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: • •
visiting our web site at www.woodheadpublishing.com contacting Customer Services (e-mail:
[email protected]; fax: +44 (0) 1223 832819; tel.: +44 (0) 1223 499140 ext. 130; address: Woodhead Publishing Limited, 80 High Street, Sawston, Cambridge CB22 3HJ, UK)
If you would like to receive information on forthcoming titles, please send your address details to: Francis Dodds (address, tel. and fax as above; e-mail: francis.
[email protected]). Please confirm which subject areas you are interested in.
© Woodhead Publishing Limited, 2011
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
© Woodhead Publishing Limited, 2011
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
© Woodhead Publishing Limited, 2011
vi
Contents
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
© Woodhead Publishing Limited, 2011
155 156
Contents
vii
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
© Woodhead Publishing Limited, 2011
174 175 176 182 184 184
186
186 187 189 199 201 203 203 203
209 209 211 213 214 224 225
viii
Contents
10
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
© Woodhead Publishing Limited, 2011
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
© Woodhead Publishing Limited, 2011
361 362 363 373 374 377 380 383 385 387
395 400 407 410 411 412
416 418 439 442 443
x
Contents
17
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
© Woodhead Publishing Limited, 2011
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
© Woodhead Publishing Limited, 2011
571 573 574 575 576 577 578 580 582 582
xii
Contents
23
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
© Woodhead Publishing Limited, 2011
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
xiv
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]
© Woodhead Publishing Limited, 2011
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]
© Woodhead Publishing Limited, 2011
xvi
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
xx
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,
© Woodhead Publishing Limited, 2011
Introduction
xxi
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
4
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
© Woodhead Publishing Limited, 2011
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.
© Woodhead Publishing Limited, 2011
6
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.
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
7
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
© Woodhead Publishing Limited, 2011
8
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
© Woodhead Publishing Limited, 2011
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
10
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds (a)
11
(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
© Woodhead Publishing Limited, 2011
12
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
13
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-
© Woodhead Publishing Limited, 2011
14
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
15
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
© Woodhead Publishing Limited, 2011
16
Advanced wound repair therapies
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).
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
17
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,
© Woodhead Publishing Limited, 2011
18
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
19
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).
© Woodhead Publishing Limited, 2011
20
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
21
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
© Woodhead Publishing Limited, 2011
22
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
23
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
© Woodhead Publishing Limited, 2011
24
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
25
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
Abd-El-Aleem, S. A., Ferguson, M. W., Appleton, I., Bhowmick, A., McCollum, C. N., & Ireland, G. W. (2001). “Expression of cyclooxygenase isoforms in normal human skin and chronic venous ulcers”, J Pathol, 195, 616–23. Abd-El-Aleem, S. A., Morgan, C., Ferguson, M. W., McCollum, C. N., & Ireland, G. W. (2005). “Spatial distribution of mast cells in chronic venous leg ulcers”, Eur J Histochem, 49, 265–72. Agren, M. S., Eaglstein, W. H., Ferguson, M. W., Harding, K. G., Moore, K., SaarialhoKere, U. K., & Schultz, G. S. (2000). “Causes and effects of the chronic inflammation in venous leg ulcers”, Acta Derm Venereol Suppl (Stockh), 210, 3–17. Agren, M. S., Steenfos, H. H., Dabelsteen, S., Hansen, J. B., & Dabelsteen, E. (1999). “Proliferation and mitogenic response to PDGF-BB of fibroblasts isolated from chronic venous leg ulcers is ulcer-age dependent”, J Invest Dermatol, 112, 463–9. AlDahlawi, S., Eslami, A., Hakkinen, L., Larjava, H. S., Hudson, L. G., Newkirk, K. M., Chandler, H. L., Choi, C., Fossey, S. L., Parent, A. E., & Kusewitt, D. F.
© Woodhead Publishing Limited, 2011
26
Advanced wound repair therapies
(2006). “The alphavbeta6 integrin plays a role in compromised epidermal wound healing”, Wound Repair Regen, 14, 289–97. Allhorn, M., Lundqvist, K., Schmidtchen, A., & Akerstrom, B. (2003). “Hemescavenging role of alpha1-microglobulin in chronic ulcers”, J Invest Dermatol, 121, 640–6. Almqvist, S., Werthen, M., Johansson, A., Tornqvist, J., Agren, M. S., & Thomsen, P. (2009). “Evaluation of a near-senescent human dermal fibroblast cell line and effect of amelogenin”, Br J Dermatol, 160, 1163–71. Alvarez, E. & Santa, M. C. (1996). “Influence of the age and sex on respiratory burst of human monocytes”, Mech Ageing Dev, 90, 157–61. Ashcroft, G. S., Horan, M. A., & Ferguson, M. W. J. (1995). “The effects of aging on cutaneous wound-healing in mammals”, J Anat, 187, 1–26. Ashcroft, G. S., Herrick, S. E., Tarnuzzer, R. W., Horan, M. A., Schultz, G. S., & Ferguson, M. W. (1997a). “Human ageing impairs injury-induced in vivo expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 proteins and mRNA”, J Pathol, 183, 169–76. Ashcroft, G. S., Horan, M. A., Herrick, S. E., Tarnuzzer, R. W., Schultz, G. S., & Ferguson, M. J. (1997b). “Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMPs) in normal skin and acute cutaneous wounds of healthy humans”, Cell Tissue Res, 290, 581–91. Ashcroft, G. S., Horan, M. A., & Ferguson, M. W. (1998). “Aging alters the inflammatory and endothelial cell adhesion molecule profiles during human cutaneous wound healing”, Lab Invest, 78, 47–58. Ashcroft, G. S., Greenwell-Wild, T., Horan, M. A., Wahl, S. M., & Ferguson, M. W. (1999). “Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inflammatory response”, Am J Pathol, 155, 1137– 46. Ashcroft, G. S., Mills, S. J., Lei, K., Gibbons, L., Jeong, M. J., Taniguchi, M., Burow, M., Horan, M. A., Wahl, S. M., & Nakayama, T. (2003). “Estrogen modulates cutaneous wound healing by downregulating macrophage migration inhibitory factor”, J Clin Invest, 111, 1309–18. Baggiolini, M. (2001). “Chemokines in pathology and medicine”, J Intern Med, 250, 91–104. Bailey, A. J. (2001). “Molecular mechanisms of ageing in connective tissues”, Mech Ageing Dev, 122, 735–55. Baird, D. M., Rowson, J., Wynford-Thomas, D., & Kipling, D. (2003). “Extensive allelic variation and ultrashort telomeres in senescent human cells”, Nat Genet, 33, 203–7. Baker, S. R., Stacey, M. C., Jopp-McKay, A. G., Hoskin, S. E., & Thompson, P. J. (1991). “Epidemiology of chronic venous ulcers”, Br J Surg, 78, 864–7. Barbul, A., Shawe, T., Rotter, S. M., Efron, J. E., Wasserkrug, H. L., & Badawy, S. B. (1989). “Wound healing in nude mice: a study on the regulatory role of lymphocytes in fibroplasia”, Surgery, 105, 764–9. Barone, E. J., Yager, D. R., Pozez, A. L., Olutoye, O. O., Crossland, M. C., Diegelmann, R. F., & Cohen, I. K. (1998). “Interleukin-1alpha and collagenase activity are elevated in chronic wounds”, Plast Reconstr Surg, 102, 1023–7. Beckman, K. B. & Ames, B. N. (1998). “The free radical theory of aging matures”, Physiol Rev, 78, 547–81.
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
27
Bell, E., Ivarsson, B., & Merrill, C. (1979). “Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro”, Proc Natl Acad Sci U S A, 76, 1274–8. Bentley, J. P. (1979). “Aging of collagen”, Trends Biotechnol, 73, 80–3. Botusan, I. R., Sunkari, V. G., Savu, O., Catrina, A. I., Grunler, J., Lindberg, S., Pereira, T., Yla-Herttuala, S., Poellinger, L., Brismar, K., & Catrina, S. B. (2008). “Stabilization of HIF-1alpha is critical to improve wound healing in diabetic mice”, Proc Natl Acad Sci U S A, 105, 19426–31. Boudreau, N. J. & Varner, J. A. (2004). “The homeobox transcription factor Hox D3 promotes integrin a5b1 expression and function during angiogenesis”, J Biol Chem, 279, 4862–8. Bradfield, P. F., Amft, N., Vernon-Wilson, E., Exley, A. E., Parsonage, G., Rainger, G. E., Nash, G. B., Thomas, A. M., Simmons, D. L., Salmon, M., & Buckley, C. D. (2003). “Rheumatoid fibroblast-like synoviocytes overexpress the chemokine stromal cell-derived factor 1 (CXCL12), which supports distinct patterns and rates of CD4+ and CD8+ T cell migration within synovial tissue”, Arthritis Rheum, 48, 2472–82. Bradley, S. F., Vibhagool, A., Kunkel, S. L., & Kauffman, C. A. (1989). “Monokine secretion in aging and protein malnutrition”, J Leukoc Biol, 45, 510–4. Brakman, M., Faber, W. R., Kerckhaert, J. A., Kraaijenhagen, R. J., & Hart, H. C. (1992). “Immunofluorescence studies of atrophie blanche with antibodies against fibrinogen, fibrin, plasminogen activator inhibitor, factor VIII-related antigen, and collagen type IV”, Vasa, 21, 143–8. Braverman, I. M. & Fonferko, E. (1982). “Studies in cutaneous aging: II. The microvasculature”, Trends Biotechnol, 78, 444–8. Browse, N. L. & Burnand, K. G. (1982). “The cause of venous ulceration”, Lancet, 2, 243–5. Buckley, C. D. (2003a). “Michael Mason prize essay 2003. Why do leucocytes accumulate within chronically inflamed joints?”, Rheumatology (Oxford), 42, 1433–44. Buckley, C. D. (2003b). “Why does chronic inflammatory joint disease persist?”, Clin Med, 3, 361–6. Burman, A., Haworth, O., Bradfield, P., Parsonage, G., Filer, A., Thomas, A. M., Amft, N., Salmon, M., & Buckley, C. D. (2005a). “The role of leukocyte-stromal interactions in chronic inflammatory joint disease”, Joint Bone Spine, 72, 10–6. Burman, A., Haworth, O., Hardie, D. L., Amft, E. N., Siewert, C., Jackson, D. G., Salmon, M., & Buckley, C. D. (2005b). “A chemokine-dependent stromal induction mechanism for aberrant lymphocyte accumulation and compromised lymphatic return in rheumatoid arthritis”, J Immunol, 174, 1693–700. Burrow, J. W., Koch, J. A., Chuang, H. H., Zhong, W., Dean, D. D., & Sylvia, V. L. (2007). “Nitric oxide donors selectively reduce the expression of matrix metalloproteinases-8 and -9 by human diabetic skin fibroblasts”, J Surg Res, 140, 90–8. Callaghan, M. J., Chang, E. I., Seiser, N., Aarabi, S., Ghali, S., Kinnucan, E. R., Simon, B. J., & Gurtner, G. C. (2008). “Pulsed electromagnetic fields accelerate normal and diabetic wound healing by increasing endogenous FGF-2 release”, Plast Reconstr Surg, 121, 130–41. Campisi, J. (1996). “Replicative senescence: an old lives’ tale?”, Cell, 84, 497–500.
© Woodhead Publishing Limited, 2011
28
Advanced wound repair therapies
Campisi, J. (1998). “The role of cellular senescence in skin aging”, J Investig Dermatol Symp Proc, 3, 1–5. Cha, J., Kwak, T., Butmarc, J., Kim, T. A., Yufit, T., Carson, P., Kim, S. J., & Falanga, V. (2008). “Fibroblasts from non-healing human chronic wounds show decreased expression of beta ig-h3, a TGF-beta inducible protein”, J Dermatol Sci, 50, 15–23. Chakravarti, B. & Abraham, G. N. (1999). “Aging and T-cell-mediated immunity”, Mech Ageing Dev, 108, 183–206. Chang, E. & Harley, C. B. (1995). “Telomere length and replicative aging in human vascular tissues”, Proc Natl Acad Sci U S A, 92, 11190–4. Chang, E., Yang, J., Nagavarapu, U., & Herron, G. S. (2002). “Aging and survival of cutaneous microvasculature”, Trends Biotechnol, 118, 752–8. Chaudhuri, V., Zhou, L., & Karasek, M. (2007). “Inflammatory cytokines induce the transformation of human dermal microvascular endothelial cells into myofibroblasts: a potential role in skin fibrogenesis”, J Cutan Pathol, 34, 146–53. Cheatle, T. (1991). “Venous ulceration and free radicals”, Br J Dermatol, 124, 508. Chiba, Y., Yamashita, Y., Ueno, M., Fujisawa, H., Hirayoshi, K., Hohmura, K., Tomimoto, H., Akiguchi, I., Satoh, M., Shimada, A., & Hosokawa, M. (2005). “Cultured murine dermal fibroblast-like cells from senescence-accelerated mice as in vitro models for higher oxidative stress due to mitochondrial alterations”, J Gerontol A Biol Sci Med Sci, 60, 1087–98. Choi, E. H., Demerjian, M., Crumrine, D., Brown, B. E., Mauro, T., Elias, P. M., & Feingold, K. R. (2006). “Glucocorticoid blockade reverses psychological stressinduced abnormalities in epidermal structure and function”, Am J Physiol Regul Integr Comp Physiol, 291, R1657–R1662. Christian, L. M., Graham, J. E., Padgett, D. A., Glaser, R., & Kiecolt-Glaser, J. K. (2006). “Stress and wound healing”, Neuroimmunomodulation, 13, 337–46. Cianfarani, F., Tommasi, R., Failla, C. M., Viviano, M. T., Annessi, G., Papi, M., Zambruno, G., & Odorisio, T. (2006). “Granulocyte/macrophage colony-stimulating factor treatment of human chronic ulcers promotes angiogenesis associated with de novo vascular endothelial growth factor transcription in the ulcer bed”, Br J Dermatol, 154, 34–41. Clark, R. A. F. 1996, “Wound repair: Overview and general considerations,” in The Molecular and Cellular Biology of Wound Repair, R. A. F. Clark, ed., Plenum Press, New York, pp. 3–50. Claudy, A. L., Mirshahi, M., Soria, C., & Soria, J. (1991). “Detection of undegraded fibrin and tumor necrosis factor-alpha in venous leg ulcers”, J Am Acad Dermatol, 25, 623–7. Coleridge Smith, P. D., Thomas, P., Scurr, J. H., & Dormandy, J. A. (1988). “Causes of venous ulceration: a new hypothesis”, Br Med J (Clin Res Ed), 296, 1726–7. Conway, K. P., Price, P., Harding, K. G., & Jiang, W. G. (2007). “The role of vascular endothelial growth inhibitor in wound healing”, Int Wound J, 4, 55–64. Cook, H., Stephens, P., Davies, K. J., Harding, K. G., & Thomas, D. W. (2000). “Defective extracellular matrix reorganization by chronic wound fibroblasts is associated with alterations in TIMP-1, TIMP-2, and MMP-2 activity”, J Invest Dermatol, 115, 225–33. Cowin, A. J., Hatzirodos, N., Holding, C. A., Dunaiski, V., Harries, R. H., Rayner, T. E., Fitridge, R., Cooter, R. D., Schultz, G. S., & Belford, D. A. (2001). “Effect of
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
29
healing on the expression of transforming growth factor beta(s) and their receptors in chronic venous leg ulcers”, J Invest Dermatol, 117, 1282–9. Davila, D. R., Edwards, C. K., Arkins, S., Simon, J., & Kelley, K. W. (1990). “Interferongamma-induced priming for secretion of superoxide anion and tumor necrosis factor-alpha declines in macrophages from aged rats”, FASEB J, 4, 2906–11. De Mattei, M., Ongaro, A., Magaldi, S., Gemmati, D., Legnaro, A., Palazzo, A., Masieri, F., Pellati, A., Catozzi, L., Caruso, A., & Zamboni, P. (2008). “Time- and dose-dependent effects of chronic wound fluid on human adult dermal fibroblasts”, Dermatol Surg, 34, 347–56. de Rigal, J., Escoffier, C., Querleux, B., Faivre, B., Agache, P., & Leveque, J. L. (1989). “Assessment of aging of the human skin by in vivo ultrasonic imaging”, Trends Biotechnol, 93, 621–5. Deie, M., Marui, T., Allen, C. R., Hildebrand, K. A., Georgescu, H. I., Niyibizi, C., & Woo, S. L. (1997). “The effects of age on rabbit MCL fibroblast matrix synthesis in response to TGF-beta 1 or EGF”, Mech Ageing Dev, 97, 121–30. Devalaraja, R. M., Nanney, L. B., Du, J., Qian, Q., Yu, Y., Devalaraja, M. N., & Richmond, A. (2000). “Delayed wound healing in CXCR2 knockout mice”, Trends Biotechnol, 115, 234–44. Dhaene, K., Van Marck, E., & Parwaresch, R. (2000). “Telomeres, telomerase and cancer: an up-date”, Virchows Arch, 437, 1–16. Dimri, G. P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E. E., Linskens, M., Rubelj, I., & Pereira-Smith, O. (1995). “A biomarker that identifies senescent human cells in culture and in aging skin in vivo”, Proc Natl Acad Sci U S A, 92, 9363–7. Eming, S. A., Krieg, T., & Davidson, J. M. (2007). “Inflammation in wound repair: molecular and cellular mechanisms”, J Invest Dermatol, 127, 514–25. Endo, T., Ogushi, F., Sone, S., Ogura, T., Taketani, Y., Hayashi, Y., Ueda, N., & Yamamoto, S. (1995). “Induction of cyclooxygenase-2 is responsible for interleukin-1 beta-dependent prostaglandin E2 synthesis by human lung fibroblasts”, Am J Respir Cell Mol Biol, 12, 358–65. Falanga, V. (2001). Cutaneous Wound Healing, First edn, Martin Dunitz Ltd, London. Falanga, V. & Eaglstein, W. H. (1993). “The ‘trap’ hypothesis of venous ulceration”, Lancet, 341, 1006–8. Faragher, R. G. & Kipling, D. (1998). “How might replicative senescence contribute to human ageing?”, Bioessays, 20, 985–91. Finkel, T. & Holbrook, N. J. (2000). “Oxidants, oxidative stress and the biology of ageing”, Nature, 408, 239–47. Galkowska, H., Olszewski, W. L., & Wojewodzka, U. (2005). “Keratinocyte and dermal vascular endothelial cell capacities remain unimpaired in the margin of chronic venous ulcer”, Arch Dermatol Res, 296, 286–95. Gamble, J. R., Harlan, J. M., Klebanoff, S. J., & Vadas, M. A. (1985). “Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor”, Proc Natl Acad Sci U S A, 82, 8667–71. Ghadially, R., Brown, B. E., Sequeira-Martin, S. M., Feingold, K. R., & Elias, P. M. (1995). “The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model”, J Clin Invest, 95, 2281–90.
© Woodhead Publishing Limited, 2011
30
Advanced wound repair therapies
Gilchrest, B. A. (1983). “In vitro assessment of keratinocyte aging”, Trends Biotechnol, 81, 184s–9s. Gilchrest, B. A. (1995). Photodamage Blackwell Science, Cambridge, MA. Gilchrest, B. A., Blog, F. B., & Szabo, G. (1979). “Effects of aging and chronic sun exposure on melanocytes in human skin”, Trends Biotechnol, 73, 141–3. Gilchrest, B. A., Szabo, G., Flynn, E., & Goldwyn, R. M. (1983). “Chronologic and actinically induced aging in human facial skin”, Trends Biotechnol, 80 Suppl, 81s–5s. Goren, I., Muller, E., Pfeilschifter, J., & Frank, S. (2006). “Severely impaired insulin signaling in chronic wounds of diabetic ob/ob mice: a potential role of tumor necrosis factor-alpha”, Am J Pathol, 168, 765–77. Grewe, M. (2001). “Chronological ageing and photoageing of dendritic cells”, Clin Exp Dermatol, 26, 608–12. Griffith, J. D., Comeau, L., Rosenfield, S., Stansel, R. M., Bianchi, A., Moss, H., & de Lange, T. (1999). “Mammalian telomeres end in a large duplex loop”, Cell, 97, 503–14. Grinnell, F., Zhu, M. (1996). “Fibronectin degradation in chronic wounds depends on the relative levels of elastase, alpha1-proteinase inhibitor, and alpha2macroglobulin”, J Invest Dermatol, 106, 335–41. Hakkinen, L., Koivisto, L., Gardner, H., Saarialho-Kere, U., Carroll, J. M., Lakso, M., Rauvala, H., Laato, M., Heino, J., & Larjava, H. (2004). “Increased expression of beta6-integrin in skin leads to spontaneous development of chronic wounds”, Am J Pathol, 164, 229–42. Harding, K. G., Morris, H. L., & Patel, G. K. (2002). “Science, medicine and the future: healing chronic wounds”, BMJ, 324, 160–3. Harman, D. (1956). “Aging a theory based on free radical and radiation chemistry”, J Gerontol, 11, 298–300. Hasan, A., Murata, H., Falabella, A., Ochoa, S., Zhou, L., Badiavas, E., & Falanga, V. (1997). “Dermal fibroblasts from venous ulcers are unresponsive to the action of transforming growth factor-beta 1”, J Dermatol Sci, 16, 59–66. Hayflick, L. (1965). “The limited in vitro life time of human diploid cell strains”, Exp Cell Res, 37, 614–36. Heilborn, J. D., Nilsson, M. F., Kratz, G., Weber, G., Sorensen, O., Borregaard, N., & Stahle-Backdahl, M. (2003). “The cathelicidin anti-microbial peptide LL-37 is involved in re-epithelialization of human skin wounds and is lacking in chronic ulcer epithelium”, J Invest Dermatol, 120, 379–89. Herouy, Y., Mellios, P., Bandemir, E., Stetter, C., Dichmann, S., Idzko, M., Hofmann, C., Vanscheidt, W., Schopf, E., & Norgauer, J. (2000). “Autologous platelet-derived wound healing factor promotes angiogenesis via alphavbeta3-integrin expression in chronic wounds”, Int J Mol Med, 6, 515–9. Herrick, S., Ashcroft, G., Ireland, G., Horan, M., McCollum, C., & Ferguson, M. (1997). “Up-regulation of elastase in acute wounds of healthy aged humans and chronic venous leg ulcers are associated with matrix degradation”, Lab Invest, 77, 281–8. Herrick, S. E., Sloan, P., McGurk, M., Freak, L., McCollum, C. N., & Ferguson, M. W. (1992). “Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers”, Am J Pathol, 141, 1085–95.
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
31
Herrick, S. E., Ireland, G. W., Simon, D., McCollum, C. N., & Ferguson, M. W. (1996). “Venous ulcer fibroblasts compared with normal fibroblasts show differences in collagen but not fibronectin production under both normal and hypoxic conditions”, J Invest Dermatol, 106, 187–93. Hieta, N., Impola, U., Lopez-Otin, C., Saarialho-Kere, U., & Kahari, V. M. (2003). “Matrix metalloproteinase-19 expression in dermal wounds and by fibroblasts in culture”, J Invest Dermatol, 121, 997–1004. Holt, D. R., Kirk, S. J., Regan, M. C., Hurson, M., Lindblad, W. J., & Barbul, A. (1992). “Effect of age on wound healing in healthy human beings”, Surgery, 112, 293–7. Hudson, L. G., Newkirk, K. M., Chandler, H. L., Choi, C., Fossey, S. L., Parent, A. E., & Kusewitt, D. F. (2009). “Cutaneous wound re-epithelialization is compromised in mice lacking functional Slug (Snai2)”, J Dermatol Sci, 56, 19–26. James, T. J., Hughes, M. A., Hofman, D., Cherry, G. W., & Taylor, R. P. (2001). “Antioxidant characteristics of chronic wound fluid”, Br J Dermatol, 145, 185–6. James, T. J., Hughes, M. A., Cherry, G. W., & Taylor, R. P. (2003). “Evidence of oxidative stress in chronic venous ulcers”, Wound Repair Regen, 11, 172–6. Jameson, J. M., Sharp, L. L., Witherden, D. A., & Havran, W. L. (2004). “Regulation of skin cell homeostasis by gamma delta T cells”, Front Biosci, 9, 2640–51. Kawanishi, S. & Oikawa, S. (2004). “Mechanism of telomere shortening by oxidative stress”, Ann N Y Acad Sci, 1019, 278–84. Kligman, A. M. & Lavker, R. M. (1988). “Cutaneous ageing: the differences between intrisic aging and photoageing”, J Cutan Aging Cosmetol Dermatol, 1, 5–12. Kohen, R. & Gati, I. (2000). “Skin low molecular weight antioxidants and their role in aging and in oxidative stress”, Toxicology, 148, 149–57. Kondo, H. & Yonezawa, Y. (1992). “Changes in the migratory ability of human lung and skin fibroblasts during in vitro aging and in vivo cellular senescence”, Mech Ageing Dev, 63, 223–33. Konturek, P. C., Brzozowski, T., Konturek, S. J., Kwiecien, S., Dembinski, A., & Hahn, E. G. (2001). “Influence of bacterial lipopolysaccharide on healing of chronic experimental ulcer in rat”, Scand J Gastroenterol, 36, 1239–47. Kramer, R. H., Fuh, G. M., Bensch, K. G., & Karasek, M. A. (1985). “Synthesis of extracellular matrix glycoproteins by cultured microvascular endothelial cells isolated from the dermis of neonatal and adult skin”, J Cell Physiol, 123, 1–9. Krasner, D. (1998). “Painful venous ulcers: themes and stories about living with the pain and suffering”, J Wound Ostomy Continence Nurs, 25, 158–68. Kurban, R. S. & Bhawan, J. (1990). “Histologic changes in skin associated with aging”, J Dermatol Surg Oncol, 16, 908–14. Kwong, L. K. & Sohal, R. S. (2000). “Age-related changes in activities of mitochondrial electron transport complexes in various tissues of the mouse”, Arch Biochem Biophys, 373, 16–22. Lal, B. K., Saito, S., Pappas, P. J., Padberg, F. T., Jr., Cerveira, J. J., Hobson, R. W., & Duran, W. N. (2003). “Altered proliferative responses of dermal fibroblasts to TGF-beta1 may contribute to chronic venous stasis ulcer”, J Vasc Surg, 37, 1285–93. Landau, Z., David, M., Aviezer, D., & Yayon, A. (2001). “Heparin-like inhibitory activity to fibroblast growth factor-2 in wound fluids of patients with chronic skin ulcers and its modulation during wound healing”, Wound Repair Regen, 9, 323–8.
© Woodhead Publishing Limited, 2011
32
Advanced wound repair therapies
Lee, H. C., Yin, P. H., Chi, C. W., & Wei, Y. H. (2002). “Increase in mitochondrial mass in human fibroblasts under oxidative stress and during replicative cell senescence”, J Biomed Sci, 9, 517–26. Liao, H., Zakhaleva, J., & Chen, W. (2009). “Cells and tissue interactions with glycated collagen and their relevance to delayed diabetic wound healing”, Biomaterials, 30, 1689–96. Lipschitz, D. A. & Udupa, K. B. (1986). “Influence of aging and protein deficiency on neutrophil function”, J Gerontol, 41, 690–4. Lobmann, R., Ambrosch, A., Schultz, G., Waldmann, K., Schiweck, S., & Lehnert, H. (2002). “Expression of matrix-metalloproteinases and their inhibitors in the wounds of diabetic and non-diabetic patients”, Diabetologia, 45, 1011–6. Loot, M. A., Kenter, S. B., Au, F. L., van Galen, W. J., Middelkoop, E., Bos, J. D., & Mekkes, J. R. (2002). “Fibroblasts derived from chronic diabetic ulcers differ in their response to stimulation with EGF, IGF-I, bFGF and PDGF-AB compared to controls”, Eur J Cell Biol, 81, 153–60. 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”, Trends Biotechnol, 111, 850–7. Loughlin, D. T. & Artlett, C. M. (2009). “3-Deoxyglucosone-collagen alters human dermal fibroblast migration and adhesion: implications for impaired wound healing in patients with diabetes”, Wound Repair Regen, 17, 739–49. Luster, A. D., Cardiff, R. D., MacLean, J. A., Crowe, K., & Granstein, R. D. (1998). “Delayed wound healing and disorganized neovascularization in transgenic mice expressing the IP-10 chemokine”, Proc Assoc Am Physicians, 110, 183–96. Mack, J. A. & Maytin, E. V. (2010). “Persistent Inflammation and Angiogenesis during Wound Healing in K14-Directed Hoxb13 Transgenic Mice”, J Invest Dermatol, 130, 856–65. Margolis, D. J., Bilker, W., Knauss, J., Baumgarten, M., & Strom, B. L. (2002). “The incidence and prevalence of pressure ulcers among elderly patients in general medical practice”, Ann Epidemiol, 12, 321–5. Mekkes, J. R., Loots, M. A., Van Der Wal, A. C., & Bos, J. D. (2003). “Causes, investigation and treatment of leg ulceration”, Br J Dermatol, 148, 388–401. Mendez, M. V., Stanley, A., Park, H. Y., Shon, K., Phillips, T., & Menzoian, J. O. (1998a). “Fibroblasts cultured from venous ulcers display cellular characteristics of senescence”, J Vasc Surg, 28, 876–83. Mendez, M. V., Stanley, A., Phillips, T., Murphy, M., Menzoian, J. O., & Park, H. Y. (1998b). “Fibroblasts cultured from distal lower extremities in patients with venous reflux display cellular characteristics of senescence”, J Vasc Surg, 28, 1040–50. Mendez, M. V., Raffetto, J. D., Phillips, T., Menzoian, J. O., & Park, H. Y. (1999). “The proliferative capacity of neonatal skin fibroblasts is reduced after exposure to venous ulcer wound fluid: A potential mechanism for senescence in venous ulcers”, J Vasc Surg, 30, 734–43. Menke, N. B., Ward, K. R., Witten, T. M., Bonchev, D. G., & Diegelmann, R. F. (2007). “Impaired wound healing”, Clin Dermatol, 25, 19–25. Mirastschijski, U., Impola, U., Jahkola, T., Karlsmark, T., Agren, M. S., & SaarialhoKere, U. (2002). “Ectopic localization of matrix metalloproteinase-9 in chronic cutaneous wounds”, Hum Pathol, 33, 355–64.
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
33
Moore, K., Ruge, F., & Harding, K. G. (1997). “T lymphocytes and the lack of activated macrophages in wound margin biopsies from chronic leg ulcers”, Br J Dermatol, 137, 188–94. Moseley, R., Stewart, J. E., Stephens, P., Waddington, R. J., & Thomas, D. W. (2004). “Extracellular matrix metabolites as potential biomarkers of disease activity in wound fluid: lessons learned from other inflammatory diseases?”, Br J Dermatol, 150, 401–13. Muggleton-Harris, A. L., Reisert, P. S., & Burghoff, R. L. (1982). “In vitro characterization of response to stimulus (wounding) with regard to ageing in human skin fibroblasts”, Mech Ageing Dev, 19, 37–43. Nagelkerken, L., Hertogh-Huijbregts, A., Dobber, R., & Drager, A. (1991). “Agerelated changes in lymphokine production related to a decreased number of CD45RBhi CD4+ T cells”, Eur J Immunol, 21, 273–81. Naka, K., Tachibana, A., Ikeda, K., & Motoyama, N. (2004). “Stress-induced premature senescence in hTERT-expressing ataxia telangiectasia fibroblasts”, J Biol Chem, 279, 2030–7. Norsgaard, H., Clark, B. F. C., & Rattan, S. I. S. (1996). “Distinction between differentiation and senescence and the absence of increased apoptosis in human keratinocytes undergoing cellular aging in vitro”, Exp Gerontol, 31, 563–70. Nwomeh, B. C., Liang, H. X., Cohen, I. K., & Yager, D. R. (1999). “MMP-8 is the predominant collagenase in healing wounds and nonhealing ulcers”, J Surg Res, 81, 189–95. Okuda, K., Khan, M. Y., Skurnick, J., Kimura, M., Aviv, H., & Aviv, A. (2000). “Telomere attrition of the human abdominal aorta: relationships with age and atherosclerosis”, Atherosclerosis, 152, 391–8. Olovnikov, A. M. (1973). “A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon”, J Theor Biol, 41, 181–90. Ongenae, K. C., Phillips, T. J., & Park, H. Y. (2000). “Level of fibronectin mRNA is markedly increased in human chronic wounds”, Dermatol Surg, 26, 447–51. Parks, W. C. (1999). “Matrix metalloproteinases in repair”, Wound Repair Regen, 7, 423–32. Passi, A., Albertini, R., Campagnari, F., & De Luca, G. (1997). “Modifications of proteoglycans extracted from monolayer cultures of young and senescent human skin fibroblasts”, FEBS Lett, 420, 175–8. Passos, J. F., Saretzki, G., Ahmed, S., Nelson, G., Richter, T., Peters, H., Wappler, I., Birket, M. J., Harold, G., Schaeuble, K., Birch-Machin, M. A., Kirkwood, T. B., & Von Zglinicki, T. (2007). “Mitochondrial dysfunction accounts for the stochastic heterogeneity in telomere-dependent senescence”, PLoS Biol, 5, e110. Patel, H. R. & Miller, R. A. (1992). “Age-associated changes in mitogen-induced protein phosphorylation in murine T lymphocytes”, Eur J Immunol, 22, 253– 60. Persoon, A., Heinen, M. M., van der Vleuten, C. J., de Rooij, M. J., Van de Kerkhof, P. C., & van Achterberg, T. (2004). “Leg ulcers: a review of their impact on daily life”, J Clin Nurs, 13, 341–54. Peterson, J. M., Barbul, A., Breslin, R. J., Wasserkrug, H. L., & Efron, G. (1987). “Significance of T-lymphocytes in wound healing”, Surgery, 102, 300–5.
© Woodhead Publishing Limited, 2011
34
Advanced wound repair therapies
Phillips, T., Stanton, B., Provan, A., & Lew, R. (1994). “A study of the impact of leg ulcers on quality of life: financial, social, and psychologic implications”, J Am Acad Dermatol, 31, 49–53. Pirila, E., Korpi, J. T., Korkiamaki, T., Jahkola, T., Gutierrez-Fernandez, A., LopezOtin, C., Saarialho-Kere, U., Salo, T., & Sorsa, T. (2007). “Collagenase-2 (MMP-8) and matrilysin-2 (MMP-26) expression in human wounds of different etiologies”, Wound Repair Regen, 15, 47–57. Podda, M. & Grundmann-Kollmann, M. (2001). “Low molecular weight antioxidants and their role in skin ageing”, Clin Exp Dermatol, 26, 578–82. Probin, V., Wang, Y., & Zhou, D. (2007). “Busulfan-induced senescence is dependent on ROS production upstream of the MAPK pathway”, Free Radic Biol Med, 42, 1858–65. Raffetto, J. D., Mendez, M. V., Marien, B. J., Byers, H. R., Phillips, T. J., Park, H. Y., & Menzoian, J. O. (2001). “Changes in cellular motility and cytoskeletal actin in fibroblasts from patients with chronic venous insufficiency and in neonatal fibroblasts in the presence of chronic wound fluid”, J Vasc Surg, 33, 1233–41. Raffetto, J. D., Vasquez, R., Goodwin, D. G., & Menzoian, J. O. (2006). “Mitogenactivated protein kinase pathway regulates cell proliferation in venous ulcer fibroblasts”, Vasc Endovascular Surg, 40, 59–66. Rao, C. N., Ladin, D. A., Liu, Y. Y., Chilukuri, K., Hou, Z. Z., & Woodley, D. T. (1995). “Alpha 1-antitrypsin is degraded and non-functional in chronic wounds but intact and functional in acute wounds: the inhibitor protects fibronectin from degradation by chronic wound fluid enzymes”, J Invest Dermatol, 105, 572–8. Rattan, S. I. & Derventzi, A. (1991). “Altered cellular responsiveness during ageing”, Bioessays, 13, 601–6. Reed, M. J., Corsa, A., Pendergrass, W., Penn, P., Sage, E. H., & Abrass, I. B. (1998). “Neovascularization in aged mice: delayed angiogenesis is coincident with decreased levels of transforming growth factor beta1 and type I collagen”, Am J Pathol, 152, 113–23. Riedel, K., Riedel, F., Goessler, U. R., Germann, G., & Sauerbier, M. (2007). “Tgfbeta antisense therapy increases angiogenic potential in human keratinocytes in vitro”, Arch Med Res, 38, 45–51. Riedel, K., Koellensperger, E., Ryssel, H., Riedel, F., Goessler, U. R., Germann, G., & Kremer, T. (2008). “Abrogation of TGF-beta by antisense oligonucleotides modulates expression of VEGF and increases angiogenic potential in isolated fibroblasts from radiated skin”, Int J Mol Med, 22, 473–80. Roubenoff, R., Harris, T. B., Abad, L. W., Wilson, P. W., Dallal, G. E., & Dinarello, C. A. (1998). “Monocyte cytokine production in an elderly population: effect of age and inflammation”, J Gerontol A Biol Sci Med Sci, 53, M20–M26. Saarialho-Kere, U., Kerkela, E., Jahkola, T., Suomela, S., Keski-Oja, J., & Lohi, J. (2002). “Epilysin (MMP-28) expression is associated with cell proliferation during epithelial repair”, J Invest Dermatol, 119, 14–21. Saarialho-Kere, U. K. (1998). “Patterns of matrix metalloproteinase and TIMP expression in chronic ulcers”, Arch Dermatol Res, 290 Suppl, S47–S54. Sardy, M. (2009). “Role of matrix metalloproteinases in skin ageing”, Connect Tissue Res, 50, 132–8. Schneider, E. L. & Mitsui, Y. (1976). “The relationship between in vitro cellular aging and in vivo human age”, Proc Natl Acad Sci U S A, 73, 3584–8.
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
35
Scott, H. J., Coleridge, S. P., & Scurr, J. H. (1991). “Histological study of white blood cells and their association with lipodermatosclerosis and venous ulceration”, Br J Surg, 78, 210–1. 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. Seibert, K., Zhang, Y., Leahy, K., Hauser, S., Masferrer, J., Perkins, W., Lee, L., & Isakson, P. (1994). “Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain”, Proc Natl Acad Sci U S A, 91, 12013–7. Sen, C. K. (2003). “The general case for redox control of wound repair”, Wound Repair Regen, 11, 431–8. Shi, J. & Miller, R. A. (1993). “Differential tyrosine-specific protein phosphorylation in mouse T lymphocyte subsets. Effect of age”, J Immunol, 151, 730–9. Shilo, S., Roy, S., Khanna, S., & Sen, C. K. (2007). “MicroRNA in cutaneous wound healing: a new paradigm”, DNA Cell Biol, 26, 227–37. Shukla, A., Rasik, A. M., & Patnaik, G. K. (1997). “Depletion of reduced glutathione, ascorbic acid, vitamin E and antioxidant defence enzymes in a healing cutaneous wound”, Free Radic Res, 26, 93–101. Sivamani, R. K., Pullar, C. E., Manabat-Hidalgo, C. G., Rocke, D. M., Carlsen, R. C., Greenhalgh, D. G., & Isseroff, R. R. (2009). “Stress-mediated increases in systemic and local epinephrine impair skin wound healing: potential new indication for beta blockers”, PLoS Med, 6, e12. Sohal, R. S. (2002). “Role of oxidative stress and protein oxidation in the aging process”, Free Radic Biol Med, 33, 37–44. Solini, A., Santini, E., Madec, S., Cuccato, S., & Ferrannini, E. (2007). “Effects of endothelin-1 on fibroblasts from type 2 diabetic patients: Possible role in wound healing and tissue repair”, Growth Factors, 25, 392–9. Song, Y. S., Lee, B. Y., & Hwang, E. S. (2005). “Dinstinct ROS and biochemical profiles in cells undergoing DNA damage-induced senescence and apoptosis”, Mech Ageing Dev, 126, 580–90. Stanley, A. C., Park, H. Y., Phillips, T. J., Russakovsky, V., & Menzoian, J. O. (1997). “Reduced growth of dermal fibroblasts from chronic venous ulcers can be stimulated with growth factors”, J Vasc Surg, 26, 994–9. Steiling, H., Munz, B., Werner, S., & Brauchle, M. (1999). “Different types of ROSscavenging enzymes are expressed during cutaneous wound repair”, Exp Cell Res, 247, 484–94. Stephens, P. 2003, “Aging of the Skin,” in Aging of Organs and Systems, 1 edn, R. Aspinall, ed., Kluwer Academic Publishers, Dordrecht, pp. 29–72. Stephens, P., Cook, H., Hilton, J., Jones, C. J., Haughton, M. F., Wyllie, F. S., Skinner, J. W., Harding, K. G., Kipling, D., & Thomas, D. W. (2003). “An analysis of replicative senescence in dermal fibroblasts derived from chronic leg wounds predicts that telomerase therapy would fail to reverse their disease-specific cellular and proteolytic phenotype”, Exp Cell Res, 283, 22–35. Subramaniam, K., Pech, C. M., Stacey, M. C., & Wallace, H. J. (2008). “Induction of MMP-1, MMP-3 and TIMP-1 in normal dermal fibroblasts by chronic venous leg ulcer wound fluid”, Int Wound J, 5, 79–86.
© Woodhead Publishing Limited, 2011
36
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Dysfunctional wound healing in chronic wounds
37
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.
© Woodhead Publishing Limited, 2011
38
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
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
40
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
41
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
© Woodhead Publishing Limited, 2011
42
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.
© Woodhead Publishing Limited, 2011
Septicemia
GI TRACT 2. Lower bowel & 3. Genital (vaginal) area • Bacteriodes fragilis • Anaerobic Strep • Clostridium novie, septicum, perfringens (gas gangrene)
The role of micro-organisms and biofilms
43
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.
© Woodhead Publishing Limited, 2011
44
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
45
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
© Woodhead Publishing Limited, 2011
46
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).
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
47
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.
© Woodhead Publishing Limited, 2011
© Woodhead Publishing Limited, 2011
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
The role of micro-organisms and biofilms
49
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
© Woodhead Publishing Limited, 2011
50
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.
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
51
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
© Woodhead Publishing Limited, 2011
52
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
53
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
© Woodhead Publishing Limited, 2011
54
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
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
55
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.
© Woodhead Publishing Limited, 2011
56
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
57
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.
© Woodhead Publishing Limited, 2011
58
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
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
59
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
© Woodhead Publishing Limited, 2011
60
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
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
61
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
© Woodhead Publishing Limited, 2011
62
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
63
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).
© Woodhead Publishing Limited, 2011
64
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
© Woodhead Publishing Limited, 2011
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
66
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
67
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.
© Woodhead Publishing Limited, 2011
68
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
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
69
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
© Woodhead Publishing Limited, 2011
70
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
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
71
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
© Woodhead Publishing Limited, 2011
72
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms Pseudomonas aeruginosa
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
© Woodhead Publishing Limited, 2011
74
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
The role of micro-organisms and biofilms
75
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.
© Woodhead Publishing Limited, 2011
76
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
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.
77 © Woodhead Publishing Limited, 2011
78
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
79
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,
© Woodhead Publishing Limited, 2011
80
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
81
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.
© Woodhead Publishing Limited, 2011
82
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
83
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
(a)
(b)
50μm
100μm (c)
(d)
100μm
50μm
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.
© Woodhead Publishing Limited, 2011
84
Advanced wound repair therapies
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,
14 wk
20 wk
100 μm
adult skin
100 μm
100 μm
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.
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
85
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
© Woodhead Publishing Limited, 2011
86
Advanced wound repair therapies (a)
50μm
(b)
10μm
(c)
10μm
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
87
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
© Woodhead Publishing Limited, 2011
88
Advanced wound repair therapies (a)
50 μm
(b)
10 μm
(c)
10 μm
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
89
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
© Woodhead Publishing Limited, 2011
90
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
91
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.
© Woodhead Publishing Limited, 2011
92
Advanced wound repair therapies
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).
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
93
(a) Exclusive diagnosis of similar diseases and differential diagnosis of hypertrophic scars and keloids Hypertrophic scars (HSs)
HSs with severe scar contractures including joint contractures
HSs with mild scar contractures
HSs without scar contractures
Large / Wide Small / Linear HSs HSs
(b) Partial surgical contracture releasing
(c) Complete surgical resection
a. Skin grafting
(d) Non-surgical multimodal therapy a. Compression therapy
b. Local flap transfer
b. Gel sheets Effective
Recurrence c. Corticosteroid injection d. Laser
(e) Surgery + adjuvant therapy
Satisfactory Unsatisfactory results improvement
(d) Conservative Repeat multimodal therapy
e. External agents f. Internal agents
a. Surgery + radiation
g. Make-up / camouflage therapy
b. Surgery + corticosteroid injection
h. Others
Effective
(f) Long-term follow-up + conservative therapies a. Gel sheets b. Taping fixation c. Compression therapy d. External agents e. Internal agents f. Make-up / camouflage therapy
Recurrence
(d) Conservative multimodal therapy
Satisfactory Unsatisfactory results improvement
Repeat
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,
© Woodhead Publishing Limited, 2011
94
Advanced wound repair therapies (a) Exclusive diagnosis of similar diseases and differential diagnosis of hypertrophic scars and keloids Keloids Small / single keloids
Large / multiple keloids
(b) Radical treatments
(c) Setting the goals of treatments with patients
(d) Surgery + adjuvant therapy
(e) Non-surgical monotherapy
(f) Mass reduction surgery
a. Corticosteroid injection b. Cryotherapy
a. Surgery + radiation
c. Laser
d. Antitumor / b. Surgery + corticosteroid injection immunosuppressive agents
Effective
Recurrence
Effective
Recurrence
Satisfactory Unsatisfactory results mass reduction
(g) Symptomatic non-surgical multimodal therapy a. Corticosteroid injection b. Cryotherapy c. Laser
Repeat
d. Antitumor / (g) Symptomatic immunosuppressive agents multimodal e. Compression therapy therapy f. Gel sheets g. External agents
(e) Monotherapy
Repeat
Other (e) monotherapy or (d) Surgery + adjuvant therapy
h. Internal agents i. Make-up / camouflage therapy j. Others
(h) Long-term follow-up + conservative therapies
Satisfactory symptom improvement
a. Gel sheets b. Taping fixation c. Compression therapy d. External agents e. Internal agents f. Make-up / camouflage therapy
Unsatisfactory results
Repeat or Consider (f) Mass reduction surgery
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.
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
95
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
© Woodhead Publishing Limited, 2011
96
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
97
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
© Woodhead Publishing Limited, 2011
98
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
99
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
© Woodhead Publishing Limited, 2011
100
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
3.8
101
Conclusions
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
References
Adzick, N. S. & Longaker, M. T. (1992) Characteristics of fetal tissue repair, New York, Elsevier. Ahn, S. T., Monafo, W. W. & Mustoe, T. A. (1991) Topical silicone gel for the prevention and treatment of hypertrophic scar. Arch Surg, 126, 499–504. Alaish, S. M., Yager, D. R. & Diegelmann, R. F. (1994) Biology of fetal wound healing: hyaluronate receptor expression in fetal fibroblasts. J Pediatr Surg, 29, 1040. Alster, T. S. (1994) Improvement of erythematous and hypertrophic scars by the 585-nm flashlamp-pumped pulsed dye laser. Ann Plast Surg, 32, 186–90. Alster, T. S. & Williams, C. M. (1995) Treatment of keloid sternotomy scars with 585 nm flashlamp-pumped pulsed-dye laser. Lancet, 345, 1198–200. Armstrong, J. R. & Ferguson, M. W. J. (1995) Otogeny of the skin and transition from scar free to scarring phenotype during wound healing in the pouch of young Mondelphis domestica. Dev. Biol., 169, 242–60. Ashcroft, G. S., Dodsworth, J., Van Boxtel, E., Tarnuzzer, R. W., Horan, M. A., Schultz, G. S. & Ferguson, M. W. (1997a) Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels. Nat Med, 3, 1209–15. Ashcroft, G. S., Horan, M. A. & Ferguson, M. W. (1997b) Aging is associated with reduced deposition of specific extracellular matrix components, an upregulation of angiogenesis, and an altered inflammatory response in a murine incisional wound healing model. J Invest Dermatol, 108, 430–7. Ashcroft, G. S., Horan, M. A. & Ferguson, M. W. (1997c) The effects of ageing on wound healing: immunolocalisation of growth factors and their receptors in a murine incisional model. J Anat, 190 (Pt 3), 351–65. Atiyeh, B. S., Costagliola, M. & Hayek, S. N. (2005) Keloid or hypertrophic scar: the controversy: review of the literature. Ann Plast Surg, 54, 676–80. Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H. & Tomic-Canic, M. (2008) Growth factors and cytokines in wound healing. Wound Repair Regen, 16, 585–601. Bauters, C., Asahara, T., Zheng, L. P., Takeshita, S., Bunting, S., Ferrara, N., Symes, J. F. & Isner, J. M. (1994) Physiological assessment of augmented vascularity induced by VEGF in ischemic rabbit hindlimb. Am J Physiol, 267, H1263–71.
© Woodhead Publishing Limited, 2011
102
Advanced wound repair therapies
Bauters, C., Asahara, T., Zheng, L. P., Takeshita, S., Bunting, S., Ferrara, N., Symes, J. F. & Isner, J. M. (1995) Site-specific therapeutic angiogenesis after systemic administration of vascular endothelial growth factor. J Vasc Surg, 21, 314–24; discussion 324–5. Beanes, S. R., Dang, C. M., Soo, C. & Lorenz, H. P. (2001a) Ontogenetic transition in the fetal wound extracellular matrix correlates with scar formation. Wound Repair Regen, 9, 151. Beanes, S. R., Dang, C. M., Soo, C., Wang, Y., Urata, M., Ting, K., Fohkalsrud, E. W., Benhaim, P., Hedrick, M. H., Adkinson, J. B. & Lorenz, H. P. (2001b) Downregulation of decorin, a transforming growth factor-beta modulator, is associated with scarless fetal wound healing. J Pediatr Surg, 11, 1666–71. Beanes, S. R., Hu, F. Y., Soo, C., Dang, C. M., Urata, M., Ting, K. & Atkinson, J. B. (2002) Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition. Plast. Reconstr. Surg., 109, 160–70. Bennett, S. P., Griffiths, G. D., Schor, A. M., Leese, G. P. & Schor, S. L. (2003) Growth factors in the treatment of diabetic foot ulcers. Br J Surg, 90, 133–46. Berman, B. & Bieley, H. C. (1996) Adjunct therapies to surgical management of keloids. Dermatol Surg, 22, 126–30. Blumenkranz, M. S., Ophir, A., Claflin, A. J. & Hajek, A. (1982) Fluorouracil for the treatment of massive periretinal proliferation. Am J Ophthalmol, 94, 458–67. Bodokh, I. & Brun, P. (1996) [Treatment of keloid with intralesional bleomycin]. Ann Dermatol Venereol, 123, 791–4. Border, W. A. & Noble, N. A. (1994) Transforming growth factor beta in tissue fibrosis. N Engl J Med, 331, 1286–92. Brem, H., Young, J., Tomic-Canic, M., Isaacs, C. & Ehrlich, H. P. (2003) Clinical efficacy and mechanism of bilayered living human skin equivalent (HSE) in treatment of diabetic foot ulcers. Surg Technol Int, 11, 23–31. Brem, H., Stojadinovic, O., Diegelmann, R. F., Entero, H., Lee, B., Pastar, I., Golinko, M., Rosenberg, H. & Tomic-Canic, M. (2007) Molecular markers in patients with chronic wounds to guide surgical debridement. Mol Med, 13, 30–9. Carmeliet, P. (2000) VEGF gene therapy: stimulating angiogenesis or angiomagenesis? Nat Med, 6, 1102–3. Carney, S. A., Cason, C. G., Gowar, J. P., Stevenson, J. H., Mcnee, J., Groves, A. R., Thomas, S. S., Hart, N. B. & Auclair, P. (1994) Cica-Care gel sheeting in the management of hypertrophic scarring. Burns, 20, 163–7. Cass, D. L., Bullard, K. M. & Sylvester, K. G. (1995) Epithelial integrin expression is rapidly upregulated in human fetal wound repair. Surg Forum, 47, 765–7. Cass, D. L., Bullard, K. M. & Sylvester, K. G. (1997) Wound size and gestational age modulate scar formation in fetal wound repair. J. Pediatr. Surg., 32, 411–15. Chen, W. Y., Grant, M. E., Schor, A. M. & Schor, S. L. (1989) Differences between adult and foetal fibroblasts in the regulation of hyaluronate synthesis. J Cell Sci, 94, 577–89. Clark, R. (1996a) Wound repair: overview and general considerations. The molecular and cellular biology of wound repair, xxiii, 3–50. Clark, R. A. F. (1996b) Wound repair overview and general considerations, New York, Plenum Press. Colwell, A. S., Longaker, M. T. & Lorenz, H. P. (2005a) Mammalian fetal organ regeneration. Adv. Biochem. Eng. Biotechnol, 93, 83–100.
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
103
Colwell, A. S., Phan, T. T., Kong, W., Longaker, M. T. & Lorenz, P. H. (2005b) Hypertrophic scar fibroblasts have increased connective tissue growth factor expression after transforming growth factor-beta stimulation. Plast Reconstr Surg, 116, 1387– 90; discussion 1391–2. Colwell, A. S., Krummel, T. M., Longaker, M. T. & Lorenz, H. P. (2006) An in vivo mouse excisional wound model of scarless healing. Plast Reconstr Surg, 117, 2292–6. Colwell, A. S., Longaker, M. T. & Lorenz, H. P. (2008) Identification of differentially regulated genes in fetal wounds during regenerative repair. Wound Repair Regen, 16, 450–9. Coolen, N. A., Schouten, K. C., Middelkoop, E. & Ulrich, M. M. (2010) Comparison between human fetal and adult skin. Arch Dermatol Res, 302(1): 47–55. Cotsarelis, G., Sun, T. T. & Lavker, R. M. (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell, 61, 1329–37. Dang, C. M., Beanes, S. R., Lee, H., Zhang, X., Soo, C. & Ting, K. (2003) Scarless fetal wounds are associated with an increased matrix metalloproteinase-to-tissuederived inhibitor of metalloproteinase ratio. Plast. Reconstr. Surg., 111, 2273–85. Darzi, M. A., Chowdri, N. A., Kaul, S. K. & Khan, M. (1992) Evaluation of various methods of treating keloids and hypertrophic scars: a 10-year follow-up study. Br J Plast Surg, 45, 374–9. Desmonliere, A. & Gabbiani, G. (1996) The role of the myofibroblast in wound healing and fibrocontractive diseases. In Clark, R. A. F. (Ed.) The molecular basis of wound repair. New York. Ellis, I. R. & Schor, S. L. (1996) Differential effects of TGF-1beta on hyaluronan synthesis by fetal and adult skin fibroblasts: implications for cell migration and wound healing. Exp Cell Res, 228, 326–33. English, R. S. & Shenefelt, P. D. (1999) Keloids and hypertrophic scars. Dermatol Surg, 25, 631–8. Espana, A., Solano, T. & Quintanilla, E. (2001) Bleomycin in the treatment of keloids and hypertrophic scars by multiple needle punctures. Dermatol Surg, 27, 23–7. Estes, J. M., Vandeberg, J., Adzick, N. S., Macgillivray, T. E., Desmonliere, A. & Gabbiani, G. (1994) Phenotypic and functional features of myofibroblasts in sheep fetal wounds. Differentiation, 56, 173–81. 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. Falanga, V., Iwamoto, S., Chartier, M., Yufit, T., Butmarc, J., Kouttab, N., Shrayer, D. & Carson, P. (2007) Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng, 13, 1299–312. Fang, B., Song, Y., Liao, L., Zhang, Y. & Zhao, R. C. (2007) Favorable response to human adipose tissue-derived mesenchymal stem cells in steroid-refractory acute graft-versus-host disease. Transplant Proc, 39, 3358–62. Ferguson, M. W. & O’kane, S. (2004) Scar-free healing: from embryonic mechanisms to adult therapeutic intervention. Philos Trans R Soc Lond B Biol Sci, 359, 839–50. Fish, P. V., Allan, G. A., Bailey, S., Blagg, J., Butt, R., Collis, M. G., Greiling, D., James, K., Kendall, J., McElroy, A., McCleverty, D., Reed, C., Webster, R. & Whitlock, G.
© Woodhead Publishing Limited, 2011
104
Advanced wound repair therapies
A. (2007) Potent and selective nonpeptidic inhibitors of procollagen C-proteinase. J Med Chem, 50, 3442–56. Fitzpatrick, R. E. (1999) Treatment of inflamed hypertrophic scars using intralesional 5-FU. Dermatol Surg, 25, 224–32. Folkman, J. (1996) New perspectives in clinical oncology from angiogenesis research. Eur J Cancer, 32A, 2534–9. Frantz, F. W., Bettinger, D. A., Haynes, J. H., Johnson, D. E., Harvey, K. M., Dalton, H. P., Yager, D. R., Diegelmann, R. F. & Cohen, I. K. (1993) The presence of bacteria in fetal wounds induces an adult-like healing response. J Pediatr Surg, 28, 428–34. Galiano, R. D., Tepper, O. M., Pelo, C. R., Bhatt, K. A., Callaghan, M., Bastidas, N., Bunting, S., Steinmetz, H. G. & Gurtner, G. C. (2004) Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells. Am J Pathol, 164, 1935–47. Garcia-Olmo, D., Garcia-Arranz, M., Herreros, D., Pascual, I., Peiro, C. & Rodriguez-Montes, J. A. (2005) A phase I clinical trial of the treatment of Crohn’s fistula by adipose mesenchymal stem cell transplantation. Dis Colon Rectum, 48, 1416–23. Garcia-Olmo, D., Herreros, D., Pascual, I., Pascual, J. A., Del-Valle, E., Zorrilla, J., De-La-Quintana, P., Garcia-Arranz, M. & Pascual, M. (2009) Expanded adiposederived stem cells for the treatment of complex perianal fistula: a phase II clinical trial. Dis Colon Rectum, 52, 79–86. Gordon, A. D., Karmacharya, J., Herlyn, M. & Crombleholme, T. M. (2001) Scarless wound healing induced by adenoviral-mediated overexpression of interleukin-10. Surg Forum, 52, 568–9. Goto, F., Goto, K., Weindel, K. & Folkman, J. (1993) Synergistic effects of vascular endothelial growth factor and basic fibroblast growth factor on the proliferation and cord formation of bovine capillary endothelial cells within collagen gels. Lab Invest, 69, 508–17. Grinnel, F. (1994) Fibroblasts, myofibroblasts, and wound contraction. J Cell Biol, 124, 401–4. Gu, D., Atencio, I., Kang, D. W., Looper, L. D., Ahmed, C. M., Levy, A., Maneval, D. & Zepeda, M. L. (2005) Recombinant adenovirus-p21 attenuates proliferative responses associated with excessive scarring. Wound Repair Regen, 13, 480–90. Ha, X., Li, Y., Lao, M., Yuan, B. & Wu, C. T. (2003) Effect of human hepatocyte growth factor on promoting wound healing and preventing scar formation by adenovirus-mediated gene transfer. Chin Med J (Engl), 116, 1029–33. Har-Shai, Y., Amar, M. & Sabo, E. (2003) Intralesional cryotherapy for enhancing the involution of hypertrophic scars and keloids. Plast Reconstr Surg, 111, 1841–52. Haverstock, B. D. (2001) Hypertrophic scars and keloids. Clin Podiatr Med Surg, 18, 147–59. Hematti, P. (2008) Role of mesenchymal stromal cells in solid organ transplantation. Transplant Rev (Orlando), 22, 262–73. Hendricks, T., Martens, M. F., Huyben, C. M. & Wobbes, T. (1993) Inhibition of basal and TGF beta-induced fibroblast collagen synthesis by antineoplastic agents. Implications for wound healing. Br J Cancer, 67, 545–50.
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
105
Heng, B. C., Liu, H. & Cao, T. (2005) Transplanted human embryonic stem cells as biological ‘catalysts’ for tissue repair and regeneration. Med Hypotheses, 64, 1085–8. Hildebrand, A., Romaris, M., Rasmussen, L. M., Heinegard, D., Twardzik, D. R., Border, W. A. & Ruoslahti, E. (1994) Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta. Biochem J, 302, 527–34. Hsu, M., Peled, Z. M., Chin, G. S., Liu, W. & Longaker, M. T. (2001) Ontogeny of expression of transforming growth factor-beta 1 (TGF-beta 1), TGF-beta 3, and TGF-beta receptors I and II in fetal rat fibroblasts and skin. Plast. Reconstr. Surg., 107, 1787–94. Hubner, G., Smola, H., Madlener, M., Fassler, R. & Werner, S. (1996) Differential Regulation of Pro-Inflammatory Cytokines During Wound Healing in Normal and Glucocorticoid-Treated Mice. Cytokines, 8(7): 548–56. Hunt, T. K. (1980) Wound healing and wound infection: theory and surgical practice, New York, Appleton-Century-Crofts. Ilan, N., Mahooti, S. & Madri, J. A. (1998) Distinct signal transduction pathways are utilized during the tube formation and survival phases of in vitro angiogenesis. J Cell Sci, 111, 3621–31. Iocono, J. A., Ehrlich, H. P., Keefer, K. A. & Krummel, T. M. (1998) Hyaluronan induces scarless repair in mouse limb organ culture. J Pediatr Surg, 33, 564–7. Iocono, J. A., Colleran, K. R., Remick, D. G., Gillespie, B. W., Ehrlich, H. P. & Garner, W. L. (2000) Interleukin-8 levels and activity in delayed-healing human thermal wounds. Wound Repair Regen, 8, 216–25. Ito, M., Liu, Y., Yang, Z., Nguyen, J., Liang, F., Morris, R. J. & Cotsarelis, G. (2005) Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat Med, 11(12): 1351–4. Jennings, R. W., Adzick, N. S., Longaker, M. T., Duncan, B. W., Scheuenstuhl, H. & Hunt, T. K. (1991) Ontogeny of fetal sheep polymorphonuclear leukocyte phagocytosis. J Pediatr Surg, 26, 853–5. Johnstone, C. C. & Farley, A. (2005) The physiological basics of wound healing. Nurs Stand, 19, 59–65. Juliano, R. L. & Haskill, S. (1993) Signal transduction from the extracellular matrix. J Cell Biol, 120, 577–85. Kennedy, C. I., Diegelmann, R. F., Haynes, J. H. & Yager, D. R. (2000) Proinflammatory cytokines differentially regulate hyaluronan synthase isoforms in fetal and adult fibroblasts. J Pediatr Surg, 35, 874–9. Kovalic, J. J. & Perez, C. A. (1989) Radiation therapy following keloidectomy: a 20-year experience. Int J Radiat Oncol Biol Phys, 17, 77–80. Krummel, T. M., Michna, B. A., Thomas, B. L., Sporn, M. B., Nelson, J. M., Salzberg, A. M., Cohen, I. K. & Diegelmann, R. F. (1988) Transforming growth factor beta (TGF-beta) induces fibrosis in a fetal wound model. J Pediatr Surg, 23, 647–52. Lane, A. L. (1986) Human fetal skin development. Pediatr Dermatol, 3, 487–91. Lee, K. S., Song, J. Y. & Suh, M. H. (1991) Collagen mRNA expression detected by in situ hybridization in keloid tissue. J Dermatol Sci, 2, 316–23. Lee, O. K., Kuo, T. K., Chen, W. M., Lee, K. D., Hsieh, S. L. & Chen, T. H. (2004) Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood, 103, 1669–75.
© Woodhead Publishing Limited, 2011
106
Advanced wound repair therapies
Leibovich, S. J. & Ross, R. (1975) The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum. Am J Pathology, 78, 71–100. Liechty, K. W., Crombleholme, T. M., Cass, D. L., Martin, B. & Adzick, N. S. (1998) Diminished interleukin-8 (IL-8) production in the fetal wound healing response. J Surg Res, 77, 80–4. Liechty, K. W., Adzick, N. S. & Crombleholme, T. M. (2000a) Diminished interleukin 6 (IL-6) production during scarless human fetal wound repair. Cytokine, 12, 671–6. Liechty, K. W., Kim, H. B., Adzick, N. S. & Crombleholme, T. M. (2000b) Fetal wound repair results in scar formation in interleukin-10 deficient mice in a syngeneic murine model of scarless fetal wound repair. J Pediatr Surg, 35, 866–72. Longaker, M. T., Chiu, E. S. & Harrison, M. R. (1989) Hyaluronic acid-stimulating activity distinguishes fetal wound fluid from adult wound fluid. Ann. Surg., 210(5): 667–72. Longaker, M. T., Whitby, D. J., Adzick, N. S., Crombleholme, T. M., Langer, J. C., Duncan, B. W., Bradley, S. M., Stern, R., Ferguson, M. W. & Harrison, M. R. (1990) Studies in fetal wound healing, VI. Second and early third trimester fetal wounds demonstrate rapid collagen deposition without scar formation. J Pediatr Surg, 25, 63–8; discussion 68–9. Longaker, M. T., Whitby, D. J., Jennings, R. W., Duncan, B. W., Ferguson, M. W. J. & Harrison, M. R. (1991) Fetal diaphragmatic wounds heal with scar formation. J Surg Res, 50, 375–85. Longaker, M. T., Whitby, D. J., Ferguson, M. W. J., Lorenz, H. P., Harrison, M. R. & Adzick, N. S. (1994) Adult skin wounds in the fetal environment heal with scar formation. Ann. Surg., 219, 65–72. Lorenz, H. P. & Adzick, N. S. (1993) Scarless skin wound repair in the fetus. West J Med, 159, 350–5. Lorenz, H. P., Longaker, M. T. & Adzick, N. S. (1992a) Fetal Wound Healing, New York, Elsevier. Lorenz, H. P., Longaker, M. T., Perkocha, L. A., Jennings, R. W., Harrison, M. R. & Adzick, N. S. (1992b) Scarless wound repair: a human fetal skin model. Development, 114, 253–9. Lorenz, H. P., Whitby, D. J., Longaker, M. T. & Adzick, N. S. (1993) Fetal wound healing. The ontogeny of scar formation in the non-human primate. Ann Surg, 217, 391–6. Lorenz, H. P., Lin, R. Y., Longaker, M. T., Whitby, D. J. & Adzick, N. S. (1995) The fetal fibroblast: the effector cell of scarless fetal skin repair. Plast Reconstr Surg, 96, 1251–9; discussion 1260–1. Lorenz, H. P., Soo, C., Beanes, S. R., Dang, C. M., Zhang, X., Atkinson, J. B. & Ting, K. (2001) Differential expression of matrix metalloproteinases and their tissuederived inhibitors in scarless fetal wound healing. Surgical Forum, 397–401. Madden, J. W. & Peacock, E. E., Jr. (1971) Studies on the biology of collagen during wound healing. 3. Dynamic metabolism of scar collagen and remodeling of dermal wounds. Ann Surg, 174, 511–20. Maguire, H. C., Jr. (1965) Treatment of keloids with triamcinolone acetonide injected intralesionally. JAMA, 192, 325–6. Maherali, N., Sridharan, R., Xie, W., Utikal, J., Eminli, S., Arnold, K., Stadtfeld, M., Yachechko, R., Tchieu, J., Jaenisch, R., Plath, K. & Hochedlinger, K. (2007)
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
107
Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell, 1, 55–70. Mallick, K. S., Hajek, A. S. & Parrish, R. K., 2nd (1985) Fluorouracil (5-FU) and cytarabine (ara-C) inhibition of corneal epithelial cell and conjunctival fibroblast proliferation. Arch Ophthalmol, 103, 1398–402. Manuskiatti, W. & Fitzpatrick, R. E. (2002) Treatment response of keloidal and hypertrophic sternotomy scars: comparison among intralesional corticosteroid, 5-fluorouracil, and 585-nm flashlamp-pumped pulsed-dye laser treatments. Arch Dermatol, 138, 1149–55. Margolis, D. J., Crombleholme, T. & Herlyn, M. (2000) Clinical protocol: Phase I trial to evaluate the safety of H5.020CMV.PDGF-B for the treatment of a diabetic insensate foot ulcer. Wound Repair Regen, 8, 480–93. Margolis, D. J., Cromblehome, T., Herlyn, M., Cross, P., Weinberg, L., Filip, J. & Propert, K. (2004) Clinical protocol. Phase I trial to evaluate the safety of H5.020CMV.PDGF-b and limb compression bandage for the treatment of venous leg ulcer: trial A. Hum Gene Ther, 15, 1003–19. Mast, B. A., Flood, L. C., Haynes, J. H., Depalma, R. L., Cohen, I. K., Diegelmann, R. F. & Krummel, T. M. (1991) Hyaluronic acid is a major component of the matrix of fetal rabbit skin and wounds: implications for healing by regeneration. Matrix, 11, 63–68. Mast, B. A., Diegelmann, R. F., Krummel, T. M. & Cohen, I. K. (1993) Hyaluronic acid modulates proliferation, collagen and protein synthesis of cultured fetal fibroblasts. Matrix, 13, 441–6. Mathew, L. K., Sengupta, S., Kawakami, A., Andreasen, E. A., Lohr, C. V., Loynes, C. A., Renshaw, S. A., Peterson, R. T. & Tanguay, R. L. (2007) Unraveling tissue regeneration pathways using chemical genetics. J Biol Chem, 282, 35202–10. McCallion, R. L., Ferguson, M. W. J. & Clark, R. A. F. (1996) Fetal Wound Healing and the Development of Antiscarring Therapies for Adult Wound Healing, New York, Plenum Press. Merkel, J. R., Dipaolo, B. R., Hallock, G. G. & Rice, D. C. (1988) Type I and type III collagen content of healing wounds in fetal and adult rats. Proc Soc Exp Biol Med, 187, 493–7. Montesano, R. & Orci, L. (1998) Transforming growth factor beta stimulates collagen-matrix contraction by fibroblasts: implications for wound healing. Proc Natl Acad Sci, 85, 4894–7. Moulin, V., Lawny, F., Barritault, D. & Caruelle, J. P. (1998) Platelet releasate treatment improves skin healing in diabetic rats through endogenous growth factor secretion. Cell Mol Biol, 44, 961–71. Muller-Eberhard, H. (1992) Complement: Chemistry and pathways. Inflammation Basic Principles and Clinical correlates. New York, Raven Press. Naeini, F. F., Najafian, J. & Ahmadpour, K. (2006) Bleomycin tattooing as a promising therapeutic modality in large keloids and hypertrophic scars. Dermatol Surg, 32, 1023–9; discussion 1029–30. Nagy, J. A., Vasile, E., Feng, D., Sundberg, C., Brown, L. F., Detmar, M. J., Lawitts, J. A., Benjamin, L., Tan, X., Manseau, E. J., Dvorak, A. M. & Dvorak, H. F. (2002) Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis. J Exp Med, 196, 1497–506.
© Woodhead Publishing Limited, 2011
108
Advanced wound repair therapies
Nath, R. K., Leregina, M., Markham, H., Ksander, G. A. & Weeks, P. M. (1994) The expression of transforming growth factor type beta in fetal and adult rabbit skin wounds. J Pediatr Surg, 29, 416–21. Niessen, F. B., Spauwen, P. H., Schalkwijk, J. & Kon, M. (1999) On the nature of hypertrophic scars and keloids: a review. Plast Reconstr Surg, 104, 1435–58. Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T. & Yamanaka, S. (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science, 322, 949–53. Olutoye, O. O., Yager, D. R., Cohen, I. K. & Diegelmann, R. F. (1996) Lower cytokine release by fetal porcine platelets: a possible explanation for reduced inflammation after fetal wounding. J Pediatr Surg, 31, 91–5. Olutoye, O. O., Barone, E. J., Yager, D. R., Cohen, I. K. & Diegelmann, R. F. (1997) Collagen induces cytokine release by fetal platelets: implications in scarless healing. J Pediatr Surg, 32, 827–30. Orive, G., Gascon, A. R., Hernandez, R. M., Igartua, M. & Luis Pedraz, J. (2003) Cell microencapsulation technology for biomedical purposes: novel insights and challenges. Trends Pharmacol Sci, 24, 207–10. Oshima, H., Rochat, A., Kedzia, C., Kobayashi, K. & Barrandon, Y. (2001) Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell, 104, 233–45. Parks, W. C. (1999) Matrix metalloproteinases in repair. Wound Repair Regen, 7, 423–32. Peacock, E. E., Madden, J. W. & Trier, W. C. (1970) Biologic basis for the treatment of keloids and hypertrophic scars. South Med J, 63, 755–60. Pepper, M. S., Ferrara, N., Orci, L. & Montesano, R. (1992) Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochem Biophys Res Commun, 189, 824–31. Pera, M. F. & Hasegawa, K. (2008) Simpler and safer cell reprogramming. Nat Biotechnol, 26, 59–60. Perez, P., Page, A., Bravo, A., Del Rio, M., Gimenez-Conti, I., Budunova, I., Slaga, T. J. & Jorcano, J. L. (2001) Altered skin development and impaired proliferative and inflammatory responses in transgenic mice overexpressing the glucocorticoid receptor. FASEB J, 15, 2030–2. Phillips, T. J., Manzoor, J., Rojas, A., Isaacs, C., Carson, P., Sabolinski, M., Young, J. & Falanga, V. (2002) The longevity of a bilayered skin substitute after application to venous ulcers. Arch Dermatol, 138, 1079–81. Quinn, K. J. (1987) Silicone gel in scar treatment. Burns Incl Therm Inj, 13 Suppl, S33–40. Ragoowansi, R., Cornes, P. G., Moss, A. L. & Glees, J. P. (2003) Treatment of keloids by surgical excision and immediate postoperative single-fraction radiotherapy. Plast Reconstr Surg, 111, 1853–9. Reiken, S. R., Wolfort, S. F., Berthiaume, F., Compton, C., Tompkins, R. G. & Yarmush, M. L. (1997) Control of hypertrophic scar growth using selective photothermolysis. Lasers Surg Med, 21, 7–12. Reish, R. G. & Eriksson, E. (2008) Scars: a review of emerging and currently available therapies. Plast Reconstr Surg, 122, 1068–78. Roberts, A. B. & Sporn, M. B. (1996) Transforming growth factor-β. In: Clark RAF editors. The molecular basis of wound repair, Plenum Press New York p. 275–308.
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
109
Robson, M. C. (1997) The role of growth factors in the healing of chronic wounds. Wound Repair Regen, 5, 12–7. Robson, M. C. (2003) Proliferative scarring. Surg Clin North Am, 83, 557–69. Rodgers, K. E., Roda, N., Felix, J. E., Espinoza, T., Maldonado, S. & Dizerega, G. (2003) Histological evaluation of the effects of angiotensin peptides on wound repair in diabetic mice. Exp Dermatol, 12, 784–90. Rosenberg, I., Cherayil, B. J., Isselbacher, K. J. & Pillai, S. (1991) Mac-2-binding glycoproteins. Putative ligands for a cytosolic beta-galactoside lectin. J Biol Chem, 266, 18731–6. Rowlatt, U. (1979) Intrauterine healing in a 20-week human fetus. Virchows Arch., 381, 353–61. Rusciani, L., Paradisi, A., Alfano, C., Chiummariello, S. & Rusciani, A. (2006) Cryotherapy in the treatment of keloids. J Drugs Dermatol, 5, 591–5. Ryan, J. M., Barry, F. P., Murphy, J. M. & Mahon, B. P. (2005) Mesenchymal stem cells avoid allogeneic rejection. J Inflamm (Lond), 2, 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. Saray, Y. & Gulec, A. T. (2005) Treatment of keloids and hypertrophic scars with dermojet injections of bleomycin: a preliminary study. Int J Dermatol, 44, 777–84. Senger, D. R., Ledbetter, S. R., Claffey, K. P., Papadopoulos-Sergiou, A., Peruzzi, C. A. & Detmar, M. (1996) Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the alphavbeta3 integrin, osteopontin, and thrombin. Am J Pathol, 149, 293–305. Shaffer, J. J., Taylor, S. C. & Cook-Bolden, F. (2002) Keloidal scars: a review with a critical look at therapeutic options. J Am Acad Dermatol, 46, S63–97. Shah, M., Foreman, D. M. & Ferguson, M. W. (1994) Neutralizing antibody to TGFbeta reduces cutaneous scarring in adult rodents. J Cell Sci, 107, 1137–57. Shah, M., Foreman, D. M. & Ferguson, M. W. (1995) Neutralization of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci, 108, 985–1002. Ship, A. G., Weiss, P. R., Mincer, F. R. & Wolkstein, W. (1993) Sternal keloids: successful treatment employing surgery and adjunctive radiation. Ann Plast Surg, 31, 481–7. Shizuru, J. A., Negrin, R. S. & Weissman, I. L. (2005) Hematopoietic stem and progenitor cells: clinical and preclinical regeneration of the hematolymphoid system. Annu Rev Med, 56, 509–38. Singer, A. F. & Clark, R. A. F. (1999) Cutaneous wound healing. N Engl J Med, 341, 738–46. Smith, P., Mosiello, G., Deluca, L., Ko, F., Maggi, S. & Robson, M. C. (1999) TGFbeta2 activates proliferative scar fibroblasts. J Surg Res, 82, 319–23. Somasundaram, K. & Prathap, K. (1970) Intra-uterine healing of skin wounds in rabbit wounds in rabbit fetuses. J Path, 100, 81–6. Soo, C., Hu, F. Y., Zhang, X., Wang, Y., Beanes, S. R., Lorenz, H. P., Hedrick, M. H., Mackool, R. F., Plass, A., Kim, S. J., Longaker, M. T., Freymiller, E. & Ting, K. (2000) Differential expression of fibromodulin, a TGF-beta modulator, in fetal skin development and scarless repair. Am J Pathology, 157, 423–33.
© Woodhead Publishing Limited, 2011
110
Advanced wound repair therapies
Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G. & Hochedlinger, K. (2008) Induced pluripotent stem cells generated without viral integration. Science, 322, 945–9. Stevenson, S., Nelson, L. D., Sharpe, D. T. & Thornton, M. J. (2008) 17beta-estradiol regulates the secretion of TGF-beta by cultured human dermal fibroblasts. J Biomater Sci Polym Ed, 19, 1097–109. Stoff, A., Rivera, A. A., Mathis, J. M., Moore, S. T., Banerjee, N. S., Everts, M., EspinosaDe-Los-Monteros, A., Novak, Z., Vasconez, L. O., Broker, T. R., Richter, D. F., Feldman, D., Siegal, G. P., Stoff-Khalili, M. A. & Curiel, D. T. (2007) Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds. J Mol Med, 85, 481–96. Stojadinovic, O., Lee, B., Vouthounis, C., Vukelic, S., Pastar, I., Blumenberg, M., Brem, H. & Tomic-Canic, M. (2007) Novel genomic effects of glucocorticoids in epidermal keratinocytes: inhibition of apoptosis, interferon-gamma pathway, and wound healing along with promotion of terminal differentiation. J Biol Chem, 282, 4021–34. Suzuma, K., Takagi, H., Otani, A. & Honda, Y. (1998) Hypoxia and vascular endothelial growth factor stimulate angiogenic integrin expression in bovine retinal microvascular endothelial cells. Invest Ophthalmol Vis Sci, 39, 1028–35. Takahashi, K. & Yamanaka, S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126, 663–76. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. & Yamanaka, S. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131, 861–72. Takeshita, S., Pu, L. Q., Stein, L. A., Sniderman, A. D., Bunting, S., Ferrara, N., Isner, J. M. & Symes, J. F. (1994a) Intramuscular administration of vascular endothelial growth factor induces dose-dependent collateral artery augmentation in a rabbit model of chronic limb ischemia. Circulation, 90, II228–34. Takeshita, S., Zheng, L. P., Brogi, E., Kearney, M., Pu, L. Q., Bunting, S., Ferrara, N., Symes, J. F. & Isner, J. M. (1994b) Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest, 93, 662–70. Tang, Y. W. (1992) Intra- and postoperative steroid injections for keloids and hypertrophic scars. Br J Plast Surg, 45, 371–3. Thielitz, A., Vetter, R. W., Schultze, B., Wrenger, S., Simeoni, L., Ansorge, S., Neubert, K., Faust, J., Lindenlaub, P., Gollnick, H. P. & Reinhold, D. (2008) Inhibitors of dipeptidyl peptidase IV-like activity mediate antifibrotic effects in normal and keloid-derived skin fibroblasts. J Invest Dermatol, 128, 855–66. Thomas, B. L., Krummel, T. M., Melang, M., Cawthorn, J. W., Nelson, J. M. & Cohen, I. K. (1988) Collagen synthesis and type expression by fetal fibroblasts in vitro. Surg Forum, 39, 642. Tolhurst, D. E. (1977) Hypertrophic scarring prevented by pressure: a case report. Br J Plast Surg, 30, 218–19. Tuan, T. L. & Nichter, L. S. (1998) The molecular basis of keloid and hypertrophic scar formation. Mol Med Today, 4, 19–24. Wagers, A. J., Christensen, J. L. & Weissman, I. L. (2002) Cell fate determination from stem cells. Gene Ther., 9, 606–12. Walder, C. E., Errett, C. J., Bunting, S., Lindquist, P., Ogez, J. R., Heinsohn, H. G., Ferrara, N. & Thomas, G. R. (1996) Vascular endothelial growth factor augments
© Woodhead Publishing Limited, 2011
Scarring and scarless wound healing
111
muscle blood flow and function in a rabbit model of chronic hindlimb ischemia. J Cardiovasc Pharmacol, 27, 91–8. Wendling, J., Marchand, A., Mauviel, A. & Verrecchia, F. (2003) 5-fluorouracil blocks transforming growth factor-beta-induced alpha 2 type I collagen gene (COL1A2) expression in human fibroblasts via c-Jun NH2-terminal kinase/activator protein-1 activation. Mol Pharmacol, 64, 707–13. Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B. E. & Jaenisch, R. (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature, 448, 318–24. Whitby, D. J. & Ferguson, M. W. J. (1991) The extracellular matrix of lip wounds in fetal, neonatal, and adult mice. Development, 112, 651–68. Whitby, D. J. & Ferguson, M. W. J. (1992) Immunohistochemical stuidies in fetal and adult wound healing, New York, Elsevier. Whitby, D. J., Longaker, M. T. & Harrison, M. R. (1991) Rapid epithelialization in fetal wounds is associated with the early deposition of tenascin. J Cell Sci, 99, 583–6. Winter, G. D. (1962) Formation of the scab and the rate of epithelization of superficial wounds in the skin of the young domestic pig. Nature, 193, 293–4. Witte, M. B., Thornton, F. J., Kiyama, T., Efron, D. T., Schulz, G. S., Moldawer, L. L. & Barbul, A. (1998) Metalloproteinase inhibitors and wound healing: a novel enhancer of wound strength. Surgery, 124, 464–70. Wu, W. S., Wang, F. S., Yang, K. D., Huang, C. C. & Kuo, Y. R. (2006) Dexamethasone induction of keloid regression through effective suppression of VEGF expression and keloid fibroblast proliferation. J Invest Dermatol, 126, 1264–71. Xu, J. & Clark, R. A. F. (1996) Extracellular matrix alters PDGF regulation of fibroblast integrins. J Cell Biol, 132, 239–49. Yoshikawa, T., Mitsuno, H., Nonaka, I., Sen, Y., Kawanishi, K., Inada, Y., Takakura, Y., Okuchi, K. & Nonomura, A. (2008) Wound therapy by marrow mesenchymal cell transplantation. Plast Reconstr Surg, 121, 860–77. Zhou, H., Wu, S., Joo, J. Y., Zhu, S., Han, D. W., Lin, T., Trauger, S., Bien, G., Yao, S., Zhu, Y., Siuzdak, G., Scholer, H. R., Duan, L. & Ding, S. (2009) Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell, 4, 381–4. Zouboulis, C. C., Blume, U., Buttner, P. & Orfanos, C. E. (1993) Outcomes of cryosurgery in keloids and hypertrophic scars. A prospective consecutive trial of case series. Arch Dermatol, 129, 1146–51. Zuk, P. A., Zhu, M., Ashjian, P., De Ugarte, D. A., Huang, J. I., Mizuno, H., Alfonso, Z. C., Fraser, J. K., Benhaim, P. & Hedrick, M. H. (2002) Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 13, 4279–95.
© Woodhead Publishing Limited, 2011
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
The discovery and development of new therapeutic treatments
4.2
113
Scar-free and scar-forming healing
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
© Woodhead Publishing Limited, 2011
114
Advanced wound repair therapies (a)
Scar
Normal skin
(b)
e
rr
Normal skin
Scar
(c) e rr
Normal skin 500 μm
Scar
© Woodhead Publishing Limited, 2011
The discovery and development of new therapeutic treatments
115
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
© Woodhead Publishing Limited, 2011
116
Advanced wound repair therapies
Regenerative healing
Scar-free healing
Scar-forming healing
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.
© Woodhead Publishing Limited, 2011
The discovery and development of new therapeutic treatments
4.3
117
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
© Woodhead Publishing Limited, 2011
© Woodhead Publishing Limited, 2011
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)
121
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
© Woodhead Publishing Limited, 2011
122
Advanced wound repair therapies Human 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 1.5 1.0 0.5 0 –0.5 –1.0 –1.5 1.5 1.0 0.5 0 –0.5 –1.0 –1.5
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
© Woodhead Publishing Limited, 2011
The discovery and development of new therapeutic treatments
123
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
© Woodhead Publishing Limited, 2011
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
Sequential microenvironments influencing molecular, cellular and tissue behaviour
124
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
© Woodhead Publishing Limited, 2011
The discovery and development of new therapeutic treatments
125
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
© Woodhead Publishing Limited, 2011
126
Advanced wound repair therapies
‘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.
© Woodhead Publishing Limited, 2011
The discovery and development of new therapeutic treatments
127
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.
© Woodhead Publishing Limited, 2011
128
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
The discovery and development of new therapeutic treatments
129
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.
© Woodhead Publishing Limited, 2011
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
Monitoring chronic wounds and determining treatment
131
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
© Woodhead Publishing Limited, 2011
132
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Monitoring chronic wounds and determining treatment
133
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).
© Woodhead Publishing Limited, 2011
134
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Monitoring chronic wounds and determining treatment
135
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
© Woodhead Publishing Limited, 2011
136
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Monitoring chronic wounds and determining treatment
137
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.
© Woodhead Publishing Limited, 2011
138
Advanced wound repair therapies (a)
(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
© Woodhead Publishing Limited, 2011
Monitoring chronic wounds and determining treatment
139
35 4B
Area [cm2]
30
3A
2B
1B
25 7A
20
5A
6A
15 10
10B
5 0
9B
8B 12B
11B 0
2
4
6 8 Volume [cm3]
14
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)
© Woodhead Publishing Limited, 2011
140
Advanced wound repair therapies
Std.Dev. [% of Area]
(a) 2.00% 1.50% 1.00% 0.50% 0.00%
0
5
10
15
20
25
30
35
10
12
14
Area [cm2]
Std.Dev. [% of Volume]
(b) 10.00% 8.00% 6.00% 4.00% 2.00% 0.00% 0
2
4
6
8
Volume [cm3]
Std.Dev. [% of Circumference]
(c)
5.00% 4.00% 3.00% 2.00% 1.00% 0.00% 0
5
10
15
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).
© Woodhead Publishing Limited, 2011
Monitoring chronic wounds and determining treatment
141
(a) 20
15
10
5
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
© Woodhead Publishing Limited, 2011
142
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Monitoring chronic wounds and determining treatment
143
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
© Woodhead Publishing Limited, 2011
144
Advanced wound repair therapies
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-
© Woodhead Publishing Limited, 2011
Monitoring chronic wounds and determining treatment
145
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:
© Woodhead Publishing Limited, 2011
146
Advanced wound repair therapies κ = (P0 − Pe)/(1 − Pe)
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
© Woodhead Publishing Limited, 2011
Monitoring chronic wounds and determining treatment
0.7
147
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).
© Woodhead Publishing Limited, 2011
148
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Monitoring chronic wounds and determining treatment
149
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
© Woodhead Publishing Limited, 2011
150
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Monitoring chronic wounds and determining treatment
151
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
© Woodhead Publishing Limited, 2011
152
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
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
156
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Functional requirements of wound repair biomaterials
157
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.
© Woodhead Publishing Limited, 2011
158
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Functional requirements of wound repair biomaterials
159
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,
© Woodhead Publishing Limited, 2011
160
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Functional requirements of wound repair biomaterials
161
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,
© Woodhead Publishing Limited, 2011
162
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Functional requirements of wound repair biomaterials
163
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
© Woodhead Publishing Limited, 2011
164
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Functional requirements of wound repair biomaterials
165
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
© Woodhead Publishing Limited, 2011
166
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Functional requirements of wound repair biomaterials
167
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
© Woodhead Publishing Limited, 2011
168
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Functional requirements of wound repair biomaterials
169
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
Achterberg V, Welling C, and Mayer-Ingold W (1996) ‘Hydroactive dressings and serum proteins: an in vitro study’, Journal of Wound Care, 5, 79–82.
© Woodhead Publishing Limited, 2011
170
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Functional requirements of wound repair biomaterials
171
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.
© Woodhead Publishing Limited, 2011
172
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Functional requirements of wound repair biomaterials
173
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.
© Woodhead Publishing Limited, 2011
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
Tissue-biomaterial interactions
7.2
175
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,
© Woodhead Publishing Limited, 2011
176
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Tissue-biomaterial interactions
177
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
© Woodhead Publishing Limited, 2011
178
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Tissue-biomaterial interactions
179
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.
© Woodhead Publishing Limited, 2011
180
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Tissue-biomaterial interactions
181
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
© Woodhead Publishing Limited, 2011
182
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Tissue-biomaterial interactions
183
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),
© Woodhead Publishing Limited, 2011
184
Advanced wound repair therapies
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).
© Woodhead Publishing Limited, 2011
Tissue-biomaterial interactions
185
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.
© Woodhead Publishing Limited, 2011
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
Polymeric materials for chronic wound and burn dressings
187
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
© Woodhead Publishing Limited, 2011
188
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Polymeric materials for chronic wound and burn dressings
189
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
© Woodhead Publishing Limited, 2011
© Woodhead Publishing Limited, 2011
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))
© Woodhead Publishing Limited, 2011
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
192
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Polymeric materials for chronic wound and burn dressings
193
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
© Woodhead Publishing Limited, 2011
194
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Polymeric materials for chronic wound and burn dressings
195
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
© Woodhead Publishing Limited, 2011
196
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Polymeric materials for chronic wound and burn dressings
197
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
© Woodhead Publishing Limited, 2011
198
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Polymeric materials for chronic wound and burn dressings
199
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
© Woodhead Publishing Limited, 2011
200
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Polymeric materials for chronic wound and burn dressings
201
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
© Woodhead Publishing Limited, 2011
202
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Polymeric materials for chronic wound and burn dressings
203
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
Agren M S (1998), ‘An amorphous hydrogel enhances epithelialisation of wounds’, Acta Dermato-Venereologica, 78, 119–122. Alvarez O (1988), ‘Moist environment for healing: matching the dressing to the wound’, Ostomy Wound Management, 21, 64–83. Bale S, Banks V, Haglestein S & Harding K (1998), ‘A comparison of two amorphous hydrogels in the debridement of pressure sores’, Journal of Wound Care, 7, 65–68.
© Woodhead Publishing Limited, 2011
204
Advanced wound repair therapies
Bergan J, Pascarella L & Mekenas L (2006), ‘Venous disorders: treatment with sclerosant foam’, The Journal of Cardiovascular Surgery, 47, 9–18. Blome-Eberwein S, Johnson R M, Miller S F, Caruso D M, Jordan M H, Milner S, Tredget E E, Sittig K M & Smith L (2010), ‘Hydrofiber dressing with silver for the management of split-thickness donor sites: A randomized evaluation of two protocols of care’, Burns, 36, 665–672. Bowler P, Jones S, Davies B & Coyle E (1999), ‘Infection control properties of some wound dressings’, Journal of Wound Care, 8, 499–502. Bowler P G (2002), ‘Wound pathophysiology, infection and therapeutic options’, Annals of Medicine, 34, 419–427. Campbell K, Woodbury M, Whittle H, Labate T & Hoskin A (2000), ‘A clinical evaluation of 3M no sting barrier film’, Ostomy Wound Management, 46, 24–30. Carter K (2003), ‘Hydropolymer dressings in the management of wound exudate’, British Journal of Community Nursing, 8, 10–16. 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, Sales-Aussias N, Zagnoli A, Denis C, Guillot B & Chosidow O (2007), ‘Dressings for acute and chronic wounds: A systematic review’, Archives of Dermatology, 143, 1297–1304. Coats T J, Edwards C, Newton R & Staun E (2002), ‘The effect of gel burns dressings on skin temperature’, Emergency Medicine Journal, 19, 224–225. Cunningham D (2005), ‘Treating venous insufficiency ulcers with soft silicone dressing’, Ostomy Wound Management, 51, 19–20. Curt D & Holger L (2005), ‘Evaluation of Tielle hydropolymer dressings in the management of chronic exuding wounds in primary care’, International Wound Journal, 2, 26–35. Douglas Q, Heather O, Hiromi S & Geoff S (2004), ‘A dressing history’, International Wound Journal, 1, 59–77. Eaglstein W H (1985), ‘Experiences with biosynthetic dressings’, Journal of the American Academy of Dermatology, 12, 434–440. Eisenbud D, Hunter H, Kessler L & Zulkowski K (2003), ‘Hydrogel wound dressings: where do we stand in 2003?’ Ostomy Wound Management, 49, 52–57. Fanucci D & Seese J (1991), ‘Multi-faceted use of calcium alginates. A painless, cost-effective alternative for wound care management’, Ostomy Wound Management, 37, 16–22. Ferrari F, Bertoni M, Bonferoni M C, Rossi S, Caramella C & Waring M J (1995), ‘Comparative evaluation of hydrocolloid dressings by means of water uptake and swelling force measurements: II’, International Journal of Pharmaceutics, 117, 49–55. Field F K & Kerstein M D (1994), ‘Overview of wound healing in a moist environment’, American Journal of Surgery, 167, 2S–6S. Finn G, Bo J, Tonny K, Sibbald R G, Rytis R, Keith H, Patricia P, Vanessa V, Peter V, Michael J, Stephan W, Rita S, Gintaris V, Terttu-Liisa A, Karel E & Monika A (2008), ‘Reducing wound pain in venous leg ulcers with Biatain Ibu: A randomized, controlled double-blind clinical investigation on the performance and safety’, Wound Repair and Regeneration, 16, 615–625. Flanagan M (1995), ‘The efficacy of a hydrogel in the treatment of wounds with non-viable tissue’, Journal of Wound Care, 4, 264–267.
© Woodhead Publishing Limited, 2011
Polymeric materials for chronic wound and burn dressings
205
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.
© Woodhead Publishing Limited, 2011
206
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Polymeric materials for chronic wound and burn dressings
207
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.
© Woodhead Publishing Limited, 2011
208
Advanced wound repair therapies
Thomas S & Hay P (1995), ‘Fluid handling properties of hydrogel dressings’, Ostomy Wound Management, 41, 54–56. Turner T D (1979), ‘Hospital usage of absrobent dressings’, The Pharmaceutical Journal, 222, 421–424. Veves A, Sheehan P, Pham H T & for the Promogran Diabetic Foot Ulcer S (2002), ‘A randomized, controlled trial of Promogran (a collagen/oxidized regenerated cellulose dressing) vs standard treatment in the management of diabetic foot ulcers’, Archives of Surgery, 137, 822–827. Vloemans A F P M, Soesman A M, Kreis R W & Middelkoop E (2001), ‘A newly developed hydrofibre dressing, in the treatment of partial-thickness burns’, Burns, 27, 167–173. Walker M, Hobot J A, Newman G R & Bowler P G (2003), ‘Scanning electron microscopic examination of bacterial immobilisation in a carboxymethyl cellulose (AQUACEL®) and alginate dressings’, Biomaterials, 24, 883–890. Waring M J & Parsons D (2001), ‘Physico-chemical characterisation of carboxymethylated spun cellulose fibres’, Biomaterials, 22, 903–912. White R & McIntosh C (2009), ‘A review of the literature on topical therapies for diabetic foot ulcers. Part 2: advanced’, Journal of Wound Care, 18, 335–341. White R & Morris C (2009), ‘Mepitel: a non-adherent wound dressing with Safetac technology’, British Journal of Nursing, 18, 58–64. Williams C (1995), ‘Mepitel’, British Journal of Nursing, 4, 51–55. Winter G D (1962), ‘Formation of the scab and the rate of epithelization of superficial wounds in the skin of the young domestic pig’, Nature, 193, 293–294. Wynne R, Botti M, Stedman H, Holsworth L, Harinos M, Flavell O & Manterfield C (2004), ‘Effect of three wound dressings on infection, healing comfort, and cost in patients with sternotomy wounds’, Chest, 125, 43–49. Zillmer R, Agren M, Gottrup F & Karlsmark T (2006), ‘Biophysical effects of repetitive removal of adhesive dressings on peri-ulcer skin’, Journal of Wound Care, 15, 187–191. Zimny S, Reinsch B, Schatz H & Pfohl M (2001), ‘Effects of felted foam on plantar pressures in the treatment of neuropathic diabetic foot ulcers’, Diabetes Care, 24, 2153–2154.
© Woodhead Publishing Limited, 2011
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
210
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Dry wound healing concept using spray-on dressings
211
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.
© Woodhead Publishing Limited, 2011
212
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Dry wound healing concept using spray-on dressings
213
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.
© Woodhead Publishing Limited, 2011
214
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Dry wound healing concept using spray-on dressings
215
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
© Woodhead Publishing Limited, 2011
216
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Dry wound healing concept using spray-on dressings
217
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.
© Woodhead Publishing Limited, 2011
218
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Dry wound healing concept using spray-on dressings
219
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
© Woodhead Publishing Limited, 2011
220
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Dry wound healing concept using spray-on dressings
221
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.
© Woodhead Publishing Limited, 2011
222
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
£68
£20
£382 £3.21
Dry wound healing concept using spray-on dressings
223
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
224
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Dry wound healing concept using spray-on dressings
225
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.
© Woodhead Publishing Limited, 2011
226
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
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
228
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Assessing the effectiveness of antimicrobial wound dressings
229
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
© Woodhead Publishing Limited, 2011
230
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Assessing the effectiveness of antimicrobial wound dressings Sample taken (usually 1ml)
231
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
© Woodhead Publishing Limited, 2011
232
Advanced wound repair therapies
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).
© Woodhead Publishing Limited, 2011
Assessing the effectiveness of antimicrobial wound dressings
233
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).
© Woodhead Publishing Limited, 2011
234
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Assessing the effectiveness of antimicrobial wound dressings
235
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
© Woodhead Publishing Limited, 2011
236
Advanced wound repair therapies Clear zone
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
© Woodhead Publishing Limited, 2011
Assessing the effectiveness of antimicrobial wound dressings
237
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.
© Woodhead Publishing Limited, 2011
238
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Assessing the effectiveness of antimicrobial wound dressings
239
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
© Woodhead Publishing Limited, 2011
240
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Assessing the effectiveness of antimicrobial wound dressings
241
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
© Woodhead Publishing Limited, 2011
242
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Assessing the effectiveness of antimicrobial wound dressings
243
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
© Woodhead Publishing Limited, 2011
244
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Assessing the effectiveness of antimicrobial wound dressings
10.7
245
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
© Woodhead Publishing Limited, 2011
246
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
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
248
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
11.1
Adhesives and interfacial phenomena in wound healing
249
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.
© Woodhead Publishing Limited, 2011
250
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
251
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.
© Woodhead Publishing Limited, 2011
252
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
253
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.
© Woodhead Publishing Limited, 2011
254
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
Adhesive failure
255
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
© Woodhead Publishing Limited, 2011
256
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
257
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
© Woodhead Publishing Limited, 2011
258
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
259
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).
© Woodhead Publishing Limited, 2011
260
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing Polyethylene:
[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.
© Woodhead Publishing Limited, 2011
262
Advanced wound repair therapies -(CH2–CH-)-
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
[ 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
© Woodhead Publishing Limited, 2011
264
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
265
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
© Woodhead Publishing Limited, 2011
266
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
267
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,
© Woodhead Publishing Limited, 2011
268
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
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
© Woodhead Publishing Limited, 2011
270
Advanced wound repair therapies γp
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
271
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
© Woodhead Publishing Limited, 2011
272
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
273
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
© Woodhead Publishing Limited, 2011
274
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
275
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
© Woodhead Publishing Limited, 2011
276
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
277
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
© Woodhead Publishing Limited, 2011
278
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
279
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
© Woodhead Publishing Limited, 2011
280
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
281
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.
© Woodhead Publishing Limited, 2011
282
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Adhesives and interfacial phenomena in wound healing
283
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.
© Woodhead Publishing Limited, 2011
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
Wound healing studies and interfacial phenomena
285
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
© Woodhead Publishing Limited, 2011
286
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
287
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
© Woodhead Publishing Limited, 2011
288
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
289
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
© Woodhead Publishing Limited, 2011
290
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
291
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
© Woodhead Publishing Limited, 2011
292
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
293
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
© Woodhead Publishing Limited, 2011
294
Advanced wound repair therapies
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,
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena Contact lens Tear fluid Cornea
Wound dressing Wound fluid Wound bed
295
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.
© Woodhead Publishing Limited, 2011
296
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
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
297
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:
© Woodhead Publishing Limited, 2011
298 • • • • •
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
299
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
© Woodhead Publishing Limited, 2011
300
Advanced wound repair therapies
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-
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
301
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
© Woodhead Publishing Limited, 2011
302
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
303
[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
46
63
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.
© Woodhead Publishing Limited, 2011
304
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
305
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
© Woodhead Publishing Limited, 2011
306
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
307
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
© Woodhead Publishing Limited, 2011
308
Advanced wound repair therapies
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-
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
309
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:
© Woodhead Publishing Limited, 2011
310 • • • • • • • • • •
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
311
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
© Woodhead Publishing Limited, 2011
312
Advanced wound repair therapies
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:
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena • • • • • •
• • • • • • •
313
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
© Woodhead Publishing Limited, 2011
314
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
315
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
References
Ahmed I I and Breslin C W (2001), ‘Role of the bandage soft contact lens in the postoperative laser in situ keratomileusis patient’, J Cataract Refract Surg, 27, 1932–1936. Asch E and Podack E (1990), ‘Vitronectin binds to activated human platelets and plays a role in platelet aggregation’, J Clin Invest, 85, 1372–1378. Berta A (1986), ‘Standardization of tear protein determinations: the effects of sampling, flow rate and vascular permeability’, in Holly F J (ed.), The Preocular Tear Film in Health, Disease, and Contact Lens Wear, Dry Eye Institute, Lubbock, TX, pp. 418–435. Broekhuyse R M (1974), ‘Tear lactoferrin: A bacteriostatic complexing protein’, Invest Ophthalmol Vis Sci, 13, 550–554. Bullen E C, Longaker M T, Updike D L, Benton R, Ladin D, Hou Z and Howard E W (1995), ‘Tissue inhibitor of metalloproteinases-1 is decreased and activated gelatinases are increased in chronic wounds’, J Invest Dermatol, 104, 236– 240. Carter M J, Tingley-Kelley K and Warriner R A, III (2009), ‘Silver treatments and silver-impregnated dressings for the healing of leg wounds and ulcers: A systematic review and meta-analysis’, Corrected proof, J Am Acad Dermatol, 10, 1016/j. jaad.2009.09.007. Cotsarelis G, Cheng S-Z, Dong G, Sun T-T, and Lavker R (1989), ‘Existence of slowcycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells’, Cell, 57, 201–209. Crosson C E, Klyce S D and Beuerman R W (1986), ‘Epithelial wound closure in the rabbit cornea’, Invest Ophthalmol Vis Sci, 27, 464–473. Cullen B, Watt P W, Lundqvist C, Silcock D, Schmidt R J, Bogan D and Light N D (2002a), ‘The role of oxidised regenerated cellulose/collagen in chronic wound
© Woodhead Publishing Limited, 2011
316
Advanced wound repair therapies
repair and its potential mechanism of action’, Int J Biochem Cell Biol, 34, 1544–1556. Cullen B, Smith R, McCulloch E, Silcock D and Morrison L (2002b), ‘Mechanism of action of PROMOGRAN, a protease modulating matrix, for the treatment of diabetic foot ulcers’, Wound Repair Regen, 10, 16–25. Cutting K F (2003), ‘Wound exudate: composition and functions’, Br J Community Nurs, 8(9 Suppl), 4–9. Davis S, Gil J, Valdes J, Perez J and Rivas Y (2009), ‘The effects of wound dressings as barriers to Pseudomonas aeruginosa colonization of full thickness wounds using a well established porcine wound model’, J Am Acad Dermatol, 60(Suppl 1), AB110. Declerck P J, De Mol M, Alessi M C, Baudner S, Paques E P, Preissner K T, MullerBerghaus G and Collen D (1988), ‘Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin)’, J Biol Chem, 263, 15454–15461. de Souza G A, de Godoy L M F and Mann M (2006), ‘Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors’, Genome Biol, 7, R72. Dillman W J and Miller I F (1973), On the adsorption of serum proteins on polymer membrane surfaces’, J Colloid Interface Sci, 44, 221–241. Drinkwater S L, Smith A and Burnand K G (2002), ‘What can wound fluids tell us about the venous ulcer microenvironment?’, Lower Extremity Wounds, 1, 184–190. Dua H S and Forrester J V (1987), ‘Clinical patterns of corneal epithelial wound healing’ Am J Ophthalmol, 104, 481–489. Duncan R L and McArthur W P (1981), ‘Lactoferrin mediated modulation of mononuclear cell activities. 1. Suppression of the immune in vitro primary antibody response’, Cell Immunol, 63, 308–320. Esmaeelpour M (2004), ‘The effect of tear collection techniques and short-term storage on the accuracy of total tear protein’, Ophthalmic Res, 36, 293. Evans M D M and Sweeney D F (2010), ‘Synthetic corneal implants’, in Chirila T (ed), Biomaterials and regenerative medicine in ophthalmology, Woodhead Publishing in Materials, CRC Press Washington, DC, pp. 65–133. Falanga V (1992), ‘Growth factors and chronic wounds: the need to understand the microenvironment’, J Dermatol, 19, 667–672. Falanga V (2004), ‘The chronic wound: impaired healing and solutions in the context of wound bed preparation’, Blood Cells, Molecules, Diseases, 32, 88–94. Farooqui S Z, Foster C S and Ma J J K (2008), ‘Central Sterile Corneal Ulceration’, http://emedicine.medscape.com/article/1196936-overview Fini, M E (1999), ‘Keratocyte and fibroblast phenotypes in the repairing cornea’, Prog Retin Eye Res, 18, 529–551. Fullard R J and Tucker D L (1991), ‘Changes in human tear protein levels with progressively increasing stimulus’, Invest Ophthalmol Vis Sci, 32, 2290–2301. Gipson I K (1992), ‘Adhesive mechanisms of the corneal epithelium’, Acta Ophthalmol, 70(s202), 13–17. Gleich L L, Rebeiz E E, Pankratov M M and Shapshay S M (1995), ‘Autologous fibrin tissue adhesive in endoscopic sinus surgery’, Otolaryngol Head Neck Surg, 112, 238–241.
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
317
Gottrup F, Jørgensen B, Karlsmark T, Sibbald R G, Rimdeika R, Harding K, Price P, Venning V, Vowden P, Jünger M, Wortmann S, Sulcaite R, Vilkevicius G, Ahokas T-L, Ettler K and Arenbergerova M (2007), ‘Less pain with Biatain-Ibu: initial findings from a randomised, controlled, double-blind clinical investigation on painful venous leg ulcers. Int Wound J, 4(Suppl 1), 24–34. Harb A and Pandya A N (2010), ‘Avoiding sutured tie-over dressings on the nasal dorsum’, Plastic Surg Int, 2010, 2 pages. Hildebrand A, Preissner K T, Miiller-Berghaus G and Teschemacher H (1989), ‘A novel β-endorphin binding protein. Complement S protein (= vitronectin) exhibits specific non-opioid binding sites for β-endorphin upon interaction with heparin or surfaces’, J Biol Chem, 264, 15429–15434. Huang A J W and Tseng S C G (1991), ‘Corneal epithelial wound healing in the absence of limbal epithelium’, Invest Ophthalmol Vis Sci, 32, 96–105. Imanishi J, Kamiyama K, Iguchi I, Kita M, Sotozono C and Kinoshita S (2000), ‘Growth factors: importance in wound healing and maintenance of transparency of the cornea’, Prog Retin Eye Res, 19, 113–129. James G A, Swogger E, Wolcott R, Pulcini E, Secor P, Sestrich J, Costerton J W and Stewart P S (2008), ‘Biofilms in chronic wounds’, Wound Repair Regen, 16, 37– 44. Jang Y-C, Tsou R, Gibran N S and Isik F F (2000), ‘Vitronectin regulates wound fibrinolysis, microvascular angiogenesis and wound contraction in mice’, Surgery, 127, 696–704. Jester J V, Petroll W M and Cavanagh H D, (1999), ‘Corneal stromal wound healing in refractive surgery: The role of myofibroblasts’, Prog Retin Eye Res, 18, 311–356. Karlgard C C S, Jones L W and Moresoli C (2002), ‘Survey of Bandage Lens Use in North America, October-December 2002’, Eye Contact Lens: Sci Clin Practice, 30, 25–30. Kijlstra A (1990), ‘The role of lactoferrin in the nonspecific immune response on the ocular surface’, Reg Immunol, 3, 193–197. Kijlstra A and Jeurissen S H M (1982), ‘Modulation of classical C3 covertase of complement by tear lactoferrin’, Immunology, 47, 263–270. Kuijpers T W, Van Lier R A, Hamann D, de Boer M, Thung L Y, Weening R S, Verhoeven A J and Roos D (1997), ‘Leukocyte adhesion deficiency type 1 (LAD-1)/variant. A novel immunodeficiency syndrome characterized by dysfunctional beta2 integrins’, J Clin Invest, 100, 1725–1733. Kuwabara T, Perkins DG and Cogan DG (1976), ‘Sliding of the epithelium in experimental corneal wounds’, Invest Ophthalmol, 15, 4–14. MacCabee M S, Trune D R and Hwang P H (2003), ‘Effects of topically applied biomaterials on paranasal sinus mucosal healing’, Am J Rhinology, 17, 203– 207. Mahomed A and Tighe B J (2006), ‘Structural aspects of the design of ocular drug delivery systems’, CLAE, 29, 202. Mahomed A, Cartwright H and Tighe B J (2003), ‘Hydrogel release systems in ocular and dermal drug delivery’, Ophthalmic Res, 35, 20. Mann A M and Tighe B J (2002), ‘The application of counter immunoelectrophoresis (CIE) in ocular protein studies Part II: Kinin activity in the lens wearing eye’, CLAE, 25, 81–88.
© Woodhead Publishing Limited, 2011
318
Advanced wound repair therapies
Mann A M and Tighe B J (2004), ‘The application of lab-on-a-chip technology in tear composition, deposited protein and tear “envelope” analysis’, Ophthalmol Res, 36(Suppl 1), 34. Mann A M and Tighe B J (2007), ‘Tear analysis and lens-tear interactions: Part I. Protein fingerprinting with microfluidic technology’, CLAE, 30, 163–173. Martin G M, Sprague C A and Epstein C J (1970), ‘Replicative life span of cultivated human cells. Effects of donor’s age, tissue and genotype’, Lab Invest, 23, 86– 92. Mertz P M, Marshall D A and Eaglstein W H (1985), ‘Occlusive wound dressings to prevent invasion and wound infection’, J Am Acad Dermatol, 12, 662–668. Mirastschijski U, Haaksma C J, Tomasek J J and Agren M S (2004), ‘Matrix metalloproteinase inhibitor GM 6001 attenuates keratinocyte migration, contraction and myofibroblast formation in skin wounds’, Exp Cell Res, 299, 465–475. Muller M (2009), ‘Cellular senescence: molecular mechanisms, in vivo significance and redox considerations’, Antiox Redox Signal, 11, 59–98. Nishiya K and Horwitz D D (1982), ‘Contrasting the effects of lactoferrin on human lymphocyte and monocyte natural killer activity and antibody-dependent cellmediated cytotoxicit’, J Immunol, 129, 2519–2523. Norde W (1986), ‘Adsorption of proteins from solution at the solid-liquid interface’, Adv Colloid Interface Sci, 25, 267–340. Norde W and Lyklema J (1978), ‘The adsorption of human plasma albumin and bovine pancreas ribonuclease at negatively charged polystyrene surfaces I. Adsorption isotherms. Effects of charge, ionic strength, and temperature’, J Colloid Interface Sci, 66, 257–265. Opdenakker G and Van Damme J (1994), ‘Cytokine-regulated proteases in autoimmune diseases’, Immunol Today, 15, 103–107. Palfreyman S, Nelson E A and Michaels J A (2007), ‘Dressings for venous leg ulcers: systematic review and meta-analysis’, BMJ, 335, 12 pages, doi: 10.1136/ bmj.39322.685405.AD Peach H C, Mann A M and Tighe B J (2002), ‘Immunoblotting and tear sampling techniques for the study of contact lens-induced variations in tear protein profiles’, Adv Expt Med Bio, 506, 967–971. Pfister R R (1994), ‘Corneal stem cell disease: Concepts, categorization, and treatment by auto- and homotransplantation of limbal stem cells’, CLAO J, 20, 64–72. Pitt W G and Cooper S L (1988), ‘Albumin adsorption on alkyl chain derivatized polyurethanes: I. The effect of C-18 alkylation’, J Biomed Mater Res, 22, 359–382. Preissner K T and Seiffert D (1998), ‘Role of vitronectin and its receptors in haemostasis and vascular remodeling’, Thromb Res, 89, 1–21. Princz M A, Sheardown H and Griffith M (2010), Corneal tissue engineering and versus synthetic artifical corneas’, in Chirila T (ed), Biomaterials and regenerative medicine in ophthalmology, Woodhead Publishing in Materials, CRC Press Washington, DC, pp. 65–133. Pytela R, Pierschbacher M D and Ruoslahti E, (1985), ‘Identification and isolation of a 140kDa cell surface glycoprotein with properties expected of a fibronectin receptor’, Cell, 40, 191–198. Sack R A, Tan K O and Tan A (1992), ‘Diurnal tear cycle: evidence for a nocturnal inflammatory constitutive tear fluid’, Invest Ophthalmol Vis Sci, 33, 626–640.
© Woodhead Publishing Limited, 2011
Wound healing studies and interfacial phenomena
319
Sack R A, Underwood P.A, Tan K O, Sutherland H and Morris C A (1993), ‘Vitronectin’, Ocular Immumol Inflamm, 1, 327–336. Salonen E M, Tervo T, Torma E, Tarkkanen A and Vaheri A (1987), ‘Plasmin in tear fluid of patients with corneal ulsers: Basis for new therapy’, Acta Ophthalmol, 65, 3–12. Schermer A, Galvin S and Sun T T (1986), ‘Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells’, J Cell Biol, 103, 49–62. Schneider E L and Mutsui Y (1976), ‘The relationship between in vitro cellular aging and in vivo human age, Proc Natl Acad Sci, 73, 3584–3588. Schönfelder U, Abel M, Wiegand C, Klemm D, Elsner P and Hipler U-C (2005), ‘Influence of selected wound dressings on PMN elastase in chronic wound fluid and their antioxidative potential in vitro’, Biomaterials, 26, 6664–6673. Sergio D, Rosenzweig M D and Holland S M (2004), ‘Phagocyte immunodeficiencies and their infections’, J Allergy Clin Immunol, 113, 620–626. Shai A and Maibach H I (2005), ‘Wound healing and ulcers of the skin. Diagnosis and therapy – the practical approach’, Springer-Verlag Berlin Heidelburg 14– 15. Shelton D N, Chang E, Whittier P S, Choi D and Funk W D (1999), ‘Microarray analysis of replicative senescence. Curr Biol, 9, 939–945. Soldati D, Rahm F and Pasche P (1999), ‘Mucosal wound healing after nasal surgery. A controlled clinical trial on the efficacy of hyaluronic acid containing cream’, Drugs Exp Clin Res, 25, 253–261. Stashak T S, Frastevedt E and Othic A (2004), ‘Update on wound dressings: Indications and best use’, Clin Tech Equine Prac, 3, 148–163. Stuchell R N, Feldman J J, Farris R L and Mandel I D (1984), ‘The effect of collection technique on tear composition’, Invest Ophthalmol Vis Sci, 25, 374–377. Suda T, Nishida T, Ohashi Y, Nakagawa S and Manabe R (1981–1982), ‘Fibronectin appears at the site of corneal stroma wounds in rabbits’, Curr Eye Res, 1, 553–556. Sundaram S, Lim F, Cooper SL 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. Szpaderska A M, Zuckerman J D and DiPietro L A (2003), ‘Differential injury responses in oral mucosal and cutaneous wounds’, J Dent Res, 82, 621–626. Tausche A-K and Sebastian G (2005), ‘Wound conditioning of a deep tissue defect including exposed bone after tumour excision using PROMOGRAN Matrix, a protease-modulating matrix. Int Wound J, 2, 253–257. Telfer N R and Moy R L (1993), ‘Wound care after office procedures’, J Dermatol Surg Oncol, 19, 722–731. Tervo T, Salonen E M, Vaheri A, Immonen I, Van Setten G B, Himberg J J and Tarkkanen (1988), ‘Elevation of tear plasmin in corneal disease’, Acta Ophthalmol, 66, 393–399. Thiagarajan P and Kelly K L (1988), ‘Exposure of binding sites for vitronectin on platelets following stimulation’, J Biol Chem, 263, 3035–3038. Thoft R A and Friend J (1983), ‘The X,Y,Z hypothesis of corneal epithelial maintenance’, Invest Ophthamol Vis Sci, 24, 1442–1443.
© Woodhead Publishing Limited, 2011
320
Advanced wound repair therapies
Tighe B J, Franklin V J, Graham C, Mann A and Guillon M (2001), ‘Ophthalmic biomaterials: the inflammatory role of vitronectin’, Anales Del Instituto Barraquer, XXX, 221–222. Trengove N J, Langton S R and Stacey M C (1996), ‘Biochemical analysis of wound fluid from nonhealing and healing chronic leg ulcers,’ Wound Repair Regen, 4, 234–239. 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. Tuominen I S, Tervo T M, Teppo A M, Valle T U Gronhagen-Riska C and Vesaluoma M H (2001), ‘Human tear fluid PDGF-BB, TNF-alpha and TGF-beta 1 vs. corneal haze and regeneration of corneal epithelium and sub-basal nerve plexus after PRK’, Exp Eye Res, 72, 631–641. Vaheri A, Bizik J, Salonen E M, Tapiovaara H, Siren V, Myohanen H and Stephens R W (1992), ‘Regulation of the pericellular activation of plasminogen and its role in tissue-destructive process’, Act Ophthalmol, 70, 34–41. Valentine, R and Wormald, P-J (2010), ‘Nasal dressings after endoscopic sinus surgery: what and why?’, Curr Opin Otolaryngology Head Neck Surg, 18, 44–48. Valleala H, Hanemaaijer R, Mandelin J, Salminen A, Teronen O and Monkkonen J and Konttinen Y T (2003), ‘Regulation of MMP-9 (gelatinase B) in activated human monocyte/macrophages by two different types of bisphosphonates’, Life Sciences, 73, 2413–2420. Vannas A, Sweeney D F, Holden B A, Sapyska E, Salonen E M and Vaheri A (1992), ‘Tear plasmin activity with contact lens wear’ Curr Eye Res, 11, 245–251. Venter T H J, Karpelowsky J S and Rodeburns H (2007), ‘Cooling of the burn wound: The ideal temperature of the coolant’, Burns, 33, 917–922. Vermeulen H, Westerbos S J and Ubbink D T (2010), ‘Benefit and harm of iodine in wound care: a systematic review’, J Hosp Inf, Corrected Proof, 10, 1016/j. jhin.2010.04.026. Vroman L (1962), ‘Effect of adsorbed proteins on the wettability of hydrophilic and hydrophobic solids’, Nature, 196, 476–477. Wahlgren M and Arnebrant T (1991), ‘Protein adsorption to solid surfaces’, Trends in Biotech, 9, 201–208. Wilson S E, Liu J J and Mohan R R (1999), ‘Stromal-epithelial interactions in the cornea’, Prog Retin Eye Res, 18, 293–309. Wilson S E, Mohan R R, Ambrosio Jr R, Hong J and Lee J (2001), ‘The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells’, Prog Reti Eye Res, 20, 625–637. Yager D R and Nwomeh B C (1999), ‘The proteolytic environment of chronic wounds’, Wound Repair Regen, 7, 433–441. Yu F-S X, Yin J, Xu K and Huang J (2010), ‘Growth factors and corneal epithelial wound healing’, Brain Res Bulletin, 81, 229–235. Zavaro A, Samra Z, Baryishak R and Sompolinsky D (1980), ‘Proteins in tears from healthy and diseased eye’, Documenta Ophthalmologica, 50, 185–199.
© Woodhead Publishing Limited, 2011
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
322
Advanced wound repair therapies
(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.
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
323
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.
© Woodhead Publishing Limited, 2011
324
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
325
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
© Woodhead Publishing Limited, 2011
326
Advanced wound repair therapies free radical vinyl polymerisation
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-
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
327
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
© Woodhead Publishing Limited, 2011
328
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
329
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.
© Woodhead Publishing Limited, 2011
330
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
331
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
© Woodhead Publishing Limited, 2011
332
Advanced wound repair therapies lipid layer
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
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
333
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.
© Woodhead Publishing Limited, 2011
334
Advanced wound repair therapies Keratan sulphate proteoglycan
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.
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
335
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
© Woodhead Publishing Limited, 2011
336
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
337
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
© Woodhead Publishing Limited, 2011
338
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
339
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
© Woodhead Publishing Limited, 2011
340
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
341
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
© Woodhead Publishing Limited, 2011
342
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
343
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
© Woodhead Publishing Limited, 2011
344
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
345
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.
© Woodhead Publishing Limited, 2011
346
Advanced wound repair therapies
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).
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
347
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
© Woodhead Publishing Limited, 2011
348
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
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
© Woodhead Publishing Limited, 2011
350
Advanced wound repair therapies Sequestration of APP
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–
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
351
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
© Woodhead Publishing Limited, 2011
352
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
353
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
© Woodhead Publishing Limited, 2011
354
Advanced wound repair therapies
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
Bahia R K (2008), ‘Skin adhesive hydrogels for the topical delivery of active agents’, Thesis, Aston University, Birmingham, UK. Bayraktar H, Akal E, Sarper O and Varnali T (2004), ‘Modeling glycosaminoglycanshyaluronan, chondroitin, chondroitin sulphate A, chondroitin sulphate C and keratan sulphate’, J Mol Structure:Theochem, 683, 121–132. Benning B K (2000), ‘Novel high water content hydrogels’, Thesis, Aston University, Birmingham, UK. Boote N D (2007), ‘Photopolymerisation of sheet hydrogels: A mechanistic study’ Thesis, Aston University, Birmingham, UK. Botos I, Scapozza L, Zhang D, Liotta L A and Meyer E F (1996), ‘Batimastat, a potent matrix metalloproteinase inhibitor, exhibits an unexpected mode of binding’, Procs Nat Acad Sci USA, 93, 2749–2754. Brulez B (2004), ‘Biomimetic prosthetic ligament and production method thereof’, WO200406705, 2004-08-12. Drotleff S, Lungwitz U, Breunig M, Dennis A, Blunk T, Tessmar J and Gopferich A (2004), ‘Biomimetic polymers in pharmaceutical and biomedical sciences’, Eur J Pharm Biopharm, 58, 385–407. Essler A and Nisbet L (2007), ‘Active wound dressing materials’, WO2007068885, to Ethicon Inc (US). Fleming M C (1999), ‘Skin adhesive hydrogels for biomedical applications’, Thesis, Aston University, Birmingham, UK. Gilbert M E, Kirker K R, Gray S D, Ward P D, Prestwich G D and Orlandi R R (2004), ‘Chrondroitin sulphate hydrogel and wound healing in rabbit maxillary sinus mucosa’, Laryngoscope, 114, 1406–1409. Gipson I K (2004), ‘Distribution of mucins at the ocular surface’, Exp Eye Res, 78, 379–388. Gozzo A J, Nunes V A, Carmona A K, Nader H B, von Dietrich C P, Silveira V L F, Shimamoto K, Ura N, Sampaio M U, Sampaio C A M and Araujo M S (2002), ‘Glycosaminoglycans affect the action of human plasma kallikrein on kininogen hydrolysis and inflammation’, Int Immunopharmacology, 2, 1861–1865. Hill D H, Ottersbach P, Djavid G, Jozefowicz M, Migonney V and Vairon J-P (2002), ‘Preparation of carboxylate-sulphonate polymers having cell proliferation-promoting properties’, United States Patent, 6365692.
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
355
Jani M, Tordai H, Trexler M, Bányai L and Patthy L (2005), ‘Hydroxamate-based peptide inhibitors of matrix metalloprotease 2’, Biochimie, 87, 4385–4392. Johnson & Johnson Vision Care (US) (2004), ‘Antimicrobial surfaces for ophthalmic devices’, WO2004094075, 2004-11-04. Karim S (2005), ‘New low molecular weight sulphated polysaccharides’, FR2871476 -2005. Kirker K R, Luo Y, Morris S E, Shelby J and Prestwich G D (2004), ‘Glycosaminoglycan hydrogel films as supplemental wound dressings for donor sites’, J Burn Care Rehabil, 25, 276–286. Kisilevsky R, Lemieux L J, Fraser P E, Kong X, Hultin P G and Szarek W A (1995), ‘Arresting amyloidosis in vivo using small-molecule anionic sulphonates or sulphates: implications for Alzheimer’s disease’, Nat Med, 1, 143–148. Kuijpers T W, Van Lier R A, Hamann D, de Boer M, Thung L Y, Weening R S, Verhoeven A J and Roos D (1997), ‘Leukocyte adhesion deficiency type 1 (LAD-1) variant. A novel immunodeficiency syndrome characterized by dysfunctional beta2 integrins’, J Clin Invest, 100, 1725–1733. Lavik E and Langer R (2004), ‘Tissue engineering: current state and perspectives’, App Microbiol Biotech, 65, 1–8. Leveugle B, Scanameo A, Ding W and Fillit H (1994), ‘Binding of heparan sulfate glycosaminoglycan to β-amyloid peptide: inhibition by potentially therapeutic polysulfated compounds’, NeuroReport, 5, 1389–1392. Lima A P C A, Almeida P C, Tersariol I L S, Schmitz V, Schmaier A H, Juliano L, Hirata I Y, Müller-Esterl W, Chagas J R and Scharfstein J (2002), ‘Heparan sulphate modulates kinin release by Trypanosoma cruzi through the activity of cruzipain’, J Biol Chem, 277, 5875–5881. Mahomed A (2005), ‘Ionic and neutral hydrogels for dermal and ophthalmic applications’, Thesis, Aston University, Birmingham, UK. Michelacci Y M (2003), ‘Collagens and proteglycans of the corneal extracellular matrix’, Braz J Med Res, 38, 1037–1046. Miksch K, Liwarska-Bizukojc E, Malachowska-Jutsz A and Kalka J (2005), ‘Acute toxicity and genotoxicity of five selected anionic and nonionic surfactants’, Chemosphere, 58, 1249–1253. Minett W T (1986), ‘Cell adhesion on synthetic polymer substrates’, Thesis, Aston University, Birmingham, UK. Mostow E N, Haraway G D, Dalsing M, Hodde J P and King D (2005), ‘Effectiveness of an extracellular matrix graft (OASIS Wound Matrix) in the treatment of chronic leg ulcers: a randomized clinical trial’, J Vasc Surg, 41, 856–862. Munro H S (2010a), ‘Modulation of proteases. Particularly in the treatment of chronic ulcerous skin lesions’, US2010183722 (A1) – 2010-07-22. Munro H S (2010b), ‘Treatment of inflammation and the complement and kinin cascades in a patient, particularly in chronic ulcerous skin lesions’, US2010143478 (A1) – 2010-06-10. Munro H S and Andrews P (2010a), ‘Compositions for use as or in wound dressings’, WO2010007436 (A2) – 2010–01-21. Munro H S and Andrews P (2010b), ‘Hydrogel composites and wound dressings’, WO2009098518 (A2) – 2009-08-13. Nalampang K, Molloy R and Suebsanit N (2006), ‘Design and preparation of synthetic hydrogels for biomedical use as wound dressings’, 4th Thailand Materials
© Woodhead Publishing Limited, 2011
356
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Sulphonated biomaterials as glycosaminoglycan mimics
357
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.
© Woodhead Publishing Limited, 2011
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
362
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Drug delivery dressings
363
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
© Woodhead Publishing Limited, 2011
364
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Drug delivery dressings
365
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
© Woodhead Publishing Limited, 2011
366
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Drug delivery dressings
367
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-
© Woodhead Publishing Limited, 2011
368
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Drug delivery dressings
369
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
© Woodhead Publishing Limited, 2011
370
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Drug delivery dressings
371
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.
© Woodhead Publishing Limited, 2011
372
Advanced wound repair therapies
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
© Woodhead Publishing Limited, 2011
Drug delivery dressings
373
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).
© Woodhead Publishing Limited, 2011
374
Advanced wound repair therapies
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.
© Woodhead Publishing Limited, 2011
Drug delivery dressings
375
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.
© Woodhead Publishing Limited, 2011
376
Advanced wound repair therapies
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).
© Woodhead Publishing Limited, 2011
Drug delivery dressings
377
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
© Woodhead Publishing Limited, 2011
378
Advanced wound repair therapies
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 prot
