 1992; 304: 930–931 11. Konig M, Vanscheidt W, Augustin M, Kapp H. Enzymatic versus autolytic debridement of chronic leg ulcers: a prospective randomised trial. Journal of Wound Care 2005; 14: 320–323 12. Marazzi M, Stefani A, Chiaratti A, Ordanini MN, Rapisarda V. Effect of enzymatic debridement with collagenase on acute and chronic hard-to-heal wounds. Journal of Wound Care 2006; 15: 222–227 13. Ramundo J, Gray M. Collagenase for enzymatic debridement: a systematic review. Journal of Wound Ostomy and Continence Nursing 2009; 36: S4– 11 14. Smith RG. Enzymatic debriding agents: an evaluation of the medical literature. Ostomy Wound Management 2008; 54: 16–34 15. Baharestani M. Exploring healthcare system paradigms and wound care practices in France. Ostomy Wound Management 1999; 45: 46–54

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16. Ramundo J, Gray M. Is ultrasonic mist therapy effective for debriding chronic wounds? Journal of Wound Ostomy and Continence Nursing 2008; 35: 579– 583 17. Granick MS, Posnett J, Jacoby M, Noruthun S, Ganchi PA, Datiashvili RO. Efficacy and cost-effectiveness of a high-powered water jet for wound debridement. Wound Repair and Regeneration 2006; 14: 394–397 18. Timmers MS, Graafland N, Bernards AT, Nelissen RG, van Dissel JT, Jukema GN. Negative pressure wound treatment with polyvinyl alcohol foam and polyhexanide antiseptic solution instillation in posttraumatic osteomyelitis. Wound Repair and Regeneration 2009; 17: 278–286 19. Palmier S, Trial C. Use of high-pressure waterjets in wound debridement. In: Surgery in Wounds (eds. Teot L, Banwell PE, Ziegler UE) pp. 72–76. SpringerVerlag. Berlin, Heidelberg, New York. 2004 20. Granick M, Boykin J, Gamelli R, Schultz G, Tenenhaus M. Toward a common language: surgical wound bed preparation and debridement. Wound Repair and Regeneration 2006; 14(suppl 1): S1–10 21. Vowden P. Autolytic debridement. In: Surgery in Wounds (eds. Teot L, Banwell PE, Ziegler UE) pp. 77–80. Springer-Verlag. Berlin, Heidelberg, New York. 2004 22. Edwards J, Stapley S. Debridement of diabetic foot ulcers. Cochrane Database of systematic reviews 2010. Issue1. Art No.: CD0035556. DOI:10.10002/14651858. CD003556.pub2 23. Cooper R. The use of honey in the management of wounds (pp. 151–161). In: Mani R, Teot L (Eds). The basic needs to achieve wound healing. Jaypee Brothers Medical Publications Ltd. New Delhi, 2011 24. Cooper R, Blair S. Challenges in modern microbiology and the role for honey (pp. 47–62). In: Cooper R, Molan P, White R (Eds.). Honey in modern wound management. HealthComm UK Limited, Aberdeen, 2009 25. Bogdanov S, Ruoff K, Oddo LP. Physiochemical methods for the characterisation of unifloral honeys. A review. Apidologie 2004; 35: S4–S7 26. Molan PC. The antibacterial activity of honey. The nature of the antibacterial activity. Bee World 1992; 73: 5–28 27. Bang LM, Buntting C, Molan PC. The effects of dilution rate of hydrogen peroxide production in honey and its implications for wound healing. Journal of Alternative and Complementary Medicine 2003; 9: 267–273 28. Eddy JJ, Gideonsen MD. Topical honey for diabetic foot ulcers. Journal of Family Practice 2005; 54: 533–535 29. Molan PC. Debridement of wounds with honey. Journal of Wound Technology 2009; 5: 12–17 30. Molan PC. The role of honey in the management of wounds. Journal of Wound Care 1999; 8: 415–418 31. Snowdon JA, Cliver DO. Micro-organisms in honey. International Journal of Food and Microbiology 1996; 31: 1–26 32. Gething G, Coqwman S. Manuka honey vs. Hydrogel – a prospective, open label, multicentre, randomised controlled trial to compare desloughing efficacy and healing outcomes in venous ulcers. Journal of Clinical Nursing 2009; 18: 466–474 33. Cazander G, Gottrup F, Jukema GN. Maggot therapy for wound healing: clinical relevance, mechanisms of action and future perspectives. Journal of Wound Technology 2009; 5: 18–23

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34. Sherman RA, Hall MJR, Thomas S. Medicinal maggots: an ancient remedy for some contemporary afflictions. Annual Revue Entomology 2000; 45: 55– 81 35. Mumcuoglu KY, Miller J, Mumcuoglu M, Friger M, Tarshis M. Destruction of bacteria in the digestive tract of the maggot of Lucilia sericata (Diptera: Calliphoridae). Journal of Medical Entomology 2001; 38: 161–166 36. Chambers L, Woodrow S, Brown AP, Harris PD, Phillips D, Hall M et al. Degradation of extracellular matrix components by defined proteinases from the greenbottle larva Lucilia sericata used for the clinical debridement of nonhealing wounds. British Journal of Dermatology 2003; 148: 14–23 37. Smith AG, Powis RA, Pritchard DI, Britland ST. Greenbottle (Lucilia sericata) larval secretions delivered from a prototype hydrogel wound dressing accelerate the closure of model wounds. Biotechnology Progress 2006; 22: 1690– 1696 38. Gupta A. A review of the use of maggots in wound therapy. Annals of Plastic Surgery 2008; 60: 224–227 39. Dumville JC, Worthy G, Bland JM, Cullum N, Dowson C, Iglesias C et al. Larval therapy for leg ulcers (VenUS II): randomised controlled trial. British Medical Journal 2009; 338: b773 40. Cazander G, van Veen KEB, Bouwman LH, Bernards AT, Jukema GN. The influence of maggot excretions on PAO1 biofilm formation on different biomaterials. Clinical Orthopaedics and Related Research 2009; 467: 536–545 41. Wolff H, Hansson C. Larval therapy – an effective method for ulcer debridement. Clinical and Experimental Dermatology 2003; 28: 134–137 42. Sherman RA, Pechter EA. Maggot therapy: a review of the therapeutic applications of fly larvae in human medicine, especially for treating osteomyelitis. Medical and Veterinary Entomology 1988; 2: 225–230 43. Steenvoorde P, Jukema GN. The antimicrobial activity of maggots: in-vivo results. Journal of Tissue Viability 2004; 14: 97–101 44. Wolff H, Hansson C. Larval therapy – an effective method for ulcer debridement. Clinical and Experimental Dermatology 2003; 28: 134–137 45. Fadaak H. Maggot debridement therapy. Burns 2003; 29: 96 (comment in Burns 2003; 29: 501–502) 46. Renner R, Treudler R, Simon JC. Maggots do not survive in pyoderma gangrenosum. Dermatology 2008; 217: 241–243 47. Steenvoorde P, Jacobi CE, Van Doorn L, Oskam J. Maggot debridement therapy of infected ulcers: patient and wound factors influencing outcome – a study on 101 patients with 117 wounds. Annals of the Royal College of Surgeons of England 2007; 89: 596–602 48. Grassberger M, Fleischmann W. The Biobag – a new device for the application of medicinal maggots. Dermatology 2002; 204: 306 49. Blake FA, Abromeit N, Bubenheim M, Li L, Schmelzle R. The biosurgical wound debridement: experimental investigation of efficiency and practicability. Wound Repair and Regeneration 2007; 15: 756–761 50. Petherick ES, O’Meara S, Spilsbury K, Iglesias CP, Nelson EA, Torgerson DJ. Patient acceptability of larval therapy for leg ulcer treatment: a randomized survey to inform the sample size calculation of a randomised trial. BMC Medical Research Methodology 2006; 6: 43

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51. Margolis DJ, Allen-Taylor L, Hoffstad O, Berlin JA. Diabetic neuropathic foot ulcers: predicting which ones will not heal. American Journal of Medicine 2003; 115: 627–631 52. International Consensus on the Diabetic Foot and Practical Guidelines on the Management and the Prevention of the Diabetic Foot. International Working Group on the Diabetic Foot. 2007, Amsterdam, the Netherlands, on CD-ROM (www.idf.org/bookshop) 53. Boulton AJ, Vileikyte L, Ragnarson-Tennvall G, Apelqvist J. The global burden of diabetic foot disease. Lancet 2005; 366(9498): 1719–1724 54. Apelqvist J, Larsson J. What is the most effective way to reduce incidence of amputation in the diabetic foot? Diabetes Metabolic Research Review 2000; 16(Suppl 1): S75–83 55. Prompers L, Huijberts M, Apelqvist J, Jude E, Piaggesi A,Bakker K, Edmonds M, Holstein P, Jirkovska A, Mauricio D, Ragnarson Tennvall G, Reike H, Spraul M, Uccioli L, Urbancic V, Van Acker K, van Baal J, van Merode F, Schaper N. High prevalence of ischaemia, infection and serious comorbidity in patients with diabetic foot disease in Europe. Baseline results from the EURODIALE study. Diabetologia 2007; 50: 18–25 56. Gershater MA, Eneroth M, Larsson J, Nyberg P, Thörne J, Apelqvist J. Complexity of Factors Related to outcome of Neuropathic and Neuroischemic/Ischemic Diabetic Foot Ulcers. Diabetologia 2009; 52: 398–407 57. Jeffcoate WJ, Chipchase SY, Ince P, Game FL. Assessing the outcome of the management of diabetic foot ulcers using ulcer-related and person-related measures. Diabetes Care 2006; 29: 1784–1787 58. Prompers L, Schaper N, Apelqvist J, Jude E, Piaggesi A, Bakker K, Edmonds M, Holstein P, Jirkovska A, Mauricio D, Ragnarson Tennvall G, Reike H, Spraul M, Uccioli L, Urbancic V, Van Acker K, van Baal J, van Merode F, Huijberts M. Prediction of outcome in individuals with diabetic foot ulcers: focus on between individuals with and without peripheral vascular disease. The EURODIALE study. Diabetologia 2008; 51: 747–755 59. Apelqvist JA. Paradigm shift is needed in Diabetic Foot Care. Wounds International 2010: 2. Online pub www.woundsinternational.com 60. Mills JL. Open bypass and endoluminal therapy: complementary techniques for revascularisation in diabetic patients with critical limb ischemia. Diabetes Metabolism Research and Reviews 2008; 24(suppl 1): 34–39 61. Eneroth M, Larsson J, Apelqvist J. Deep foot infections in patients with diabetes and foot ulcer: an entity with different characteristics, treatments, and prognosis. Journal of Diabetes Complications 1999; 13: 254–263 62. Lipsky B, Berent AR, Embil J, de Lalla F. Diagnosing and treating diabetic foot infections. Diabetes Metabolism Research and Reviews 2004; 20(Suppl 1): 56– 64 63. Lipsky BA. Report from the international consensus on diagnosing and treating the infected diabetic foot. Diabetes Metabolism Research and Reviews 2004; 20(Suppl 1): 68–77 64. Berend AR, Peters EJG, Bakker K, Embil J, Eneroth M, Hinchliffe R, Jeffcoate WJ, Lipsky BA, Senneville E, The J, Valk GD. Diabetic foot osteomyelitis: a progress report on diagnosis and a systematic review of treatment. Diabetes Metabolism Research and Reviews 2008; 24(Suppl 1): 145–S161

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65. Caselli A, Latin V, Lapenna A, Di Carlo S, Pirozzi F, Benvenuto A, Uccioli L. Transcutaneous oxygen tension monitoring after successful revascularisation in diabetic patients with ischaemic foot ulcers. Diabetic Medicine 2005; 22: 460–465 66. Bus A, Valk GD, van Deursen RW, Armstrong DA, Caravaggi C, Hlavacek P, Bakker K, Cavanagh PR. The effectiveness of footwear and offloading interventions to prevent and heal foot ulcers and reduce plantar pressure in diabetes: a systematic review. Diabetes Metabolism Research and Reviews 2008; 24(Suppl 1): 162–180 67. Hinchliffe RJ, Valk GD, Apelqvist J, Armstrong DG, Bakker K, Game FL, Hartemann A, Heurtier M, Londahl M, Price PE, van Houtum WH, Jeffcoate WJ. A systematic review of the effectiveness of interventions to enhance the healing of chronic ulcers of the foot in diabetes. Diabetes Metabolism Research and Reviews 2008; 24(Suppl 1): 119–144 68. Blanke W, Hallern B. Sharp wound debridement in local anesthesia using EMLA cream: 6 years experience in 1084 patients. European Journal of Emergency Medicine 2003; 10: 229–231

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Index

absorbency, 193 absorbent tampons, 589 accuracy, 131 acellular dermal matrix see acellular human cadaver dermis acellular human cadaver dermis, 498–9 acellular therapies development, 476–85 ECM-based grafts selection available for wound repair, 478–9 engineered ECM-based scaffolds, 480–2 extracellular matrix, 477, 480 naturally derived ECM-based scaffolds, 482–5 acetate, 367 acrylic acid bis-(3-sulphopropyl)-ester potassium salt, 328 acrylic adhesives, 265 Acticoat, 417 Acticoat Absorbent with SILCRYST, 441 Acuvue, 311 Acuvue Advance, 303 adeno-associated virus, 404 adenovirus, 403 adhesion, 247, 250–1 adhesion proteins, 87 adhesive materials adhesion, adhesivity and interfacial behaviour, 247–53 adhesion and adhesivity, 250–1 generalised liquid/solid wetting parameters, 250 interfacial interactions, 247–9 interfacial tension and biological interfaces, 252–3 bioadhesion, 253–7 cohesive/adhesive failure, 255 human skin representative values, 256 interfacial phenomena in wound healing, 247–81 surgical adhesives and tissue sealants, 273–80 collagen, gelatin and albumin-based adhesives, 277–9 cyanoacrylates, 275–6

fibrin sealants, 276–7 ideality and practicality, 274–5 mussel adhesives and genetic engineering, 279 protein-based adhesives, 277 synthetic adhesives, 279–80 wound healing, 257–73 hydrocolloid dressing materials, 267–70 hydrogel dressing materials, 270–3 hydrophobic pressure-sensitive skin adhesives, 258–67 adhesivity, 250–1 adipose-derived stem cells, 557–8, 560 adipose tissue, 558 adult stem cells, 554–5 frequently utilised sources, 556–61 adipose-derived stem cells, 557–8, 560 bone marrow-derived stem cells, 556 cutaneous stem cells, 560–1 umbilical cord-derived stem cells, 557, 558, 559 adult wound healing, 77 vs foetal wound healing, 78, 89–92 differences in cell type and function, 89–91 differences in cytokine profiles, 91–2 fibroblasts, 90–1 gene expression profiles, 92 interleukins, 92 neutrophils, 90 platelets, 89–90 transforming growth factor-beta, 91 Advanced BioHealing, 473, 482, 503–4 advanced therapies, 505–17 delivering value, 509–17 connecting technology with the medical need, 511–17 role of science in product strategy and use, 509–11 similarities and differences when targeting healing in chronic wounds types, 513–14 marketplace, 517–18 second generation, 505–9 AHCD see acellular human cadaver dermis

633 © Woodhead Publishing Limited, 2011

634

Index

albumin-based adhesives, 277–9 alginate dressings, 197–8, 200 alginates, 163, 380–3, 441 alginic acid, 197 Algosteril, 432, 441 alkyl benzene sulphonate, 337 Alloderm, 485, 495, 498, 501 allografts, 467, 469 alternating current, 571, 576 illustration, 572 Altrika Ltd, 472 AM Scientifics Ltd, 484 American Society of Gene and Cell Therapy, 411 ampicillin, 364 angiogenesis, 5 ANN see artificial neural network anterior ocular surfaces, 311–12 Antheraea mylitta, 531 antibacterial activity assays, 365 antibiotics, 363, 440, 612, 626 antimicrobial wound dressings, 416–43 assessing effectiveness in vitro, 227–44 agar plate with colonies, 229 commercially available dressings, 228 bacterial barrier testing, 237–41 test method, 238–41 common types, 420–1 currently available dressings/formulations types, 418–38 evidence on clinical efficacy, 419, 422–32 acute, chronic or infected wounds, 432 burn wounds, 430–1 clinical evidence on silver and other antimicrobial dressings, 422–9 leg ulcers, 431–2 future trends, 442–3 new technology in ‘antimicrobial’ formulation, 443 potential new molecules, 442–3 historical perspective, 416–17 laboratory in vitro and in vivo (animal) efficacy, 432–8 in vitro laboratory and in vivo animal studies, 434–8 log reduction testing, 229–33 calculation and interpretation of results, 231–3 principles, 229–31 standards, 230 other considerations, 241–4 choice of test method, 241–2 future trends, 242–4 rationale of application effects, 417–18 types, 439–42 antibiotics and antiseptics, 440 metal with antimicrobial activity, 439–40 others, 441–2

zone of inhibition, 234–7 calculation of results, 236 other considerations, 236–7 principle, 234–5 time points, 235–6 antimicrobials, 363–7 systemic, 363 antiseptics, 440, 576 Apligraf, 473–4, 475, 495, 500, 504, 505, 506, 507, 511, 517, 518 Aquacel Ag, 417, 430, 432 Aranz Medical Ltd, 132, 150 ArthroCare Corporation, 313 articular cartilage, 334–5 artificial neural network, 146, 148 ATCC 55730, 72 Atmos Fritzsching & Co, 589 atomic force microscopy (AFM), 365 autografts, 467 Avance, 417 avotermin current treatments for scar management, 451–2 agents in development for dermal scarring reduction, 452 approaches to manage scarring postsurgery, 451 future trends, 458–9 medical need for therapies, 450 new biological approaches, 452–8 new class of prophylactic medicines, 455–8 preclinical studies, 453–5 scarring reduction, 450–9 TGFβ isoforms, 452–3, 454 duration and magnitude of effect on scar-forming healing response, 454 vs placebo and standard care showing scar improvement images, 457 vs placebo showing scar improvement, 458 Bacillus megaterium, 526 bacteria, 183 bacterial barrier testing, 237–41 test method, 238–41 typical test rig set up, 241 bacterial burden, 165 bacteriophages, 405 ballistic gene delivery, 406–7 bandage lenses (BLs), 311 basic fibroblast growth factor, 368, 378 Baxter Healthcare Corporation, 313 becaplermin, 97, 370–1, 496 bFGF see basic fibroblast growth factor biguanides, 617 bilayered cellular matrix (BCM), 506 bioactive dressing, 377 bioadhesion, 253–7

© Woodhead Publishing Limited, 2011

Index Biobags, 621 Biobran, 482 Biobrane, 158 bioburden, 165 biofilms, 51, 60–9, 613–14, 619 chronic wounds, 60–9 delayed wound healing factors, 61 interactive cooperative community, 51–60 8 factors in 3-D architecture, 57 chemical, physical and environmental properties, 57–9 chronic wounds electron micrograph, 60 definitions, 51–2 electron micrograph of phenotype, 55 electron micrographs, 59–60 environmental Eh and pH biofilm gradient, 58 microbial components, 59 microbial stage cycle, 54 mono to complex biofilm development, 55 structure, 52–7 universal cyclic growth of microbes, 53 and micro-organisms, dysfunctional wound healing, 39–75 biofilms in chronic wounds, 60–9 microbiology, 40–51 therapeutic or restorative microbiology/ modeling, 69–75 therapeutic or restorative microbiology/ modeling, 69–75 development, 72–5 lactobacillus effect, 73, 74 probiotic impact on biofilm, 71 probiotics intervention, 71–2 Triphasic Wound Model PLUS, 69–71, 70 in vitro to in vivo SWITCH, 74 biointegration, 180–1 biological methods gene delivery, 401–6 non-viral vectors, 404–6 viral vectors, 401–4 adeno-associated virus, 404 adenovirus, 403 Herpes simplex virus, 404 retrovirus, 401, 403 biologically derived scaffolds, 524–39 collagen-derived scaffolds, 531–2 elastin-derived scaffolds, 532–4 future trends, 539 keratin-derived scaffolds, 534–5 polyhydroxyalkanoate-derived scaffolds, 524, 526–7 monomer units chemical structures, 526 polymers thermal and mechanical properties, 525 polysaccharide-derived scaffolds, 535–9 cellulose, 537–8

635

chitin, 537 chitosan, 535–7 hyaluronan, 538–9 resilin-derived scaffolds, 534 silk-derived scaffolds, 527–31 proteins as biologically derived polymers, 528–9 biomaterials, 174 surface interactions, 176–82 biointegration, 180–1 biological interactions, 178–9 response to tissue environment, 181–2 tissue response, 182–4 carcinoma, 183–4 immunogenic reaction, 183 infection, 183 inflammation, 182 toxicity, 183 biomimetic biomaterials, 321–2 proteoglycans as models in wound healing, 322–4 biomimetic models, 330–5 articular cartilage and intervertebral disc, 334–5 key components of extracellular matrix, 335 cornea, 330–4 keratan sulphate proteoglycan and dermatan sulphate proteoglycan, 334 repeating sulphated glycosaminoglycan units, 333 tear film, 332 natural systems, 330 biomimetic principles stereochemical disposition chondroitin sulphate and related repeat units, 339 ring substituents in chondroitin sulphate, 339 sulphonated biomaterials, 335–41 other body sites, 340–1 stereochemical considerations, 338–40 structural, biological and toxicological considerations, 335–8 Biostep, 380 Biosurface Technologies, 495, 502 biphasic asymmetric pulsed current, 579 bleomycin, 96–7 Blue Sky Medical, 590, 592, 593 Bombyx mori, 527 bone marrow-derived stem cells, 556 Boyle’s Law, 593, 594, 595 bradykinin, 301–2 broad spectrum antibiotics, 612 bupivacaine, 157, 366 calcium alginate dressings, 159, 197–8 Calgary Biofilm device see MBEC Assay Cambrex, 474

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636

Index

Cambrex Bio Science, 475 carbon dioxide plasma, 371 carboxymethylcellulose, 373, 381, 382 fibres, 163, 198, 200 carcinoma, 183–4 CarrasorbH, 441 caveolar endocytosis, 401 cefazolin, 364 cell-based products, 501–5 bi-layered skin equivalent, 504–5 cultured dermal replacement, 502–4 cultured epithelial sheets, 501–2 cell senescence, 293 Cellerix, 508 cellular therapies, 98, 475–6 development, 469–76 cellular engineered skin replacements selection, 470–1 cultured dermal constructs, 472–3 cultured epidermal sheets, 469, 472 engineered skin equivalents, 473–5 limitations, 475–6 cellulose, 537–8 monomer units chemical structures, 536 CelTx, 505 Centre for Medicare and Medicaid Services, 592 cetrimide, 382 charge repulsion, 342–4 chemical adhesion, 251 chemokine, 14–16 deficiencies, 22–3 chitin, 509–10, 537 monomer units chemical structures, 536 chitosan, 366, 383, 441–2, 509–10, 535–7 monomer units chemical structures, 536 chlorhexidine, 367, 382 salts, 367 chlorhexidine digluconate, 367 chloroxylenol, 382 chronic venous leg ulcers, 512, 514–16 chronic wounds, 130–1, 155–6, 165 background material, 149–50 wound colour, 149 wound size, 149–50 biofilms, 60–9, 61 bioreactor and wound environment, 66–9 critical colonization/climax community, 62–3 delayed wound healing factors, 61 hypothesis application and utilisation to planktonic ratio, 66–9 integrating biofilm stage and planktonic transmission, 68 MBEC based planktonic phenotypes, 67 microbial phenotypes, planktonic or biofilm significance ratio, 68

microbial progression and biofilm formation relationship, 65 Percival and Thomas dual hypothesis, 50, 63–6 theory amplification, 67 wound continuum, 61–2 dry wound healing using spray-on dressings, 209–25 case studies, 214–24 key properties of an ideal wound dressing, 211–13 protein-based spray-on dressings, 213–14 dysfunctional wound healing, 3–25 clinical images, 11 normal skin, 3–5 skin ageing, 6–10 skin wounds, 10–24 monitoring and determining treatment, 130–50 polymeric materials for wound dressings, 186–203 advanced moisture-retentive, 187–9 future trends, 203 infection control, 199–201 polymeric materials in moist wound healing, 189–99 sulphonated biomaterials and biomimetic behaviour, 341–51 cell-surface effects and neutrophil function, 346 heparin/heparin mimicry, 348–51 interfacial tension and hydrophobic shielding, 342 ionic equilibria and selectivity, 344–5 kinin effects, GAGs and pain inhibition potential, 347–8 matrix metalloprotease absorption and inhibition, 345 osmotic drive and charge repulsion, 342–4 vitronectin/plasmin effects, 346–7 theories, 12–13 cellular events, 13 wound colour measurements, 139–49 approaches, 146–8 digital cameras, 144–5 lighting, 142–4 logistic regression, ANN and SVM against clinician performance, 149 motivation, 139–42 wound colour, 145–6 wound inflammation detection, 148–9 wound size measurements, 131–9 definitions, 131–4 MAVIS measurement device, 135–9 motivation, 131 techniques, 134–5 ciprofloxacin, 364, 379, 382, 383

© Woodhead Publishing Limited, 2011

Index clindamycin, 364 Clostridium perfringens, 609, 612 Cochrane Collaboration, 615 cohesion, 247 cohesive failure, 254 collagen, 80, 85, 90, 211, 277–9, 369, 377–80, 480, 531–2, 535 collagen matrix, 181–2 collagenase, 181 collateral damage, 66–9 Comfeel, 441 commercialisation engineered tissue products, 495–519 advanced therapies in the marketplace, 517–18 advanced therapies second generation, 505–8 cell-based products, 501–5 delivering value in advanced therapies, 509–17 engineered templates and scaffolds, 496–8 lessons from the first generation, 505 processed tissues, 498–501 compression therapy, 95, 158–9 COMSTAT, 70 confocal laser scanning microscopy (CLSM), 70 contact lens interfacial phenomena, 302–4 interfacial tear envelope dynamics material influence, lotrafilcon A vs galyfilcon A, 303 material influence over time, wear, lotrafilcon B, 303 Contreet, 417 controlled lighting, 142–3 Cook Biomedical, 501 cord lining stem cells, 557 cornea, 295–8, 330–4 anatomical structure, 296 limbus, 297 non-keratinised, 298–300 corneal model wound fluid and the tear film collection, 304–9 biological wound fluid collection, 306 tear film collection, 305–6 wound exudate collection, 307–8 wound fluid models, 308–9 wound healing and biomaterial studies, 294–302 cornea and dermal wounds, 295 corneal wound healing, 295–8 corneal wound healing biomarkers, 300–2 non-keratinised cornea, 298–300 wound healing studies and interfacial phenomena, 284–315

637

biomaterials in mucosal wound healing, 309–14 interfacial phenomena in ocular surface contact lens studies, 302–4 wound dressing biomaterials, 284–94 corneal wound healing, 295–8 biomarkers, 300–2 corneal anatomical structure, 296 corneal limbus, 297 corticosteroids, 95 Covidien, 484 COX-1, 15 COX-2, 15–16 CR Bard, 481 critical colonisation, 62–3 critical surface tension, 257 cryotherapy, 95 cultured epithelial autografts, 469, 472 Cultured Skin Substitute (CSS), 474 cupping, 588 cutaneous stem cells, 560–1 cyanoacrylates, 275–6 cytokines, 79, 97–8 Cyzac, 473 Dacron-mesh silver nylon stocking, 580 Dahlquist criterion, 254, 258 debridement background, 607–9 black heel not needing debridement, 609 black toe associated with peripheral vascular disease, 608 diabetic foot, 624–7 non-viable tissue complications, 609–13 dry gangrene at the wrist, 612 hernia wound infected with Meleney’s synergistic gangrene, 611 necrotic pressure sore with infection and sepsis, 611 thigh wound after debridement and healing, 613 non-viable tissue in wounds, 606–27 organisation, 614 presence of biofilm, 613–14 scoring the effectiveness, 623–4 timing and types, 614–23 autolytic debridement using dressings, 618–19 chemical debridement, 616–17 debridement using honey, 619–20 enzymatic debridement, 617 high pressure therapy (hydrojets), 617–18 hydrotherapy and irrigation techniques, 617 Larva therapy (maggot debridement therapy), 620–3 mechanical debridement (wet-to-dry), 616

© Woodhead Publishing Limited, 2011

638

Index

several wounds needing different anaesthetic approaches, 615 surgical debridement, 616 topical negative pressure therapy, 623 debridement performance index (DPI), 624 Debritom, 618 denaturing gradient gel electrophoresis (DGGE), 46 Dermagraft, 473, 482, 496, 500, 503, 507, 517 Dermal Regeneration Template, 495 DermaMatrix, 485 dermis, 7–8 desbrider, 607 DFU see diabetic foot ulcers diabetic foot, 624–7 diabetic foot ulcers, 516 differential gene expression theory, 10 diffusive adhesion, 251 digital cameras, 144–5 direct current, 571 illustration, 572 direct injection, 406 disk sensitivity assay, 237 DNA PCR, 46 Donnan equilibria, 344 Doppler ultrasound, 608 doxycycline, 364 Doyle, 313 dressings antimicrobial, 416–43 currently available dressings/ formulations types, 418–38 future trends, 442–3 types, 439–42 Drosophila CG15920 gene, 534 DRT see Dermal Regeneration Template drug delivery dressings, 361–86 alginates, 380–3 collagen, 377–80 defined, 361 future trends, 385–6 honey, 383–5 hydrocolloids, 373–4 hydrogels, 374–7 role in wound management, 362–3 topically delivered therapeutic compounds, 363–73 antimicrobials, 363–7 growth factors, 367–71 less common therapeutic agents, 372–3 dry wound healing spray-on dressings for chronic wounds, 209–25 case studies, 214–24 key properties of an ideal wound dressing, 211–13 protein-based spray-on dressings, 213–14 dynamic range, 145

dysfunctional angiogenesis, 23–5 dysfunctional dermal repair, 18–23 chemokine deficiencies, 22–3 extracellular matrix alterations, 19 fibroblast responses, 18–19 growth factors/cytokines alteration, 21 oxidative stress imbalance, 21–2 protease imbalance, 20–1 wound fluid, 21 dysfunctional re-epithelialisation, 16–18 dysfunctional wound healing chronic wounds, 3–25 clinical images, 11 normal skin, 3–5 skin ageing, 6–10 skin wounds, 10–24 micro-organisms and biofilms role, 39–75 biofilms in chronic wounds, 60–9 interactive cooperative community, 51–60 microbiology, 40–51 therapeutic or restorative microbiology/ modeling, 69–75 Ebers Papyrus, 588 ECM see extracellular matrix Ecologic Hypothesis, 64 EGF see epidermal growth factor elastin, 532–4 elastin fibres, 533 electrical simulation antibacterial effects, 575–6 current of injury, 573–4 high voltage pulsed current effect, 576–7 low intensity direct currents effect, 577–9 other types applied to wounds, 578–9 combination with the application of heat, 578–9 electrical current other types, 579 physiological effects, 574–5 other effects, 575 tissue oxygenation improvement, 574–5 wound angiogenesis, 574 wound healing, 571–82 direct, alternating and pulsed current, 572 electroporation, 407 electrostatic adhesion, 251 electrostatic attraction, 176 ELISA, 369 embryonic stem cells, 99, 553–4 EMLA, 614 engineered tissue products advanced therapies in the marketplace, 517–18 advanced therapies second generation, 505–8 cell-based products, 501–5

© Woodhead Publishing Limited, 2011

Index bi-layered skin equivalent, 504–5 cultured dermal replacement, 502–4 cultured epithelial sheets, 501–2 commercialisation, 495–519 delivering value in advanced therapies, 509–17 connecting technology with the medical need, 511–17 role of science in product strategy and use, 509–11 similarities and differences when targeting healing in chronic wounds types, 513–14 engineered templates and scaffolds, 496–8 lessons from the first generation, 505 processed tissues, 498–501 engineered tissues acellular therapies development, 476–85 ECM-based grafts selection, 478–9 engineered ECM-based scaffolds, 480–2 extracellular matrix, 477, 480 naturally derived ECM-based scaffolds, 482–5 cellular therapies development, 469–76 cellular engineered skin replacements selection, 470–1 cultured dermal constructs, 472–3 cultured epidermal sheets, 469, 472 engineered skin equivalents, 473–5 limitations, 475–6 traditional approaches, 466–8 requirements details for engineered skin replacement development, 468 wound microenvironment, 464–6 growth factors subset and cytokines, 465 healing process overview, 465 wound repair, 463–87 EpiCel, 469, 472, 495, 502 epidermal growth factor, 4, 368, 397 epidermis, 560 ageing, 6–7 Epidex, 469, 472, 476 erythromycin, 364, 382 Escherichia coli, 576 etafilcon A, 311 ethylendiaminetetraacetic acid (EDTA), 380 Euroderm GmbH, 469 EURODIALE study, 625 European Medicines Agency (EMEA), 455 European Tissue Repair Society (ETRS), 607 EUSOL, 616 expression cassette, 405 extra polymeric substance (EPS), 52 extracellular matrix, 4, 80, 90, 322, 477 alterations, 19 changes, 7–8 wound repair, 477, 480

639

Eykona Technologies Ltd, 150 EZ-Derm, 484 F-127 Poloxamer, 70 Fibracol, 481 fibrin, 165–6 fibrin cuff hypothesis, 12 fibrin sealants, 274, 276–7 fibrinogen, 176 fibroblast growth factor, 397, 464, 466 fibroblasts, 18–19, 79, 80, 90–1, 98 Fibrocell, 506 fibromodulin, 85, 86, 99 fibronectin, 179, 300, 480–1 fibroplasia, 4–5 fibroproliferative scarring, 81–2 hypertrophic scars, 81 keloids, 82 pathogenesis, 82 FloSeal Matrix Hemostatic Sealant, 313 5-fluorouracil, 96 foam dressings, 192–3 hydropolymer, 193–4 Focus Night&Day, 303, 311 foetal skin, 84–7 adhesion proteins, 87 collagen content, 85 early scar formation in E18 fetal rat wounds, 88–9 scarless healing of E16 fetal rat wounds, 86–7 histology, 84 hyaluronic acid, 85 proteoglycan extracellular matrix modulators, 85–7 foetal wound healing scarless healing, 82–9 E16 fetal mouse wounds, 83 fetal cutaneous wound healing, 82–4 fetal skin, 84–7 scarless repair in fetal skin, 87–9 vs adult wound healing, 78, 89–92 differences in cell type and function, 89–91 differences in cytokine profiles, 91–2 fibroblasts, 90–1 gene expression profiles, 92 interleukins, 92 neutrophils, 90 platelets, 89–90 transforming growth factor-beta, 91 Forticell Biosciences Inc., 474 Fourier transform infrared spectroscopy, 177–8 free radical damage theory, 10 GAGs see glycosaminoglycans galvanotaxis, 573–4 galyfilcon A, 303

© Woodhead Publishing Limited, 2011

640

Index

gauze, 162 gelatin, 277–9 gelatin-resorcinol-formaldehydeglutaraldehyde (GRFG) glues, 278 gelatin-resorcinol-formaldehyde (GRF) adhesive, 278 Gelfoam, 313 Gelita-Spon Gelatin Sponge and Film, 313 gene expression, 92 gene therapy biological methods, 401–6 non-viral vectors, 404–6 viral vectors, 401–4 chemical methods cationic liposomes, 407 delivery methods, 400–7 chemical methods, 407 common techniques, 402 ethical issues, 410–11 future trends, 411–12 growth factors, 397–9 characteristics, 398–9 history, 396–7 physical methods, 406–7 ballistic gene delivery, 406–7 direct injection, 406 electroporation, 407 microseeding, 406 skin as a target for therapy, 400 wound repair, 395–412 genes with wound healing effects, 408–9 growth factors gene transfer to full thickness wound in porcine experimental model, 410 genetic engineering, 279 genomic microarray analysis, 92 gentamycin, 382 Genzyme, 495, 502 Genzyme Biosurgery, 469 German patent, 589 Global Scar Comparison Scale, 455 gluconate, 367 glutaraldehyde, 378 glycosaminoglycan mimics sulphonated biomaterials in wound healing, 321–54 biomimetic biomaterials, 321–2 biomimetic models, 330–5 biomimetic principles, 335–41 hydrogels based on sulphonated monomers, 353 polymers and biomimesis, 324–30 possible modes of biomimetic behaviour, 341–51 products based on sulphonated polystyrene, 352 products derived from natural proteoglycan fragments, 352

proteoglycans, 322–4 synthetic and biologic dressings, 353–4 glycosaminoglycans, 330, 333, 347–8, 497, 532 gold, 406 GR-DIAL glue, 278 GraftJacket, 485, 500 grafts classification according to sources, 467 see also specific grafts granulation tissue, 80 growth factor inducible element (FIRE), 409 growth factors, 97–8, 367–71 wound healing, 397–9 characteristics, 398–9 see also specific growth factors GSCS see Global Scar Comparison Scale gutless, 403 HealthPoint, 483, 507 hematopoietic stem cell (HSCs), 553 HemCon bandage, 442 heparin, 348–51 heparin mimicry, 348–51 heparin sulphate, 350 interference with regulating action of β-site amyloid precursor protein, 350 structure, 349 hepatocyte, 99 Herpes simplex virus, 404 hexafluoroisopropanol, 530 high molecular weight kininogen, 347–8 high voltage pulsed current, 576–7 hMSCs see human mesenchymal stem cells homografts, 467 honey, 383–5, 619–20 raw, 620 human dermal fibroblasts (HDF), 560 human hair keratin (HHK), 535 human mesenchymal stem cells, 555 Human Microbiome study, 44 Humanitarian Device Exemption (2007), 502 hyaluronan, 481, 538–9 monomer units chemical structures, 536 hyaluronic acid, 85, 90, 99 hydrocolloid dressings, 196, 200 structure and properties, 267–70 subsets and properties, 269 hydrocolloids, 163, 373–4, 618 hydrofibre dressings, 198–9, 200–1 hydrogel dressings structure and properties, 270–3 changes in polar and dispersive components, 270 subsets and properties, 269 hydrogels, 194–6, 270–3, 329–30, 370, 374–7, 618 hydrogen peroxide, 375, 384 hydrophilicity, 177

© Woodhead Publishing Limited, 2011

Index hydrophobic shielding, 342 2-hydroxyethyl methacrylate, 327 hyperemic treatment, 588–9 hypertrophic scars, 81 ICX-SKN, 507 Ideal Gas Laws, 593 IL see interleukins IL-6, 92 IL-8, 92 IL-10, 92, 123 ilodecakin, 123 immune response, 13–16 altered growth factor/cytokine/chemokine levels, 14–16 immunogenicity, 183 immunoreactivity, 183 induced pluripotent stem cells, 100 inducible nitric oxide synthase (iNOS), 409 infected cell proteins (ICP), 404 infection, 141–2, 183, 292–3 prevention and control, 164–7 inflammation, 13–16, 79–80, 142, 182 altered growth factor/cytokine/chemokine levels, 14–16 inflammatory cell populations, 13–14 insulin, 575 insulin growth factor 1, 409 insulin like growth factor-1, 464 Integra, 481, 561 Integra DRT, 497 Integra Life Sciences, 481, 495, 505, 506 interactive dressings see hydrocolloids Intercellular Adhesion Molecules (ICAM), 401 Intercytex, 473, 507 interfacial interactions, 247–9 interfacial phenomena use and relevance of corneal model, 284–315 biomaterials in mucosal wound healing, 309–14 corneal model, 294–302 ocular surface contact lens studies, 302–4 wound dressing biomaterials, 284–94 wound fluid and the tear film collection, 304–9 interfacial tension, 248, 249, 252–3, 342 interfollicular epidermis, 560 interleukins, 92 internal plasticisation, 260 intervertebral disc, 334–5 inverse cross polarisation, 136 Invotec International, 313 iodine, 440, 576 iodine delivery systems, 365 Iodosorb, 417 ISO 11664-4:2008, 144

641

Jackson-Pratt Mini Snyder flat drain, 591 Jelonet Paraffin Gauze Dressing, 313 Johnson & Johnson, 313, 481, 496, 509 Junod’s boot, 589 illustration, 590 Juvidex, 98 Juvista, 98, 123, 453 Kaltostat, 441 kappa values, 146 keloids, 82, 94 Kendall Healthcare Co., 313 KeraCure, 508 KeraPac, 508 keratin, 534–5 keratinocyte growth factor, 4, 409 keratinocytes, 98 keratoprostheses (KPro), 312 Kinetic Concepts Inc., 495, 592 kinins, 301, 347–8 Klapp Cup, 589 Kremlin Kit, 592 Kringle 1 (K1), 368 kurtosis, 148 LabChip kit, 302 Laboratory of Tissue Repair and Gene Therapy, 410 lactic acid, 597 lactobacillus reuteri, 72 Landis–Starling equation, 596 Larva therapy see maggot debridement therapy laser eye surgery, 312 laser in situ epithelial keratomileusis (LASEK), 312 laser in situ keratomileusis (LASIK), 312 lasers, 95–6 Lavasept, 617 lectins, 401 leg ulcers, 130, 131, 431–2 Leptospernum scoparium, 384 Leptosporidium honeys, 385 ‘leukocyte trapping’ hypothesis, 12 Lever Brothers, 336 lidocaine hydrochloride, 376 LifeCell, 485, 495, 500, 501, 505, 506 lighting, 142–4 lipoplexes, 407 living skin equivalent, 473 log reduction, 229 calculation and interpretation of results, 231–3 individual steps, 232 principles, 229–31 individual steps, 232 schematic, 231 testing, 229–33 standards, 230

© Woodhead Publishing Limited, 2011

642

Index

L’Oreal, 476 lotrafilcon A, 303 lotrafilcon B, 302, 303 low intensity direct currents, 577–9 lower critical solution temperature (LCST), 376 lysyl oxidase, 87 3M, 481 MacBeth, 144 macrophages, 464 maggot debridement therapy, 620–3 use, 623 manuka see Leptospernum scoparium marrow stromal cells, 556 mass spectroscopy, 365 matrix metalloproteinase, 5, 80, 87, 162, 287, 288, 345 MAVIS see Measurement of Area and Volume Instrument System MBEC Assay, 243 mean, 147 Measurement of Area and Volume Instrument System, 135–9 instrument description, 135–7 MAVIS-III instrument, 136 instrument performance, 138–9 area and volume of 12 calibration artefacts, 139 precision of area, volume, and circumference, 140 precision values of wound area, 141 making measurements, 137–8 stereo image of excision wound, 138 taking images, 137 mechanical adhesion, 251 median, 147 Medline, 481 Medtronic Xomed, 313 Merocel, 313 MeroGel, 313 mesenchymal stem cells, 99–100, 554, 556 metal sorption analysis, 365 methacrylates, 263 methacrylic acid, 327, 328 methicillin-resistant S. aureus (MRSA), 367 methicillin-susceptible S. aureus (MSSA), 367 microamperage direct current, 576 microbiological broth dilution method, 382 microbiology, 40–51 anatomic vs microbial barriers, 41–4 aero-tolerance, 43 four reservoirs of Homo sapiens, 42 four traditional anatomical barriers, 42 NIH human microbiome study, 44 biofilm/plantonic phenotype ratio, 49–51 Percival/Thomas Dual Hypothesis, 50 planktonic ratio, 51

definitions and pathophysiology, 44–5 biphasic wound infection, 45 Eh/pH early and late colonisation, 44–5 disease, statistics and background, 40–1 microbial location, 45–9 denaturing gradient gel electrophoresis (DGGE), 47 planktonic (Pp) organisms, 45–9 traditional and non-culture technique comparison, 48 wound isolates, 49 microorganisms and biofilms, dysfunctional wound healing, 39–75 biofilms in chronic wounds, 60–9 interactive and cooperative community, 51–60 microbiology, 40–51 therapeutic or restorative microbiology/ modeling, 69–75 microseeding, 406 Miller Technique, 598 minocyclin, 364 MMP-1, 20–1 MMP-2, 20–1 MMP-3, 20–1 MMP-8, 20–1 MMP-9, 20–1 MMP-13, 20–1 moist wound healing, 214–15 inflamed periwound area and moderately exudating wound, 215 moisture vapour transmission, 193 rate, 163, 265–6 molecular therapy see gene therapy Mölnlycke Health Care, 481 monocytes, 79 monomers, 325–6 mucosal surfaces, 310 mucosal wound healing biomaterials, 309–14 anterior ocular surfaces, 311–12 nasal wound healing, 312–14 mucus, 331–2 Mueller-Hinton agar, 234 multiple organ dysfunction syndrome (MODS), 610 multiple organ failure (MOF), 610 mussel adhesives, 279 Myskin, 472 n-vinyl pyrrolidone, 327 Nanog, 557 narrow spectrum antibiotics, 612 nasal wound healing, 312–14 Nasopore, 313 near infrared spectroscopy, 608 nefilcon A, 311

© Woodhead Publishing Limited, 2011

Index negative pressure history, 588–93 Junod’s Boot, 590 pathophysiologic mechanisms of action, 594–8 science of negative pressure, 593–4 search for the perfect negative pressure technology, 598–602 setup in the contracted state with suction applied, 595 setup in the relaxed state, 595 wound therapy, 499, 587–602 neoangiogenesis, 80 neomycin sulphate, 367 neutrophils, 79, 90, 346 non-viable tissue complications, 609–13 dry gangrene at the wrist, 612 hernia wound infected with Meleney’s synergistic gangrene, 611 necrotic pressure sore with infection and sepsis, 611 thigh wound after debridement and healing, 613 debridement, 606–27 background, 607–9 diabetic foot, 624–7 organisation, 614 presence of biofilm, 613–14 scoring the effectiveness, 623–4 timing and types, 614–23 normal skin time course, 4 tissue formation, 4–5 angiogenesis, 5 fibroplasia, 4–5 re-epithelialisation, 4 wound contraction, 4–5 tissue remodelling, 5 vascular and inflammatory responses, 3–4 wound healing, 3–5 Oasis, 483–4, 501 odour management, 167–8 O2Optix, 302, 303 optical aberrations, 145 OrCel, 474, 475, 506, 508 Organogenesis, 473, 495, 504 ornithine transcarboxylase deficiency (OTCD), 396 Ortec International, 506 osmotic drive, 342–4 oximetry, 575 PAI see plasminogen activator inhibitor PAI-1, 289, 300, 301, 346 pain, 156–61 pain inhibition, 347–8 pain relief, 293–4

643

PDGF see platelet derived growth factor Percival and Thomas dual hypothesis, 50, 63 peripheral arterial disease (PAD), 624–5 Permacol, 484 Permaderm, 474, 475 Persil, 336 phagocytosis, 16 Pharmacia and Upjohn Co., 313 PHAs see polyhydroxyalkanoate Photometrix Imaging Ltd, 150 photorefractive keratectomy (PRK), 312 physical methods gene delivery, 406–7 ballistic gene delivery, 406–7 direct injection, 406 electroporation, 407 microseeding, 406 pixel noise, 145 plasmids, 401, 406, 407 plasmin, 346–7 plasminogen activator inhibitor, 289 platelet derived growth factor, 90, 97, 115–16, 368, 378, 397, 464 platelet derived growth factor-B, 408 platelets, 89–90, 464 platinum electrodes, 576 Poiseuille’s Rule, 593, 594, 595 poly(2-hydroxyethyl methacrylate), 253 polyacrylates, 262–3 polycaprolactone (PCL), 183 poly(D,L)-lactide, 157 polyethylene glycols (PEGs), 279–80 polyethylene oxide (PEO), 178 Polyganics BV, 313 polyHEMA, 326, 327 polyhexamethylene biguanide (PHMB), 292–3 polyhydroxyalkanoate, 524, 526–7 monomer units chemical structures, 526 poly(hydroxyethyl methacrylate), 376 polylactic acid (PLLA), 183 polylactic-co-glycolic acid (PLGA), 184 polymer adsorption, 178 polymerase chain reaction (PCR), 243, 244 polymeric wound dressings advanced moisture-retentive, 187–9 ideal wound dressing, 189 chronic wound and burns, 186–203 commercially available dressings, 202 future trends, 203 infection control, 199–201 polymeric materials in moist wound healing, 189–99 calcium alginate, 197–8 classes, 190–1 foam dressings, 192–3 hydrocolloids, 196 hydrofibre dressings, 198–9 hydrogels, 194–6 hydropolymer foam dressings, 193–4

© Woodhead Publishing Limited, 2011

644

Index

polyurethane transparent films, 189–92 silicone, 199 polymethyl methacrylate (PMMA), 184 polyurethane adhesives, 280 polyurethane films, 189–92 poly(vinyl ether) PSAs, 265 porcine wound model, 410 povidone, 580 povidone-iodine (PVP-I), 440 precision, 131–2 pressure-sensitive adhesives, 254, 258 structure and properties, 258–67 polyacrylates and methacrylates, 263 polymer structure and organisation, 260 polyvinyl ethers, 262 selection of candidate PSAs, 261 subsets and properties, 269 surface property criteria, 259 pressure ulcers, 516–17 Prevascar, 123 Primatrix, 484, 485, 501 pro-inflammatory cytokines, 24 probiotics, 71–2 proflavine, 382 Promogran Prisma, 380 Promograns, 164, 287–8, 509 Prontosan, 617 prophylactic scar reduction, 123–5 protease, 20–1 protein adsorption, 176, 178 protein-based adhesives, 277 protein-based dressings, 211 protein solubility, 177 proteoglycan extracellular matrix modulators, 85–7 proteoglycans, 330, 332–3 PSAs see pressure-sensitive adhesives Pseudomonas aeruginosa, 166, 576, 619 pulsed current, 572–3 illustration, 572 Puracol, 481 PVA+ Sponge Packing, 313 quartz crystal microbalance, 178 radiation, 96 radioactive labelling, 177 Ralstonia eutropha, 526 Rapid Rhino, 313 re-epithelialisation, 4 reactive oxygen species (ROS), 164 recombinant human epidermal growth factor (rh-EGF), 368, 378 reepithelialisation, 80 Regranex see becaplermin Renovo, UK, 453 reprogramming, 555 resilin, 534

retrovirus, 401, 403 Rocket Rhino, 313 Salmonella enteriditis, 619 scaffolds biomaterial processed from biologicallyderived polymers, 524–39 collagen-derived scaffolds, 531–2 elastin-derived scaffolds, 532–4 future trends, 539 keratin-derived scaffolds, 534–5 polyhydroxyalkanoate-derived scaffolds, 524, 526–7 polysaccharide-derived scaffolds, 535–9 resilin-derived scaffolds, 534 silk-derived scaffolds, 527–31 scar-forming healing, 124 scar-free healing, 124 scar management, 112 new therapeutic treatments, 112–26 pre-clinical studies to clinical efficiency, 122–3 prophylactic scar improvement therapies, 123–5 mechanisms and properties, 124 scar-free and scar-forming healing, 113–16 collagen cutaneous scar, 115 vertebrates, 116 scarring mechanisms and potential treatments, 117–22 experimental incisional wound models, 121 gene expression in models of incisional wounds and scars, 122 in vivo models, 118–20 scar tissue, 112 scarless wound healing, 77–101 adult vs fetal wound healing, 78, 89–92 fibroproliferative scarring, 81–2 future trends, 97–100 process, 78–81 scarless fetal wound healing, 82–9 treatment options for scars, 92–7 scarring, 77, 112 new therapeutic treatments, 112–26 mechanisms and potential treatments, 117–22 pre-clinical studies to clinical efficiency, 122–3 prophylactic scar improvement therapies, 123–5 scar-free and scar-forming healing, 113–16 wound healing, 77–101 adult vs fetal wound healing, 78, 89–92 fibroproliferative scarring, 81–2 future trends, 97–100 process, 78–81

© Woodhead Publishing Limited, 2011

Index scarless fetal wound healing, 82–9 treatment options for scars, 92–7 scarring reduction avotermin, 450–8 current treatments for scar management, 451–2 future trends, 458–9 medical need for therapies, 450 new biological approaches, 452–8 scars, 77 formation, 77, 80 treatment options, 92–7 5-fluorouracil, 96 bleomycin, 96–7 compression therapy, 95 corticosteroids, 95 cryotherapy, 95 hypertrophic scars, 93 keloids, 94 lasers, 95–6 radiation, 96 surgery, 93–4 topical silicone gel, 96 Schirmer strip, 305, 306 semi-occlusive dressings, 200, 201 sepsis, 610 sericin, 527 Serratia marcescens, 238–9 Severe Combined Immunodeficiency (SCID), 396 Shippert Medical Technologies Corporation, 313 Silhouette system, 132 silicone, 199 silicone adhesives, 266 silk proteins, 527 silkworm silk, 527 silver, 166, 237, 365, 367, 416, 576 burn wounds, 430–1 clinical evidence on silver and other antimicrobial dressings, 422–9 metal with antimicrobial activity, 439–40 silver nitrate, 416, 430, 440 silver sulphadiazine, 365, 366, 367, 379, 382, 383, 430 skin ageing dysfunctional wound healing onset ageing skin functional result, 6 dermis ageing, 7–8 epidermis ageing, 6–7 mechanisms, 8–10 differential gene expression theory, 10 dysfunctional wound healing onset, 6–10 free radical damage theory, 10 replicative senescence and telomere loss, 8–10 Skin Ethnic, 476 skin ulcer, 11

645

Smith & Nephew, 313, 380, 473, 482, 503 sodium 2-acrylamido 2-methyl propane sulphonic acid, 328 somatic stem cells see adult stem cells specialised proteins, 330 split-thickness skin grafts, 467, 468, 472, 475 spray-on dressings case studies, 214–24 leg ulcer caused by industrial injury, 214–16 dry chronic wound healing, 209–25 protein-based spray-on dressings, 213–14 ideal wound dressing, 211–13 freedom from particulate contamination, 213 gaseous exchange, 211 maintenance of humidity at wound surface, 211 non-adherence, 212 pH maintenance of the wound, 213 protection from trauma, 212 provision of thermal insulation, 212–13 leg ulcer caused by accident in the home, 216–20, 222 complete healing after 10 weeks, 222 inflamed skin and heavily exudating wound, 220 less inflamed wound and periwound, 221 moist wound healing vs dry wound healing for both legs, 222 leg ulcer caused by industrial injury, 214–16, 218 build-up of film on wound, 216 complete healing after 67 days, 217 inflamed periwound area and moderately exudating wound, 215 moist wound healing vs dry wound healing costs, 218 periwound area and wound site reduction, 217 pressure ulcer in hospitalised patient, 220–4 complete healing after 217 days, 224 granulation and remodelling, 223 granulation and visible healthy tissue, 224 sacrum of patient bedridden for 3 years, 223 standard deviation, 147 Staphylococcus aureus, 242, 416, 576, 609 stem cells, 99–100 adult stem cells, 554–61 frequently utilised sources, 556–61 clinical applications, 561–2 current skin substitutes available for wound coverage, 554 embryonic stem cells, 553–4

© Woodhead Publishing Limited, 2011

646

Index

properties, 552 therapies for wound repair, 552–62 StrataDerm, 506–7 Stratatech, 506 Strattice, 501 stromelysin-1, 372 structural proteins, 330 subatmospheric pressure, 592 sulphonated biomaterials biomimetic behaviour in chronic wounds, 341–51 cell-surface effects and neutrophil function, 346 heparin/heparin mimicry, 348–51 interfacial tension and hydrophobic shielding, 342 ionic equilibria and selectivity, 344–5 kinin effects, GAGs and pain inhibition potential, 347–8 matrix metalloprotease absorption and inhibition, 345 osmotic drive and charge repulsion, 342–4 vitronectin/plasmin effects, 346–7 volume swell vs ionic strength, 343 biomimetic models, 330–5 articular cartilage and intervertebral disc, 334–5 characteristics of some natural systems, 330 cornea, 330–4 biomimetic principles, 335–41 other body sites, 340–1 stereochemical considerations, 338–40 structural, biological and toxicological considerations, 335–8 glycosaminoglycan mimics in wound healing, 321–54 biomimetic biomaterials, 321–2 biomimetic models, 330–5 hydrogels based on sulphonated monomers, 353 products based on sulphonated polystyrene, 352 products derived from natural proteoglycan fragments, 352 proteoglycans, 322–4 synthetic and biologic dressings, 353–4 polymers and biomimesis, 324–30 2-hydroxyethyl methacrylate, 327 acrylic acid bis-(3-sulphopropyl)-ester potassium salt, 328 conventional hydrogels, 329–30 functional groups, 326–8 methacrylic acid, 328 methyl methacrylate polymerisation, 326 monomer structures, 325–6

sodium 2-acrylamido 2-methyl propane sulphonic acid, 328 structural considerations, 324–5 support vector machine, 146, 148 Suprasorb A, 441 Suprasorb C, 164 surface-free energy see surface tension surface tension, 248 surface topography, 176 surgery, 93–4 surgical adhesives, 254, 258, 273–80 collagen, gelatin and albumin-based adhesives, 277–9 cyanoacrylates, 275–6 fibrin sealants, 276–7 ideality and practicality, 274–5 mussel adhesives and genetic engineering, 279 protein-based adhesives, 277 synthetic adhesives, 279–80 Surgical Materials Testing Laboratory (SMTL), 168 Surgicel, 313 SVM see support vector machine Synthes, 485 synthetic adhesives, 279–80 Systagenix, 287, 380 systemic antimicrobials see antibiotics systemic inflammatory response syndrome (SIRS), 610 Tabotamps, 164 tasar silkworm see Antheraea mylitta tear collection, 305–6 tear envelope, 304 Teepol, 337 Tegagel, 481 TEI Biosciences, 484, 501 Telfa, 313 telomere hypothesis, 8–10 senescence, 9 tetracycline, 382 TGF-β1, 82, 90, 91, 98, 115 TGF-β2, 82, 91, 98, 115 TGF-β3, 83, 91, 116 TIMP-1, 20–1 TIMP-2, 20–1 tissue, 174 tissue-biomaterial interactions, 174–84 biomaterial surface interactions, 176–82 biointegration, 180–1 biological interactions, 178–9 biomaterial response to tissue environment, 181–2 definitions, 174 tissue response to biomaterial, 182–4 carcinoma, 183–4 immunogenic reaction, 183 infection, 183

© Woodhead Publishing Limited, 2011

Index inflammation, 182 toxicity, 183 tissue-derived inhibitors (TIMPs), 87 tissue equivalent, 504 tissue formation, 80 tissue inhibitors of matrix metalloproteinases (TIMPs), 5, 287, 288 tissue remodelling, 5, 80–1 tissue sealants, 254, 258, 273–80 collagen, gelatin and albumin-based adhesives, 277–9 cyanoacrylates, 275–6 fibrin sealants, 276–7 ideality and practicality, 274–5 mussel adhesives and genetic engineering, 279 protein-based adhesives, 277 synthetic adhesives, 279–80 tobramycin, 379 topical antimicrobials, 612 topical silicone gel, 96 toxicity, 183 Transcyte, 482, 496, 502–3, 508 transforming growth factor-α, 4 transforming growth factor-β, 4, 85, 91, 98, 368, 379, 397, 452–3, 454, 464, 575 duration and magnitude of effect on scarforming healing response, 454 ‘trap’ hypothesis, 12 trauma, 212 Triphasic Wound Model PLUS, 69–71 development, 72–5 graphic representation, 70 Trypanosoma cruzi, 348 tryptone soya broth, 240 Type I collagen, 80 Type III collagen, 80 umbilical cord-derived stem cells, 557, 558, 559 epithelial and mesenchymal cells, 558 mesenchymal cells characteristics, 559 uncontrolled lighting, 143–4 vancomycin, 364 vascular endothelial growth factor, 97, 397 VEGF see vascular endothelial growth factor VEGF-A, 97–8 VEGF-C, 97 venous leg ulcer, 11–12, 504 chronic, 512, 514–16 Versajet, 618 viable but non-culturable (VBNC) bacteria, 45–6 Vigilon, 481 viral gene delivery, 401 virus-like particles (VLP), 405–6 vitronectin, 288–9, 346–7

647

VLU see venous leg ulcer VULCAN trial, 431 water vapour transmission rate (WVTR), 163 wetting, 247 whirlpool debridement, 617 wound colour, 139–49 approaches, 146–8 holistic, 146–7 kurtosis, 148 mean and median, 147 segmentation, 146 standard deviation, 147 digital cameras, 144–5 lighting, 142–4 controlled, 142–3 uncontrolled, 143–4 motivation, 139–42 detection of infection, 141–2 tissue classification, 139–40 wound colour, 145–6 wound inflammation detection, 148–9 logistic regression, ANN and SVM, 149 wound continuum, 61–2 wound contraction, 4–5 wound dressing biomaterials functional aspects of design, 289–94 cell senescence, 293 cellular interaction, 291–2 infection control, 292–3 pain relief, 293–4 temperature, 290–1 wound fluid management, 290 interfacial aspects of compatibility and wound response, 284–94 biological aspects, 287–9 biomaterial aspects, 285–6 wound dressings, 258 materials, 156–61 wound exudate, 161–4, 213 wound exudate collection, 307–8 wound fluid, 21 wound fluid models, 308–9 wound healing, 112, 155, 362 adhesives and interfacial phenomena, 247–81 adhesion, adhesivity and interfacial behaviour, 247–53 adhesive materials overview, 257–73 bioadhesion, 253–7 surgical adhesives and tissue sealants, 273–80 adult vs fetal wound healing, 78, 89–92 differences in cell type and function, 89–91 differences in cytokine profiles, 91–2 electrical simulation, 571–82 antibacterial effects, 575–6

© Woodhead Publishing Limited, 2011

648

Index

current of injury, 573–4 high voltage pulsed current effect, 576–7 low intensity direct currents effect, 577–9 other types applied to wounds, 578–9 physiological effects, 574–5 fibroproliferative scarring, 81–2 hypertrophic scars, 81 keloids, 82 pathogenesis, 82 future trends, 97–100 growth factors and cytokines, 97–8 small molecule compounds, 98–9 stem cells, 99–100 process, 78–81 inflammation, 79–80 tissue formation, 80 tissue remodelling, 80–1 scarless fetal wound healing, 82–9 fetal cutaneous wound healing, 82–4 fetal skin, 84–7 scarless repair in fetal skin, 87–9 scarring and scarless, 77–101 sulphonated biomaterials as glycosaminoglycan mimics, 321–54 biomimetic biomaterials, 321–2 biomimetic models, 330–5 biomimetic principles, 335–41 hydrogels based on sulphonated monomers, 353 polymers and biomimesis, 324–30 possible modes of biomimetic behaviour, 341–51 products based on sulphonated polystyrene, 352 products derived from natural proteoglycan fragments, 352 proteoglycans, 322–4 synthetic and biologic dressings, 353–4 treatment options for scars, 92–7 5-fluorouracil, 96 bleomycin, 96–7 compression therapy, 95 corticosteroids, 95 cryotherapy, 95 hypertrophic scars, 93 keloids, 94 lasers, 95–6 radiation, 96 surgery, 93–4 topical silicone gel, 96 use and relevance of corneal model, 284–315 biomaterials in mucosal wound healing, 309–14 corneal model, 294–302 interfacial phenomena in ocular surface contact lens studies, 302–4

wound dressing biomaterials, 284–94 wound fluid and the tear film collection, 304–9 wound inflammation, 148–9 wound microenvironment, 464–6 growth factors subset and cytokines, 465 wound healing process overview, 465 wound pain, 156–61 wound repair, 78–9 engineered tissues, 463–87 acellular therapies development, 476–85 cellular therapies development, 469–76 traditional approaches, 466–8 wound microenvironment, 464–6 molecular and gene therapies, 395–412 ethical issues, 410–12 future trends, 411–12 gene delivery methods, 400–7 stages, 464, 466 stem cell therapies, 552–62 adult stem cells frequently utilised sources, 556–61 clinical applications, 561–2 see also wound healing wound repair biomaterials functional requirements, 155–69 exudate management, 161–4 future trends, 168–9 infection prevention and control, 164–7 odour management, 167–8 wound pain and dressing materials, 156–61 wound size, 131–9, 149–50 definitions, 131–4 area, 132 calibration artefact, 133 circumference, 132 volume, 133–4 MAVIS measurement device, 135–9 instrument description, 135–7 instrument performance, 138–9 taking images, 137–8 motivation, 131 techniques, 134–5 length and area measurements, 134 volume measurements, 134–5 wound therapy negative pressure, 587–602 history, 588–93 pathophysiologic mechanisms of action, 594–8 science of negative pressure, 593–4 search for the perfect negative pressure technology, 598–602 wounds non-viable tissue debridement, 606–27 background, 607–9 debridement in the diabetic foot, 624–7 debridement organisation, 614

© Woodhead Publishing Limited, 2011

Index debridement timing and types, 614–23 non-viable tissue complications, 609–13 presence of biofilm, 613–14 scoring the debridement effectiveness, 623–4 Wright Medical Technology, 485, 500 Xelma, 481 xenografts, 467

649

zone of inhibition, 234–7 calculation of results, 236 other considerations, 236–7 principle, 234–5 pictorial view, 236 problems and possible modifications, 235 time points, 235–6

© Woodhead Publishing Limited, 2011