Regenerative Medicine and Biomaterials for the Repair of Connective Tissues

Regenerative medicine and biomaterials for the repair of connective tissues

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Regenerative medicine and biomaterials for the repair of connective tissues Edited by Charles Archer and Jim Ralphs

Published by Woodhead Publishing Limited, Abington Hall, Granta Park, Great Abington, Cambridge CB21 6AH, UK www.woodheadpublishing.com Woodhead Publishing India Private Limited, G-2, Vardaan House, 7/28 Ansari Road, Daryaganj, New Delhi ± 110002, India www.woodheadpublishingindia.com Published in North America by CRC Press LLC, 6000 Broken Sound Parkway, NW, Suite 300, Boca Raton, FL 33487, USA First published 2010, Woodhead Publishing Limited and CRC Press LLC ß 2010, Woodhead Publishing Limited 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. Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress. Woodhead Publishing Limited ISBN 978-1-84569-417-3 (book) Woodhead Publishing Limited ISBN 978-1-84569-779-2 (e-book) CRC Press ISBN 978-1-4398-0110-9 CRC Press order number: N10011 The publishers' 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 publishers ensure that the text paper and cover board used have met acceptable environmental accreditation standards. Typeset by Godiva Publishing Services Limited, Coventry, West Midlands, UK Printed by TJ International Limited, Padstow, Cornwall, UK

Contents

Contributor contact details

1

The structure and regenerative capacity of synovial joint tissues

1

Introduction Structure and function of synovial joint Joint tissues and their biomechanical properties Resident mesenchymal progenitor cells in synovial joint tissues Conclusions and future trends Sources of further information and advice References

1 2 4 16 26 28 29

A.-M. S AÈ AÈ M AÈ N E N , University of Turku, Finland, J. P. A. A R O K O S K I , University of Kuopio and Kuopio University Hospital, Finland, J. S. J U R V E L I N , University of Kuopio, Finland and I. K I V I R A N T A , University of Helsinki, Finland 1.1 1.2 1.3 1.4 1.5 1.6 1.7

2

The myofibroblast in connective tissue repair and regeneration B. H I N Z , University of Toronto, Canada

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

xiii

Introduction Myofibroblasts: humble tissue construction workers Know thy enemy: a quick guide to identify the myofibroblast Origins of the myofibroblast Mesenchymal stem cells (MSC) and the myofibroblast phenotype: regeneration, repair or risk? What drives myofibroblast differentiation? Lessons to be learned from the myofibroblast for the effective use of mesenchymal stem cells (MSC) Conclusions and future trends References

39 39 41 44 46 49 55 60 62 63

vi

Contents

Part I Cartilage repair and regeneration 3 3.1 3.2 3.3 3.4 3.5 3.6

4

The structure of articular cartilage

E. B. H U N Z I K E R , University of Bern, Switzerland Introduction General structure and function of articular cartilage Dual function of immature articular cartilage during postnatal growth Physiological mechanism underlying the evolution of a mature from an immature articular cartilage structure Inter-species differences in articular cartilage structure, and structure±function correlations in humans References

Measuring the biomechanical properties of cartilage cells

D. L. B A D E R and M. M. K N I G H T , Queen Mary University of London, UK 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 5.6

6

83 84 89 95 98 101

106

Introduction Measurement of chondrocyte biomechanics Intracellular biomechanics Biomechanical conditioning of chondrocytes Future trends Acknowledgements References

106 107 116 117 129 130 130

Understanding tissue response to cartilage injury

137

Introduction Clinical in vivo cartilage injury Animal models of cartilage injury In vitro cartilage injury Conclusions References

137 138 143 146 149 149

F. D E L L ' A C C I O , Barts and The London School of Medicine and Dentistry, UK and T . L . V I N C E N T , Kennedy Institute of Rheumatology, UK

Understanding osteoarthritis and other cartilage diseases T. A I G N E R , Medical Center Coburg, Germany, N. S C H M I T Z , University of Leipzig, Germany and S. S OÈ D E R , University of Erlangen-Nurnberg, Germany

6.1

83

Introduction

155

155

Contents 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9

7

The normal joint Major cartilage pathology and pathobiology In vivo cartilage repair Grading/scoring systems for cartilage degeneration Grading/scoring of cartilage repair Sources of further information and advice Future trends References

156 157 165 168 170 173 174 174

Using animal models of cartilage repair to screen new clinical techniques

178

C. W. M c I L W R A I T H , Colorado State University, USA

7.1 7.2 7.3 7.4 7.5 7.6

8

Introduction Review of models in non-equine species Early equine models of cartilage repair Current models of cartilage repair in the equine femoropatellar and femorotibial joints Current status of animal models of cartilage repair References and further reading

Cartilage tissue repair: autologous osteochondral mosaicplasty

L. H A N G O D Y , Uzsoki Hospital, Hungary, G. K I S H , Saint George Medical, USA, T. K O R E N Y , PeÂcs Medical School, Hungary, L. R. H A N G O D Y , Semmelweis Medical School, Hungary and L. M OÂ D I S , Debrecen Medical School, Hungary 8.1 8.2 8.3 8.4 8.5 8.6 8.7

9

Introduction The development of the mosaicplasty resurfacing technique: animal and other studies Surgical technique: pre-operative planning Surgical instruments and choice of surgical technique Arthroscopic mosaicplasty Conclusions References

Cartilage tissue repair: autologous chondrocyte implantation M. B R I T T B E R G , University of Gothenburg, Sweden

9.1 9.2 9.3

vii

Introduction Chondrogeneic cell implantation Articular or other types of chondrocytes, allogeneic or autologous chondrocytes?

178 179 181 185 194 196

201

201 201 202 204 204 206 219 220

227 227 228 228

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Contents

9.4 9.5 9.6 9.7 9.8 9.9 9.10 9.11 9.12 9.13

Autologous chondrocytes Human clinical use and studies with autologous chondrocyte implantation Other joints besides the knee joint Clinical follow-up results Imaging evaluation of the cartilage repair Randomised controlled studies Chondrocyte implantation and osteoarthritis (OA) Conclusions and future trends Sources of further information and advice References

10

Cell sheet technologies for cartilage repair

10.1 10.2 10.3 10.4 10.5 10.6 10.7

Introduction Overview of present clinical applications Challenge for cartilage repair Properties of chondrocyte sheets Future trends in cartilage repair Regulations regarding regenerative medicine in Japan References

251 252 253 258 262 262 263

11

Cell therapies for articular cartilage repair: chondrocytes and mesenchymal stem cells

266

M. S A T O , Tokai University School of Medicine, Japan

R. ANDRIAMANALIJAONA, University of Caen, France

11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 11.10 11.11 11.12 11.13

Introduction The chondrocyte: a unique cell The macromolecular network and biomechanical properties of cartilage Phenotypic changes Cell therapy for articular cartilage repair: chondrocytes and mesenchymal stem cells (MSCs) The use of chemical compounds to enhance matrix production Strategies to maintain the chondrogenic phenotype: the use of three-dimensional systems Use of exogenous growth factors to promote chondrogenic phenotype Use of gene therapy to deliver chondrogenic factors Control of chondrocyte phenotype and chondrogenesis by hydrostatic pressure Use of low oxygen tension in cartilage repair Conclusions Acknowledgements

230 233 239 239 242 243 244 245 246 246

251

266 267 271 273 273 277 279 281 283 284 285 289 291

Contents 11.14

12 12.1 12.2 12.3 12.4 12.5 12.6 12.7

13

13.1 13.2 13.3 13.4 13.5

References and further reading

ix 292

Scaffolds for musculoskeletal tissue engineering

301

Introduction Cell types utilized for tissue regeneration Scaffolds for engineering musculoskeletal tissue Tissue remodeling Matrix stimulation and cell±cell communications in tissue regeneration Future trends and perspectives References

301 302 306 310

H. L I and J. H. E L I S S E E F F , Johns Hopkins University, USA

Outcome measures of articular cartilage repair

M. E. T R I C E , Johns Hopkins University School of Medicine, USA Introduction Patient-based (subjective) outcome measures Process-centered (objective) outcome measures Conclusions References

314 318 319

330

330 333 338 344 344

Part II Repair of tendons and ligaments 14 14.1 14.2 14.3 14.4 14.5

15

15.1 15.2 15.3 15.4

The structure of tendons and ligaments

M. B E N J A M I N , Cardiff University, UK

Introduction Basic aspects of cell and extracellular matrix (ECM) structure Specialised regions of tendons and ligaments Conclusions References

Tendon biomechanics

M. K J á R , Bispebjerg Hospital and University of Copenhagen, Denmark, S. P. M A G N U S S O N , University of Copenhagen, Denmark and A . M A C K E Y , Bispebjerg Hospital, Denmark Introduction Biochemical adaptation of tendon to loading Biomechanics of human tendon References and further reading

351 351 354 362 368 369

375

375 376 382 388

x

16

16.1 16.2 16.3 16.4 16.5 16.6 16.7 16.8

Contents

Tendon injury and repair mechanisms

N. M A F F U L L I , Barts and The London School of Medicine and Dentistry, UK, and U. G. L O N G O , P. S H A R M A and V. D E N A R O , Campus Biomedico University, Italy

394

Introduction: tendon injury Tendinopathy Genetics Tendon rupture Pain in tendinopathy Tendon healing following acute injuries Conclusions References

394 394 398 400 406 407 410 410

Tissue engineering for ligament and tendon repair

419

17.1 17.2 17.3 17.4 17.5 17.6

Introduction Tissue engineering approaches for ligament and tendon repair Reconstruction of ligaments and tendons Future trends Sources of further information and advice References

419 420 427 428 430 430

18

Cell-based therapies for the repair and regeneration of tendons and ligaments

17

M. L E E and B. M. W U , University of California, Los Angeles, USA

R. K. W. S M I T H , The Royal Veterinary College, UK 18.1 18.2 18.3 18.4 18.5 18.6 18.7 18.8 18.9 18.10 18.11

Introduction The rationale behind the use of cells to treat tendon and ligament injuries Cell choice for tendon and ligament treatment Mixed cell populations Allogenic versus autologous sources Proposed beneficial actions of stem cells on tendon healing Stem cell-induced tenogenesis in vitro Stem cell-induced tenogenesis in vivo Conclusions Sources of further information and advice References

436 436 437 438 441 442 442 442 443 447 447 447

Contents

19

Scaffolds for tendon and ligament tissue engineering

J. C. H. G O H and S. S A H O O , National University of Singapore, Singapore 19.1 19.2 19.3 19.4 19.5 19.6

xi

452

Criteria and requirements for tendon/ligament tissue engineering scaffolds Biomaterials for tendon and ligament tissue engineering Scaffold architecture Functional scaffolds Future trends References

452 453 455 460 462 463

Index

469

Contributor contact details

(* = main contact) Editors Professor Charles Archer and Dr Jim Ralphs School of Biosciences Cardiff University Cardiff CF10 3AX UK E-mail: [email protected]; [email protected] Chapter 1 Anna-Marja SaÈaÈmaÈnen* University of Turku Department of Medical Biochemistry and Genetics Institute of Biomedicine Kiinamyllynkatu 10 FIN-20520 Turku Finland E-mail: [email protected]

Jari P. A. Arokoski University of Kuopio and Kuopio University Hospital Institute of Clinical Medicine and Department of Physical and Rehabilitation Medicine PO Box 1627 FIN-70211 Kuopio Finland E-mail: [email protected] Jukka S. Jurvelin Department of Physics University of Kuopio PO Box 1627 FIN-70211 Kuopio Finland E-mail: [email protected] Ilkka Kiviranta University of Helsinki Department of Orthopaedics and Traumatology Topeliuksenkatu 5 B FIN-00260 Helsinki Finland E-mail: [email protected]

xiv

Contributor contact details

Chapter 2 Dr. Boris Hinz Laboratory of Tissue Repair and Regeneration Matrix Dynamics Group Faculty of Dentistry University of Toronto Room 241, Fitzgerald Building 150 College Street Toronto, ON M5S 3E2 Canada E-mail: [email protected]

Chapter 5 F. Dell'accio Centre of Experimental Medicine and Rheumatology William Harvey Research Institute Barts and The London School of Medicine and Dentistry II floor John Vane Building Charterhouse Square London EC1M 6BQ UK E-mail: f.dell'[email protected]

Chapter 3 E. B. Hunziker Center of Regenerative Medicine for Skeletal Tissues Department of Clinical Research University of Bern Bern Switzerland E-mail: [email protected]

T. L. Vincent* Kennedy Institute of Rheumatology 65 Aspenlea Road London W6 8LH UK E-mail: [email protected]

Chapter 4 D. L. Bader* and M. M. Knight Medical Engineering Division School of Engineering and Materials Science Queen Mary University of London Mile End Road London E1 4NS UK E-mail: [email protected]

Chapter 6 Thomas Aigner* Medical Center Coburg Institute of Pathology Ketschendorferstr. 33 96450 Coburg Germany E-mail: [email protected] Nicole Schmitz Institute of Pathology University of Leipzig Liebigstr. 26 04103 Leipzig Germany E-mail: [email protected]

Contributor contact details Stephan SoÈder Institute of Pathology University of Erlangen-Nurnberg Krankenhausstrasse 810 91054 Erlangen Germany Chapter 7 C. W. McIlwraith Barbara Cox Anthony University Chair Orthopedic Research Laboratory Colorado State University 300 West Drake Ft Collins, CO 80525 USA E-mail: [email protected] Chapter 8 LaÂszlo Hangody* Medical and Health Science Center Faculty of Medicine University of Debrecen Debrecen Hungary and Department of Orthopaedics Uzsoki Hospital Budapest Hungary E-mail: [email protected] Gary Kish Wound Care Center Portsmouth Regional Hospital Portsmouth New Hampshire USA and

xv

Medical and Health Science Center Faculty of Medicine Department of Anatomy, Histology and Embryology University of Debrecen Debrecen Hungary E-mail: [email protected] TamaÂs Koreny Faculty of Medicine Institute of Musculoskeletal Surgery Department of Traumatology and Hand Surgery University of PeÂcs PeÂcs Hungary E-mail: [email protected] LaÂszlo Rudolof Hangody Faculty of General Medicine Doctoral School Semmelweis University Budapest Hungary E-mail: [email protected] LaÂszlo MoÂdis Medical and Health Science Center Faculty of Medicine Department of Anatomy, Histology and Embryology University of Debrecen Debrecen Hungary E-mail: [email protected]

xvi

Contributor contact details

Chapter 9 M. Brittberg Cartilage Research Unit University of Gothenburg Endoscopium Department of Orthopedics Kungsbacka Hospital S-434 80 Kungsbacka Sweden E-mail: [email protected]

Chapter 13 Michael E. Trice Johns Hopkins University School of Medicine Johns Hopkins Bayview Medical Center 4940 Eastern Avenue Baltimore, MD 21224 USA E-mail: [email protected]

Chapter 10 Associate Professor Masato Sato Department of Orthopaedic Surgery, Surgical Science Tokai University School of Medicine 143 Shimokasuya Isehara Kanagawa 259-1193 Japan E-mail: [email protected]

Chapter 14 Professor Michael Benjamin School of Biosciences Cardiff University Museum Avenue Cardiff CF10 3AX UK E-mail: [email protected]

Chapter 11 R. Andriamanalijaona Normandie Incubation Centre d'Innovation Technologique 17, rue Claude Bloch -BP 55027 14076 Caen cedex 5 France E-mail: [email protected] Chapter 12 Hanwei Li and Jennifer H. Elisseeff* Department of Biomedical Engineering Johns Hopkins University Clark Hall 106 3400 N. Charles Street Baltimore, MD 21218 USA E-mail: [email protected]; [email protected]

Chapter 15 Professor Michael Kjñr* Institute of Sports Medicine and Centre for Healthy Ageing Bispebjerg Hospital Denmark E-mail: [email protected] and Head of Institute of Sports Medicine Faculty of Health Sciences University of Copenhagen Bispebjerg Bakke 23 DK-2400 Copenhagen NV Denmark

Contributor contact details Professor S. P. Magnusson Professor of Musculoskeletal Rehabilitation University of Copenhagen Bispebjerg Bakke 23 DK-2400 Copenhagen NV Denmark E-mail: [email protected] Dr Abigail Mackey Institute of Sports Medicine Bispebjerg Hospital Denmark Chapter 16 N. Maffulli* Centre for Sports and Exercise Medicine Barts and The London School of Medicine and Dentistry Mile End Hospital 275 Bancroft Road London E1 4DG UK E-mail: [email protected] U. G. Longo, P. Sharma and V. Denaro Department of Orthopaedic and Trauma Surgery Campus Biomedico University Via Alvaro del Portilo, 200 00128 Trigoria Rome Italy

xvii

Chapter 17 M. Lee* and B. M. Wu Department of Bioengineering University of California, Los Angeles 5121 Engineering V 420 Westwood Plaza Los Angeles, CA 90095 USA E-mail: [email protected] Chapter 18 R. K. W. Smith Department of Veterinary Clinical Sciences The Royal Veterinary College Hawkshead Lane North Mymms Hatfield AL9 7TA UK E-mail: [email protected] Chapter 19 J. C. H. Goh* and Sambit Sahoo Department of Orthopaedic Surgery Division of Bioengineering National University of Singapore Singapore E-mail: [email protected]

1

The structure and regenerative capacity of synovial joint tissues ÈA È MA È N E N , University of Turku, Finland, A.-M. SA J . P . A . A R O K O S K I , University of Kuopio and Kuopio University Hospital, Finland, J . S . J U R V E L I N , University of Kuopio, Finland and I . K I V I R A N T A , University of Helsinki, Finland

Abstract: This chapter provides an introduction to the structure, function, and biomechanical properties of synovial joint and its tissues with special emphasis to articular cartilage. Structural elements are described at the cellular level. Major extracellular matrix components, their organization and relationship with biomechanical properties are described. Also, a short introduction to basic methodology to measure biomechanical parameters is presented. In addition, the studies demonstrating presence of human endogenous multi-potent mesenchymal stem/stromal cells (MSCs) and mesenchymal progenitors in synovial joint and associated tissues are reviewed. Possible implications of endogenous MSCs in tissue repair potential are discussed. Key words: synovial joint, biomechanics, multi-potent mesenchymal stromal cell, tissue regeneration.

1.1

Introduction

The purpose of this chapter is to introduce the reader to the structure and function of synovial joint and associated structures. First, the macroscopic structure of synovial joint compartments, cellular composition, tissue organization, and description of the major extracellular components will be reviewed in articular cartilage, subchondral bone, tendon and ligaments, synovial membrane, and meniscus. Second, the interrelationship of extracellular matrix composition with biomechanical properties of the tissues will be discussed. In addition, the basic methodology used for measuring the biomechanical parameters of joint tissues, with emphasis on articular cartilage, will be introduced. Third, the presence of resident mesenchymal stromal cells (MSCs) and progenitors in synovial joint tissues will be described, and differences in properties of MSCs derived from different intra-articular and extra-articular tissues will be discussed. MSCs represent the intrinsic repair potential in these tissues, but they also have a significant input in regulating tissue homeostasis by secreting several

2

Regenerative medicine and biomaterials in connective tissue repair

growth factors, cytokines and bioactive factors. The progress of stem cell research during the last ten years has increased our understanding of their function in tissue regeneration. However, knowledge of the role of MSCs in the repair processes of many joints tissues is still deficient.

1.2

Structure and function of synovial joint

The synovial joint is a functional unit with mechanically interacting structural components (Fig. 1.1). The development of synovial joints arises from the mesenchymal cells (Archer et al. 2003; Khan et al. 2007). Hyaline cartilage itself forms the cartilaginous model of the developing skeleton. It is replaced by bone in a process known as endochondral ossification (Mackie et al. 2008). Articular cartilage (AC) covers the ends of the bones and synovial fluid lubricates and nourishes the cartilaginous tissue. Ligaments bind the skeletal elements together and a fibrous capsule encapsulates the joint. The synovial joint (e.g. knee joint) may also contain meniscal structures internally. Each joint tissue, including bone, muscle, AC, ligaments, and tendons, has its unique

1.1 Schematic presentation of the anatomy of the knee joint. A sagittal view.

The structure and regenerative capacity of synovial joint tissues

3

structure and functional properties, and changes in any component may lead to anabolic or catabolic responses in another joint component. The knee joint, joining femur and tibia in the lower limb, is the biggest synovial joint in the body. It consists of three articulating bones (femur, tibia and patella) covered by hyaline cartilage, the quadriceps and hamstring muscles, collateral and cruciate ligaments that hold the joint together, patellar tendon and the menisci. In principle, the knee is constructed of two joints, i.e. the patellofemoral joint and the tibio-femoral joint. The knee joint structures enable compression, rolling and sliding between the contacting bones. Also, the joints transmit loads of the upper body, reaching tibio-femoral loads of eight times the body weight (Kuster et al. 1997) and patello-femoral joint loads seven times the body weight (Nisell 1985) during normal daily activities, such as downhill walking and jogging. Based on the experimental analysis during simulated walking cycle, maximum tibio-femoral contact stresses of 14 MPa were recorded (Thambyah et al. 2005). This could be considered potentially dangerous for AC, knowing that there appears to be a critical threshold stress (15±20 MPa) that causes cell death and rupture of collagen network in vitro (D'Lima et al. 2001; Torzilli et al. 2006). It is suggested that the amount of loadinduced cell death is a function of the duration and magnitude of the applied load. In a healthy synovial joint, the friction coefficient between contacting articular surfaces is low; typical values between 0.01 and 0.04 have been estimated in a human hip (Unsworth et al. 1975). Several lubrication theories have been proposed, including hydrodynamic, squeeze film, and weeping and boosted mechanisms for lubrication. Each of them specifically addresses the role of intrinsic fluid and synovial fluid. Based on the operational demand, more than one lubrication mechanism is needed to provide the low friction within the synovial joint. For lubrication, the highly important mechanism is the interstitial fluid pressurization within the cartilage matrix (Ateshian 2009). However, during static loading, the boundary type of lubrication is facilitated by the molecules such as hyaluronan (HA), glycoproteins, and surface active phospholipids found in the synovial fluid (Katta et al. 2008). Functional adaptation is known as conditioning of the structure, composition and functional properties of the joint tissues to mechanical loads they are exposed to (Hyttinen et al. 2001; Tammi et al. 1987). In a healthy joint, this will lead to optimized joint function. However, mechanical conditioning may fail, leading to overloading of joint structures and, subsequently, to harmful changes in the tissues. Further, this will create an imbalance between tissue properties and functional demands, leading potentially to progressive degeneration of the structures in question. In osteoarthritis (OA), the pathological process may be triggered by changes in any joint component and no consensus has been found for the initial pathological mechanism. However, mechanical factors have been considered critical in the initiation and progress of OA. Changes in cartilage,

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Regenerative medicine and biomaterials in connective tissue repair

such as early superficial depletion of proteoglycans (Arokoski et al. 2000; Helminen et al. 2000) has been considered as a primary mechanism for the OA process, but alternative theories address the role of initial subchondral changes, including bone stiffening (Radin and Rose 1986; Burr and Radin 2003), in the pathological degeneration of cartilage tissue.

1.3

Joint tissues and their biomechanical properties

1.3.1

Articular cartilage

Articular cartilage is highly specialized connective tissue, and it is aneural, alymphatic, and generally considered to be avascular. Its nourishment depends on the synovial fluid and subchondral bone. The thickness of AC varies from some micrometers to a few millimeters in different cartilage areas within the joint, in different joints, and animal species. Biomechanically, the primary function of the AC is to provide a covering material that protects the subchondral bone and provides a smooth, lubricated surface that facilitates movements with little friction between the articulating surfaces. Cartilage has the ability to reduce the nominal contact pressures and to increase joint congruence. Traditionally, AC is divided into three pseudo-stratified zones that are separated by a tidemark from the deepest calcified zone (Fig. 1.2). This classification is based on collagen fibril orientation and the organization and morphology of chondrocytes at different depths throughout AC. The zones can be characterized as follows: · Zone 1: Superficial (tangential) zone; adjacent to the joint cavity occupying 5±10% of the matrix volume. It is characterized by relatively low proteoglycan (PG) content. The collagen fibrils are closely packed and the orientation of fibrils is predominantly tangential (parallel) to the surface. The cells are discoidal with their long axes parallel to the surface. · Zone II: Middle (transitional or intermediate) zone; occupies up to 45% of the matrix volume. It is characterized by a significant increase in the PG content. The collagen fibrils are randomly oriented. The cells are spheroidal and equally spaced. · Zone III: Deep (radial) zone; occupies up to 45% of the matrix volume and has the highest PG content. The distance between collagen fibrils increases and they are arranged perpendicularly (radially) to the articular surface. Spheroidal cells often form columns aligned with the radial collagen fibrils. · Zone IV: Calcified zone; adjacent to the subchondral bone occupies 5±10% of the matrix volume. There are a few chondrocytes, the matrix is mineralized with crystals of calcium salts and the PG content is low. The collagen fibrils are radially aligned. A borderline, called the `tidemark', separates the calcified zone from the deep zone.

The structure and regenerative capacity of synovial joint tissues

5

1.2 A schematic presentation of the structure of articular cartilage illustrating the different zones and regions of articular cartilage and subchondral bone. Collagen fibrils are oriented tangentially in the superficial zone and radially in the middle and deep zones of the articular cartilage. Chondrocytes in the superficial zone are discoidal and in the deep zone spheroidal and often form columns. Tidemark at the mineralization front separates deep zone and calcified zone. Subchondral bone plate and trabecular bone provide support to the articular cartilage and is vascularized.

This zonal pattem is present in adult AC of most species, although the relative proportion of each zone varies. The extracellular matrix surrounding each chondrocyte can be subdivided into three discrete zones, which are the pericellular, territorial, and interterritorial matrices. Chondrocytes Cell volume, 2±10% of the total tissue volume, in AC is low in comparison with other tissues. Chondrocytes are the only cell type in AC. As being entrapped within this highly organized matrix and isolated in the lacunae that is called as chondron, mature chondrocytes have essentially no migratory potential, although some in vitro motility has been shown (Poole 1997; Morales 2007). Chondrocytes are ultimately responsible for the integrity, organization, and maintenance of the extracellular matrix (ECM) (Bhosale and Richardson 2008).

6

Regenerative medicine and biomaterials in connective tissue repair

Three subpopulations of chondrocytes with different morphologies have been identified in AC (Kouri et al. 1996). Type 1 cells found in superficial and upper middle zones of AC represent typical chondrocytes. Type 2 cells represent secretory, fibroblast-like cells that are more abundant in the non-fibrillated osteoarthritic samples than in healthy cartilage. Type 3 cells are degenerating chondrocytes found throughout the OA cartilage although more frequently in the fibrillated tissue. Extracellular matrix Articular cartilage consists of two principal phases: a solid organic ECM, which is predominantly composed of collagen fibrils and PGs, and a mobile interstitial fluid phase. Water and inorganic salts make up about 70±80% of the tissue wet weight. The distribution of the chondrocytes and ECM constituents varies throughout the thickness of the cartilage. The structure of cartilage ECM is reviewed in more detail in Chapter 3. Type II collagen is the most abundant protein in the ECM of AC, where it forms a three-dimensional fibrous network of alpha1 (II) collagen homotrimers assembled into copolymeric bundles with type IX and XI collagens (Eyre 2002). Also minor amounts of type III, VI, XII and XIV collagens are found in cartilage. Type X collagen is normally restricted in the hypertrophic zone in AC cartilage and growth plate. The three-dimensional network of collagens is filled with PGs that are macromolecules consisting of a central core protein to which one or more glycosaminoglycan (GAG) chains and oligosaccharides are attached (Knudson and Knudson 2001; Roughley 2006). On mass basis, aggrecan is the most abundant PG in cartilage (Fig. 1.3). The core protein of the aggrecan can be divided into N-terminal HA binding region with a G1 globular domain, small interglobular domain, another N-terminal globular domain G2, keratan sulfate attachment domain KS, two chondroitin sulfate attachment domains CS1 and CS2, and a lectin binding C-terminal globular domain G3 (Fig. 1.3). G1 domain interacts with link protein to stabilize the binding of aggrecan molecules into large aggregates with HA. The GAGs are unbranched carbohydrates made up of repeating disaccharide units with negatively charged sulfate and carboxyl groups responsible for the water binding properties of the aggrecan molecule that regulate the elastic properties of the cartilage (Cowin and Doty 2007a). Chondrocytes synthesize also numerous other non-collagenous ECM components (Roughley 2001, 2006). Small leucine-rich repeat proteoglycan (SLRP) family members decorin, biglycan, fibromodulin, lumican, and others have functions in regulating the collagen fibrillogenesis, activity and distribution of different cytokines and growth factors, as well as in cell signaling (Schaefer and Iozzo 2008). Several other PGs and glycoproteins are found in the extracellular space, e.g., cartilage oligomeric protein (COMP), versican and tenascin, or on

The structure and regenerative capacity of synovial joint tissues

7

1.3 Schematic presentation of the major extracellular matrix components of articular cartilage. (a) Aggrecan molecules attach to hyaluronan with link protein to form large (> 1  106 Da) proteoglycan aggrecates which are entrapped within the network of type II collagen fibrils. (b) Characteristic structural domains of aggrecan. Hyaluronan (HA); two interglobular domains (IGD) G1 and G2; keratan sulfate binding region (KS), chondroitin sulfate binding region 1 (CS1), chondroitin sulfate binding region 2 (CS2), and a Cterminal globular domain (G3) (adapted from Qu 2007).

the chondrocyte surface, e.g., perlecan, syndecans, integrins and growth factor receptors (van der Kraan et al. 2002; Chiquet-Ehrismann and Tucker 2004; Melrose et al. 2008; Shakibaei et al. 2008). These factors and their relationship in tissue function, homeostasis and degeneration have been reviewed by, for example, Gentili and Cancedda (2009), Goldring et al. (2008) and Goldring and Marcu (2009), and are more thoroughly reviewed in the other chapters of this book. The poor intrinsic repair capacity of adult AC results from avascular nature of the tissue. Lacking the blood vessels, the damage in AC produces no inflammatory reaction, which would lead to chemotactic recruitment of the repair cells, typical in the repair process of other tissues. Also, synthesis and turnover of type II collagen in adult AC are exceptionally low, the half-life of the collagen molecules being over one hundred years (Maroudas et al. 1992; Verzijl et al. 2000). Hence, any marked injuries in the collagen network are likely to initiate a cascade leading to progressive degradation of AC (Aigner and StoÈve 2003).

1.3.2

Subchondral bone

Subchondral bone is formed via endochondral ossification of the cartilage template at the secondary ossification centers of the bone epiphyses during joint development (Burr 2004; Mackie et al. 2008). Subchondral bone provides support to AC, and biomechanically it has an essential role on cartilage homeostasis. Subchondral bone consists of the subchondral bone plate (SBP) to which the subchondral trabecular bone (STB) is attached (Fig. 1.2) (Imhof et al. 2000; Burr

8

Regenerative medicine and biomaterials in connective tissue repair

2004). Both SBP and STB are formed of bone lamellae. Subchondral bone is composed of two types of lamellae: concentric lamellae around the osteons and flat lamellae. SBP, like compact bone, is composed of subunits called osteons consisting of concentric lamellae surrounding the central (Haversian) canal. STB and the inner surface of SBP are covered by osteoblasts and osteoclasts. Physiologically and mechanistically distinct STB is highly vascularized and it provides another route for cartilage nutrition in addition to synovial fluid (Imhof et al. 2000). The subchondral bone structure, i.e., SBP thickness and STB density vary with region in the joint (Oettmeier et al. 1992). Based on Wolff's law, both the bone density and the organization of bone trabeculae correlate with the magnitude and direction of compressive and tensile stresses of loading (Wolff 1892). There are five different bone cell types ± osteocytes, osteoprogenitors, bone lining cells, osteoblasts, and osteoclasts. Osteoblasts and osteoclasts form bone remodeling units that maintain the integrity of the bone and balance between deposited and resorbed bone (Cowin and Doty 2007b; Bartl and Frisch 2009). Bone matrix is composed of organic and inorganic components. Up to 88% of organic matrix is collagen, mainly type I collagen, which forms an organized template for the matrix mineralization by deposition of hydroxyapatite and apatite. In addition, organic matrix contains up to 12% of the dry weight of osteocalcin, osteonectin, several phosphoproteins, lipids and proteoglycans. Bone marrow of the trabecular bone maintains a heterogeneous population of various multi-potent mesenchymal stromal cells that provide progenitors for differentiation of osteochondral and other mesenchymal cell lineages as well as trophic environment for hematopoiesis.

1.3.3

Meniscus

Menisci are semi-lunar discs between tibia and femur in the knee joint protecting AC from excess shocks by distributing loads and stabilizing joints during movement (Fig. 1.4(a)) (Setton et al. 1999a; Sweigart and Athanasiou 2001). Meniscus tissue is composed of outer, dense connective tissue and inner fibrocartilage regions (Fig. 1.4(b)) (Verdonk et al. 2005). The outer region is vascularized dense fibrous connective tissue that connects to the internal knee joint capsule. Its matrix is maintained by fibroblast-like fibrochondrocytes which produce type I collagen fibrils. Elastin fibers as bridge-like connections between collagen fibrils have been suggested as contributing to the recovery after deformation (Ghosh and Taylor 1987). The inner region of the meniscus is avascular, aneural, and alymphatic tissue, which is why, similar to cartilage, its repair capacity is lower than that of the outer region. Cells in the inner region are chondrocyte-like fibrochondrocytes, and its matrix contains many similar components common to cartilage and tendon. Type II collagen forms about 60% and type I collagen about 40% of the total collagen in the inner region. Minor

The structure and regenerative capacity of synovial joint tissues

9

1.4 Schematic presentation of the menisci in the knee joint. (a) Macroscopic view of the proximal tibia with menisci, and insertion sites of ligaments. (b) Vascularized outer region and avascular inner region of the meniscus. (c) Schematic organization of the collagen fibers in different layers of the meniscus. Collagen fibers at the superficial layer on both tibial and femoral sides are thin and intersect at various angles. Below that on both sides are lamellar layers where collagen fibers are arranged into lamella-like bundles that are radially arranged in the external circumference of the anterior and posterior segments of bundles, and intersect at various angles in the other regions. In the central main layer the bundles of collagen fibers are oriented in a circular manner, with a few interwoven radial tie fibers in the internal circumference. At the external circumference, also loose connective tissue from the capsule penetrates the central main layer (arrow) ((b) adapted from Verdonk et al. 2005 and (c) adapted from Petersen and Tillmann 1998).

amounts of type III, V and VI collagens have been found in both regions (Sweigart and Athanasiou 2001). Collagen fibers are radially and circumferentially organized to provide appropriate structure to resist tensile forces. Based on the collagen fiber orientation and thickness, three different layers can be recognized in the inner fibrocartilage region (Fig. 1.4(c)) (Petersen and Tillmann 1998; Sweigart and Athanasiou 2001). Aggrecan is the major PG responsible for the maintenance of viscoelastic properties of the tissue, and its concentration is highest in the inner and middle parts of the meniscus, decreasing towards the periphery. It forms a spatially organized network in contrast to cartilage, where it is more diffusely distributed (Valiyaveettil et al. 2005). Perlecan and SLRPs decorin, biglycan, fibromodulin, and keratocan, and small amounts of adhesion molecules such as fibronectin and thrombospondin are also found in the meniscus (Melrose et al. 2005, 2008). PGs residing particularly at the surface zone are thought to contribute to the smooth

10

Regenerative medicine and biomaterials in connective tissue repair

frictionless movement of the menisci over the articular surfaces (Melrose et al. 2005, 2008).

1.3.4

Tendon and ligaments

Tendons are specialized dense connective tissue structures connecting bones and muscles, while transmitting forces and allowing joint movements (Kannus 2000). Tenoblasts in the developing and young tendon, and tenocytes, elongated and dispersed fibroblast-like cells in the adult tendon, form about 90±95% of the cells in tendon. The remaining cells are chondrocyte-like cells at regions of pressure and insertion sites, entheses, and synoviocytes on tendon surface, and vascular cells in the endo- and epitenon regions (Benjamin et al. 2006; Cowin and Doty 2007c; Kannus 2000; Riley 2008). The tendon matrix is maintained by tendon cells that are embedded within the long collagen fibrils running parallel to each other and arranged into bundles in a staggered fashion (Fig. 1.5). Type I collagen is the major collagen component of the ECM, but it contains also `minor' collagens and elastin, glycoproteins and adhesive group molecules, e.g., fibronectin, thrombospondin, tenascin C, and undulin (Kannus 2000). Aggrecan and versican are the large PGs providing, together with HA the properties to resist compressive and tensile forces during movements. SLRPs such as fibromodulin, biglycan, and decorin are found to regulate collagen fibrillogenesis, bone morphogenic protein (BMP) activity or stem cell niche organization (Yoon and Halper 2005; Zhang et al. 2005; Bi et al. 2007). Ligaments are dense connective tissue structures connecting articulating bones and giving stability to the joints (Duthon et al. 2006; Cowin and Doty 2007c; Petersen and Zantop 2007). Although they are anatomically distinct from

1.5 Collagen fibril organization in (a) tendon and (b) ligament (adapted from Nordin and Frankel 1989).

The structure and regenerative capacity of synovial joint tissues

11

tendon, they have an overlapping gene expression profile and matrix composition (Rumian et al. 2007). Spindle shaped fibroblasts maintain the ECM that is mainly composed of type I collagen fibers but also contains some other collagens, e.g., type II collagen in the endotendon, type III collagen in the reticular fibers, type IV collagen in the vascular basement membranes, and type VI collagen as a gliding component between functional fibrillar units (Duthon et al. 2006). Parallel organization of collagen fibrils into bundles together with PGs, glyco-conjugates and elastic components results in the formation of a unique, complex elastic network capable of withholding varying multiaxial stresses and tensile strains (Cowin and Doty 2007c). The degree of collagen fibril organization is lower than in tendon (Fig. 1.5) which also has consequences in the biomechanical properties (see below).

1.3.5

Synovial membrane and synovial fluid

Synovial membrane or synovium secretes joint lubricating components into the synovial fluid, nourishes the joints, and removes debris from the synovial space. It is composed of two layers, the intimal layer and the loose connective tissue layer (FitzGerald and Bresnihan 1995; Iwanaga et al. 2000; Sutton et al. 2009). Three basic cell types, type A synoviocytes, type B synoviocytes, and dendritic cells, are found in the intimal layer. Type A synoviocytes, or macrophage-like synoviocytes, are likely to originate from bone marrow and can be considered as resident or tissue macrophages that are mainly phagocytic with large Golgi complex and lysosomes (Iwanaga et al. 2000). Type B synoviocytes, or fibroblast-like synoviocytes, manufacture collagen, fibronectin, HA, and PG 4 (also known as lubricin, megacaryocyte stimulating factor (MSF) and superficial zone protein (SZP)) into synovial fluid to maintain joint lubrication (Rhee et al. 2005; Elsaid et al. 2007; Wann et al. 2009). They differ from other deeper, subintimal fibroblasts in that they contain characteristic lamellar bodies, and produce also surfactant protein A and VCAM-1 (FitzGerald and Bresnihan 1995; Vandenabeele et al. 2003). Dendritic cells form less than 1% of the synovial cells (FitzGerald and Bresnihan 1995). They are potent antigen presenting cells that have a pro-inflammatory role in initiation of the immune responses in rheumatoid arthritis (RA), where they are the effectors of cartilage destruction and a major source for inflammatory cytokine TNF which indirectly induces cartilage collagenolysis (Lutzky et al. 2007). Below the intimal layer there is a loose connective tissue layer that contains fibroblasts, macrophages, adipocytes, mast cells, nerve fibres, vascular endothelial cells, granulocytes, and lymphocytes (FitzGerald and Bresnihan 1995). It is well vascularised, innervated and supplied by lymphatic vessels (Sutton et al. 2009). Synovial fluid is the joint lubricant and shock absorber for AC. Synovial fluid is a blood plasma dialysate, which contains HA and glycoproteins, synthesized

12

Regenerative medicine and biomaterials in connective tissue repair

by type B synovial lining cells (Fam et al. 2007). HA contributes to the high viscosity and lubricating properties of synovial fluid and is currently used also as a therapy for OA. Recently the synthesis and active secretion of HA were coupled to the movements and use of the joint (Ingram et al. 2008). In addition to substances secreted by the lining cells, synovial fluid contains plasma proteins that originate from the blood vessels vascularizing synovium (Fam et al. 2007). Cellular components are present in small amounts in normal synovial fluid, including different leukocytes: lymphocytes, monocytes, synovial lining cells, and polymorphonuclear cells (Fam et al. 2007). The rate and mechanism of passage of substances going through synovium depend on the size of the molecules. Gases and crystalloids diffuse rapidly in both directions. Larger proteins are taken out of the synovial fluid by way of lymphatics. Macrophages phagocytose cellular debris and particles that are too large to be removed otherwise (Iwanaga et al. 2000). The turnover time for synovial fluid volume is estimated to be about one hour in rabbit and normal human knees, while that for HA is much longer, 17±30 hours in rabbit knee joints (Levick 1987; Ingram et al. 2008). It is now commonly believed that synovial macrophages are responsible for producing the proinflammatory cytokines into the joint space and drive the inflammatory responses with stimulation of cartilage cytokines and matrix degrading proteases under pathological conditions such as OA or RA (Blom et al. 2007; Sutton et al. 2009).

1.3.6

Biomechanical properties of joint tissues

The mechanical properties of tissues can be determined from the load± deformation behavior in compression, tension, bending, or shear geometry. The 3- or 4-point bending tests have actively been used for bone samples and tension tests for tendons and ligaments. For mechanical testing of AC, compression, tension, and shear techniques have traditionally been applied. In compression, unconfined compression, confined compression, and indentation geometries (Fig. 1.6) and stress±relaxation or creep (Fig. 1.7) test protocols are generally in use. To calculate the true material properties of joint tissues, theoretical analysis of the measurements is needed. In most classical models, soft tissues are simplified as homogeneous isotropic linearly elastic materials. The relationship between the stress and strain is described as linear and two independent elastic constants are needed to describe the material, i.e., the elastic (Young's) modulus (E) and Poisson's ratio () (Table 1.1). Consequently, this model is inadequate for characterizing time-dependent mechanical behavior of soft tissues, especially that of AC. However, it has been used actively to calculate the instantaneous (dynamic) or equilibrium (static) modulus for AC (Hayes et al. 1972; Jurvelin et al. 1990).

The structure and regenerative capacity of synovial joint tissues

13

1.6 Schematic presentation of the typical measurement configurations in use for mechanical testing of the articular cartilage. (a) Unconfined compression: the tissue is compressed between two smooth metallic plates allowing fluid flow in the lateral direction. (b) Confined compression: the tissue is placed in a metallic chamber and compressed with a porous filter allowing fluid flow axially through the filter. (c) Indentation: the tissue is compressed with a cylindrical plane-ended or spherical-ended indenter allowing fluid flow in both lateral and axial directions (from Saarakkala 2007).

The joint tissues are all viscoelastic in their mechanical behavior, i.e., the mechanical response, depends significantly on the rate of loading. Depending on the tissue type, the viscoelasticity may originate from the intrinsic property of the solid tissue, or from the interstitial fluid flow within the tissue under load. Under loads with volumetric dilatation, these two viscoelastic mechanisms are difficult to separate. The latter, called poroelasticity, is especially well recognized in AC. A traditional model for AC, taking the interstitial fluid movement into account, is the linear isotropic biphasic model (Mow et al. 1980). In addition to elastic parameters of the solid matrix, knowledge of the tissue permeability is needed for characterizing the time-dependent behavior of the tissue. To extract the model parameters, experimental mechanical measurements are conducted and, subsequently, the theoretical model is fitted to the experimental data. As an extension of the biphasic model, fibril reinforced models have been introduced (Soulhat et al. 1999; Korhonen et al. 2003; Wilson et al. 2004). In these models, the compression±tension nonlinearity is taken into account by inclusion of the collagen fibril network. The material parameters of the fibrilreinforced model are Young's modulus and Poisson's ratio of the drained porous

1.7 (a) In a creep measurement, cartilage tissue deformation (strain) is recorded under a constant load (stress) applied at t0. (b) In a stress-relaxation measurement, the cartilage tissue load (stress) is recorded under a constant deformation (strain) applied at t0 (from Saarakkala 2007).

14

Regenerative medicine and biomaterials in connective tissue repair

Table 1.1 Basic equations for the determination of isotropic elastic parameters of cartilage Parameter

Equation

Stress ()

ˆ

dF dA

Strain ()

ˆ

L0 ÿ L L

Young's modulus (E) (unconfined compression)

Eˆ

a a

Poisson's ratio ()

ˆ

l a

Shear modulus ()

ˆ

E 2…1 ‡ †

Aggregate modulus (HA) (confined compression)

HA ˆ

Young's modulus (E) (indentation)

Eˆ

…1 ÿ  2 †a  2h 

Shear modulus () (indentation)

ˆ

…1 ÿ †a  4h 

F A L L0 a and a l a h …a=h; †

1ÿ E …1 ‡ †…1 ÿ 2†

reaction force area of the surface in which the force is acting initial thickness thickness after compression/tension axial stress and strain lateral strain indenter radius cartilage thickness theoretical scaling factor due to finite and variable cartilage thickness (Hayes et al.1972; Jurvelin et al.1990)

matrix, permeability, and Young's modulus of the fibril network. Triphasic theory is an extension of the biphasic model incorporating three phases: an incompressible solid, an incompressible fluid, and a monovalent ionic phase (Lai et al. 1991). The model assumes that the total stress of the tissue is composed of the fluid stress, solid stress and chemical potentials. This model can be used to accurately include the effect of cartilage swelling. Owing to tissue heterogeneity and anisotropy, as well as to mimic realistic loading geometries, the model implementations for nonlinear behavior of AC are most often conducted numerically using finite-element analysis. The structure, composition, and properties of all joint components have evolved on the grounds of their biological and mechanical function. In a healthy

The structure and regenerative capacity of synovial joint tissues

15

joint, uncalcified cartilage and meniscus (elastic modulus in compression of 0.1± 1 MPa), calcified cartilage (elastic modulus ~0.3 GPa), and subchondral bone (elastic modulus >1 GPa) establish a structural and functional continuum with optimal mechanical properties (Mente and Lewis 1994; Setton et al. 1999a; Arokoski et al. 2000; Helminen et al. 2000). The tibio-femoral contact stresses may be very high (>10 MPa), compared with typical Young's modulus of 1 MPa for solid matrix of normal cartilage, indicating that hydrostatic pressure within cartilage must serve as a primary mechanism for successful load bearing. Further, intrinsic fluid pressurization contributes significantly to low friction between articulating surfaces (Ateshian 2009). In OA, critical loss of fluid pressurization mechanism of load support takes place. Tendons, with densely packed collagen fibers, show typically very high tensile modulus (>1 GPa) and strength (>100 MPa). Tendons are highly elastic with minor viscoelastic effects and their nonlinear tensile behavior is related to gradual alignment and stretching of the fibers (Ker 2007). Ligaments, owing to lower collagen content and highly woven collagen structure, are less stiff and strong than tendons. Articular cartilage exhibits significant compression±tension nonlinearity. Compressive equilibrium modulus of healthy cartilage in unconfined compression is ~1 MPa; however, under highly dynamic loads hydraulic stiffening produces a modulus that is much higher, and comparable to that of tensile modulus (5±25 MPa, Setton et al. 1999b). Owing to inhomogeneous structure, e.g., depth-dependent increase of PG content, the compressive modulus and permeability increase and decrease, respectively, along cartilage depth (Schinagl et al. 1997; Boschetti et al. 2004). The tensile modulus is highest in the superficial cartilage zone, where the direction of the collagen fibers is parallel to articular surface, and the collagen content is highest. Deeper in the tissue, the more random orientation produces lower tensile stiffness (Kempson et al. 1973). In joint tissues, each structural component and their interactions contribute to overall mechanical characteristics of the tissue. In cartilage, PGs, owing to the swelling stress they produce, and their effect to tissue permeability, are considered important for mechanical characteristics in compression, while collagen is the primary structure resisting tension (Huang et al. 2001). However, joint tissues can be considered to be biological composites, and the structural interactions critically control the mechanical behavior as well. Therefore, these sophisticated structures have remained difficult to replicate using tissue engineering methods, making tissue repair of, for example, AC, challenging. It is well shown that proper collagen cross-linking is essential for a functional matrix in both native and engineered cartilage (Broom 1984; BastiaansenJenniskens et al. 2008). Further, mechanical properties of AC are sensitively modulated by the changes in structural integrity of the tissue (Fig. 1.8).

16

Regenerative medicine and biomaterials in connective tissue repair

1.8 Minor degenerative changes in matrix, based on the scoring the histological integrity of the cartilage using Mankin score, can lead to inferior mechanical properties (e.g. dynamic modulus) of AC (from Laasanen et al. 2003).

1.4

Resident mesenchymal progenitor cells in synovial joint tissues

Remarkable progress during the last ten years of stem cell research has increased our understanding of how stem cells can be induced and manipulated to form repair tissue. Adult stem cells, especially bone marrow MSCs, which are a highly variable population of multi-potent mesenchymal stem cells and progenitors, have been actively characterized due to their great potential in regenerative medicine (reviewed by, for example, Barry and Murphy 2004; Keating 2006; Caplan 2007; Phinney and Prockop 2007; Abdallah and Kassem 2008, 2009; Chen and Tuan 2008; Jones and McGonagle 2008; NoÈth et al. 2008; Arthur et al. 2009). Currently, MSCs and progenitors have been found residing virtually in all organs and tissues (Sakaguchi et al. 2005; da Silva Meirelles et al. 2006; Chamberlain et al. 2007; El Tamer and Reis 2009). While the aim of this whole book is to gather together current understanding of the normal biology, disease pathogeneses, and different therapeutic approaches of connective tissue disorders, especially those related to joint and associated tissues, it is important also to be aware of the endogenous stem cells and progenitors residing in these tissues. Therefore, inventory of resident stem cells and progenitors in human synovial and associated tissue joints (Table 1.2), and some of their properties will be briefly discussed in this chapter.

1.4.1

Mesenchymal stromal cells

Stem cells have a potential to self-renew, proliferate, and differentiate into multiple cell types. Adult multi-potent stem cells have a more limited differ-

The structure and regenerative capacity of synovial joint tissues

17

entiation potential in comparison to embryonic stem cells. Early embryonic stem cells (derived from the inner layer of blastocyst) are totipotent and can give rise to all germ layers. Pluripotent embryonic stem cells lack differentiation potential to placental cells, but can differentiate to form other tissues. Two major classes of multi-potent stem cells are found in adult bone marrow, hematopoietic stem cells, and nonhematopoietic stromal cells. MSCs are multi-potent nonhematopoietic cells that can differentiate into mesodermal lineages (Fig. 1.9(a)). They represent a small percentage of the total population of nucleated cells in the bone marrow, where the majority of the cells consist of hematopoietic stem cells and hematopoiesis supporting stromal cells (Pittenger et al. 1999). MSCs are defined as highly clonogenic cells having potential for self-renewal and differentiation into multiple mesenchymal tissues (Pittenger et al. 1999). Johnstone was the first to induce chondrogenic differentiation of mesenchymal progenitor cells isolated from rabbit bone marrow (Johnstone et al. 1998). In contrast to the hematopoietic stem cells, no single mesenchymal stem cell specific marker has been found so far. They appear to be a rather heterogeneous population and most of the cells seem to be progenitors rather than true stem cells. To clarify the confusion in the nomenclature and to attempt to standardize the research in this field, the International Society for Cellular Therapy has made two statements (Fig. 1.9(b)). First, they recommend the term `multi-potent mesenchymal stromal cell' instead of the mesenchymal stem cell, the acronym still remaining the same `MSC' for both (Horwitz et al. 2005). Second, multipotent MSCs should fulfill the minimal criteria of expressing certain surface antigens characteristic for mesenchymal cells. In addition, MSCs should not express some hematopoietic and epithelial cell surface antigens, and they should also have a capacity to differentiate under appropriate conditions to chondrogenic, osteogenic, and adipogenic lineages (Dominici et al. 2006). Several controversial issues prevailing in adult stem cell research including nomenclature, and other MSC characteristics, were critically discussed in a recent review by Darwin Prockop (2009).

1.4.2

Role of mesenchymal progenitor cells in joint homeostasis

Being resident in subchondral bone, bone marrow stromal cells may give rise to a spontaneous AC repair that is seen when cartilage lesions extend to the underlying bone, resulting in a formation of cartilage repair tissue. Already in the 1940s, before the era of the modern arthroplasty surgery, abrasion of the osteoarthritic joint surfaces and drilling several holes 6 mm in diameter to the subchondral bone were used as a therapy for OA (Magnuson 1941; Pridie 1959). However, functionally impaired fibrocartilagenous repair tissue did not give satisfactory results. Currently microfracture is a frequently used technique for the repair of AC lesions of the knee. In this `marrow stimulating' technique an

1.9 Definition of adult mesenchymal stem/stromal cell (MSC). (a) Mesenchymal tissues contain MSCs and progenitor cells that under defined conditions have a capacity to differentiate into multiple connective tissue cell types. (b) The minimal criteria for defining the term `multipotent mesenchymal stromal cell' as suggested by the International Society for Cellular Therapy: (1) they must be plastic adherent, (2) express certain cell surface antigens, and (3) have a capacity to differentiate to at least chondrocytic, osteogenic and adipogenic lineages (Horwitz et al. 2005; Dominici et al. 2006) (adapted from SÌÌmÌnen et al. 2008).

The structure and regenerative capacity of synovial joint tissues

19

awl is used to penetrate the subchondral bone to produce small holes in cartilage defects, allowing marrow cells to migrate to the cartilage lesion site (Steadman et al. 2002). Recent studies have shown that microfracture provides effective short-term functional improvement of knee function, but often results in formation of suboptimal fibrocartilage (Knutsen et al. 2007; Mithoefer et al. 2009). The principal role of adult MSCs and progenitors has been considered to maintain physiological balance in the organism by serving a cellular reserve for tissue remodeling and rejuvenation, but they can do more than just respond to stimuli and differentiate. Newly committed progenitor cells have been shown to secrete several growth factors and cytokines (Haynesworth et al. 1996), and immunosuppressive factors, e.g., HLA-G that interfere with the immune recognition system (Selmani et al. 2009; Siegel et al. 2009). Caplan and Dennis (2006) introduced a term `trophic mediator' to MSCs, and defined the trophic effects as `those chemotactic, mitotic, and differentiation-modulating effects, which emanate from cells as bioactive factors that exert their effects primarily on neighboring cells and whose effects never result in differentiation of the producer cell'. Stem cells are maintained in so-called stem cell niches at specific sites in the tissues (Gregory et al. 2005; Jones and Wagers 2008; Walker et al. 2009). Stem cell niches have been characterized in tendon, AC, and zone of Ranvier, where PGs or their GAG sulfation patterns have been suggested as having important roles in maintaining and organizing the niches, and regulating the local BMP activity (Bi et al. 2007; Hayes et al. 2008; Karlsson et al. 2009). Hence, there seems to be a complex and bidirectional regulation system of the stem cell response to stimuli for differentiation and secretion of bioactive factors, thereby influencing tissue homeostasis (Fig. 1.10) (Caplan and Dennis 2006; Caplan 2009).

1.4.3

Articular cartilage

Articular cartilage was earlier thought to lack stem cells or progenitors but several recent studies have demonstrated their existence in the tissue (Table 1.2). A few years ago it was first shown that young bovine AC contains a multi-potent progenitor cell population in the superficial zone with differentiation plasticity into various connective tissues, including bone, tendon, and perimysium (Dowthwaite et al. 2004). Several studies have shown the presence of multipotent progenitors with limited expandability in AC of healthy young individuals (reviewed by Tallheden et al. 2006). In OA cartilage, increased number of cells with MSC phenotype has been found (Alsalameh et al. 2004; Fickert et al. 2004; Hiraoka et al. 2006). Adult human AC contained a small population of cells that coexpressed surface antigens CD105 and CD166 (ALCAM, activated leukocyte adhesion molecule) (Alsalameh et al. 2004). These markers have been proposed to define a population of MSCs in bone

Table 1.2 Studies demonstrating presence of multipotent progenitor cells in human synovial joint and associated tissues Reference

Multipotency1

Articular cartilage Barbero et al. (2003)

ACO

Fickert et al. (2004) Alsalameh et al. (2004)

ACO ACO

Koelling et al. (2009)

ACO

Thornemo et al. (2005)

ACO

Synovial membrane and synovial fluid De Bari et al. (2001) ACO Vandenabeele et al. (2003) ± Sakaguchi et al. (2005)

ACO

Mochizuki et al. (2006)

ACO

Jones et al. (2008)

ACO

Morito et al. (2008)

ACO

Notes2 Plasticity of dedifferentiated clonal chondrocytes was tested. TGF -1, FGF-2 and plateletderived growth factor BB enhanced C and O but reduced A differentiation capacity. (AC) Osteoarthritic cartilage contained 5% of CD9+/CD90+/CD166+ multi-potent MSCs. (AC) 3.49% of CD105+/CD166+ MSCs in normal articular cartilage and frequency is increased in osteoarthritic cartilage. (AC) Migratory chondrogenic progenitors (type 2 cells with fibroblast like morphology) were found in osteoarthritic cartilage. (AC) Osteoarthritic cartilage contained multi-potent progenitors (3.6% of all cells). (AC) Single cell derived multi-potent MSC clones were isolated from knee joint SM. (AD) Morphologically SF MSCs resembled type B synoviocytes why they likely originate from synovial lining. (AD) MSCs derived from several tissues were compared; synovial MSCs were superior in chondrogenesis. (SM AD PE BM MU) Osteogenic and chondrogenic potential was highest in the synovium-derived populations (SM AS AD) SF MSC prevalence increased 7-fold in early OA. MSCs likely originated from synovium. Chondrogenic potential was more consistent in SF than BM MSCs. (SF BM) SF MSC prevalence increased 100-fold after ACL injury and they aligned to the rupture site. (SF SM BM)

Ligaments and tendon Huang et al. (2008)

ACO

Cheng et al. (2009)

ACO

de Mos et al. (2007) Bi et al. (2007)

ACO ACO

Intra-articular fat pads English et al. (2007)

ACO

Khan et al. (2008)

C

Wickham et al. (2003)

ACO

Other De Bari et al. (2006) Williams et al. (1999) Zheng et al. (2007) Segawa et al. (2009)

ACMO ACMO CMO ±

1

ACL and total knee replacement surgery samples. Variation in tripotency and differentiation and proliferation rate between patients. (ACL) MSCs were isolated from cruciate ligaments and BM. Phenotype was similar in all MSC populations. (ACL PCL BM) Hamstring tendon contained cells with intrinsic differentiation potential. (TE) Tendon MSCs are maintained in a fibromodulin and biglycan modulated niche. They differentiated to form entheses-like tissue. (TE) Hoffa's fat pad contained MSCs that maintained chondrogenic phenotype long time. (AC AD BM) Chondrogenic cells were isolated from infrapatellar fat pad, and expansion in FGF-2 enhanced chondrogenic potential. (AD) Infrapatellar fat pad contained multi-potent MSCs. (AD) Periosteum contained multi-potent MSCs. (PE) Skeletal muscle contained multi-potent mesenchymal progenitor cells. (MU) Myoendothelial cells isolated from skeletal muscle were multi-potent. (MU) Gene expression profiles of intra-articular tissue colony forming MSCs (SM, ME, LI) were closer to each other and articular chondrocytes than to extra-articular tissue MSCs (BM, AD, MU). PRELP was a characteristic highly expressed gene among intra-articular tissue MSCs. (SM ME LI MU AD BM)

Tested differentiation capacity to adipogenic (A), chondrogenic (C), osteogenic (O), or myogenic (M) lineages. Cell source(s) used for MSC isolation are in parentheses: articular cartilage (AC), synovial membrane (SM), synovial fluid (SF), adipose synovium (AS), adipose tissue (AD), anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), tendon (TE), meniscus (ME), skeletal muscle (MU), periosteum (PE).

2

22

Regenerative medicine and biomaterials in connective tissue repair

1.10 Mesenchymal stem cells are maintained in stem cell niches and function as trophic mediators and reservoir for tissue remodeling and repair. Proteoglycans, particularly glycosaminoglycan sulfation patterns of aggrecan and perlecan in cartilage (Hayes et al. 2008), and small leucine rich repeat proteoglycans (SLRPs) fibromodulin (Fmn) and biglycan (Bgn) in tendon (Bi et al. 2007), have been suggested to have important functions in regulating the organization and/or growth factor presentation in the niches (principle of the graph adapted from Caplan and Dennis 2006).

marrow stroma and have properties similar to mesenchymal progenitor cells (Majumdar et al. 1998). As the presence of CD105+/CD166+ progenitor cells was significantly increased in OA cartilage, they were speculated to have a role in pathogenesis of OA (Alsalameh et al. 2004). As CD105 is endoglin, a TGF receptor III, its expression in MSC population is likely to enhance the chondrogenic potential due to responsiveness to exogenous TGF , which is used for induction of chondrogenesis. Fate and differentiation regulating factor Notch1 has been regarded as a progenitor marker. Surprisingly high numbers of Notch1 positive cells have been found both in healthy and OA cartilage. Over 70% of the cells in primary culture of cells isolated from AC with induction in OA were Notch1 positive (Hiraoka et al. 2006). In normal human AC, taking all cartilage zones together, over 45% of the cells expressed progenitor markers Notch1, a stem cell marker Stro-1 and vascular endothelial molecule VCAM-1, with the highest expression in the superficial zone (Grogan et al. 2009). Most of the cells in chondrocyte clusters in OA cartilage also expressed all these progenitor markers, suggesting responses to OA pathogenesis. In another study with adult bovine knee AC, Notch1 expression did not correlate with multi-potent properties of the progenitors, thus questioning the value of Notch1 as an early progenitor marker, and suggesting that the actual progenitor cell population is much smaller in adult AC (Karlsson et al. 2008). Supporting this, normal and OA cartilage contained 0.14% so-called side-population (SP) cells identified by their negative staining for Hoechst 33342 dye, that differentiated into chondrocytes and osteocytes, but

The structure and regenerative capacity of synovial joint tissues

23

not adipocytes, thus likely representing a more primitive osteoprogenitor population than Notch1, Stro-1, and VCAM-1 expressing cells (Grogan et al. 2009). Recently, migratory chondrogenic progenitor cells from fibrocartilagenous repair tissue were identified during the later stages of human OA (Khan et al. 2009; Koelling et al. 2009). These cells resemble type 2 cells with a secretory phenotype (see `Chondrocytes' in Section 1.3.1), originally described by Kouri et al. (1996) in OA cartilage. Clonogenic cells were isolated after they migrated out from the cartilage explants onto the plastic. Amazingly, these cells also were able to migrate into the deeper zones of the OA cartilage explant from the surface in tissue culture, as tracked by GFP marker gene. Morphologically similar cells were identified migrating through breaks in the tidemark of OA cartilage by electron microscopic studies. These cells expressed transcription factors Sox9 and runx2, differentiated into osteoblasts and adipocytes, and their chondrogenic potential was enhanced after downregulation of runx2, suggesting that they are derived from the osteoprogenitor lineage (Koelling et al. 2009). Although historically AC has been considered to have a poor intrinsic repair capacity, and progenitor cells appear to be able to spontaneously induce only improper fibrocartilagenous repair tissue, their presence in cartilage opens up novel possibilities for the future developments of cartilage repair in the late stages of OA.

1.4.4

Synovium

Synovial fibroblast-like cells were isolated from adult human synovial membrane (De Bari et al. 2001; Vandenabeele et al. 2003). Their progenitor nature was studied by five independent clones that were all capable of chondrogenic, osteogenic, and adipogenic differentiation. These cells contained specific lamellar bodies, and expressed surfactant protein A, both characteristic to type B synoviocytes, thus suggesting that they may originate from synovial lining. Distinct expression profiles between MSCs derived from intra-articular tissues (synovium, meniscus, and ligament) and extra-articular tissues (muscle, adipose tissue, and bone marrow) were observed in patient matched analysis of 47 000 human transcripts (Segawa et al. 2009). Intra-articular tissue MSCs and articular chondrocytes expressed significantly more PRELP, ECRG4 and OGN while extra-articular tissues expressed higher levels of DSP, NRG1, SERPINB2, LFNG, NOG, and NEF3. Comparison of the synovium-derived progenitor cells with those derived from bone marrow, periosteum, skeletal muscle, and subcutaneous adipose tissue in patient matched studies have indicated their superiority in chondrogenic differentiation (Sakaguchi et al. 2005). Synovial MSCs colony forming unit (CFU) was 100-fold of that in the bone marrow (Morito et al. 2008). Their superiority in proliferation rate and chondrogenic

24

Regenerative medicine and biomaterials in connective tissue repair

differentiation was also supported by in vivo studies where MSCs from bone marrow, adipose tissue, and muscle were transplanted into cartilage defects together with periosteal patch in rabbits (Koga et al. 2008). Also, Fan et al. (2009) summarizes the superiority of chondrogenic properties of synovial MSCs over bone marrow MSCs, including higher expression of hyaluronan receptor CD44 and UDPGD, an enzyme vital for hyaluronan synthesis, and they also already express low levels of cartilage genes of COMP, aggrecan, type IX and XI collagen, and have a higher proliferation capacity than MSCs from other tissues. Synovium also originates from a common pool of progenitors with cartilage (Archer et al. 2003). These characteristics are advantageous in clinical applications, including that synovial membrane also easily self-regenerates, thereby allowing biopsies to obtain cells for autologous transplantation. Their high tendency to produce fibrocartilagenous tissue rather than hyaline cartilage remains yet a challenge to be overcome.

1.4.5

Synovial fluid

Jones et al. (2004) identified a presence of MSC population in synovial fluid of OA patients. Later they showed that also normal synovial fluid has a resident MSC population that increases at early OA (Jones et al. 2008). These cells are highly clonogenic and multi-potent both in young bovine and normal human joints. Synovial fluid cells represent a more homogenous pool in comparison with bone marrow MSCs. They were highly clonogenic cells with consistent chondrogenic capacity and were less adipogenic than bone marrow MSCs. Synovial fluid cells expressed higher levels of CD44 (hyaluronan receptor, a putative marker of enhanced chondrogenesis) than bone marrow MSCs, and lacked expression of CD271 (a low-affinity nerve factor receptor, characteristic marker of human bone marrow MSCs). Thus they are likely to originate from synovium (dislodged synovial fragments) or from the superficial AC layer rather than from bone marrow via circulation. Intra-articular ligament injury induced increased prevalence of synovial fluid MSCs and their adhesion onto the injured ligament (Morito et al. 2008). Also these cells were suggested to originate from synovium, as they were more similar to synovial MSCs than those derived from bone marrow, as compared by their morphologic and gene expression features. Reasons for synovial fluid MSC increase during ligament injury may be due to vessel injury-related promotion of cytokines and chemokines or to inflammation as was suggested by Jones et al. (2008).

1.4.6

Tendon and ligaments

Salingcarnboriboon et al. (2003) established mouse tendon-derived cell lines exhibiting pluripotent stem cell-like property with differentiation potential to

The structure and regenerative capacity of synovial joint tissues

25

osteogenic, chondrogenic and adipogenic lineages, but these cells were not characterized for their surface antigens. Bi and collegues (2007) identified a stem/progenitor cell population with universal stem cell characteristics in mouse and human tendon. These cells resided in a unique niche where small PGs biglycan and fibromodulin were found to be critical components in organizing this niche and regulating the local BMP activity. Multi-potent MSCs have also been isolated from ligaments showing diversity in the differentiation potential between six independent clones (Huang et al. 2008). Only one clone out of six was tripotent, and when compared with bone marrow MSCs, anterior cruciate ligament (ACL) derived clones expressed more type I and III collagens, suggesting higher potential for ligament fibroblasts. Tripotent MSC populations with typical MSC properties and similar phenotype to bone marrow MSCs were also isolated and expanded from ACL and posterior cruciate ligament (PCL) (Cheng et al. 2009).

1.4.7

Meniscus

In a comparative patient matched study, progenitor cells from several intraarticular and extra-articular tissues, including menisci, were isolated and their differentiation potential and other MSC properties compared (Segawa et al. 2009). This study revealed distinct gene expression profiles between these tissue groups, thus suggesting that intra-articular sources may be more favourable for chondrogenic differentiation and cartilage repair than MSCs from extra-articular tissues. The existence of multi-potent MSCs in meniscus tissue has thus been presented, but to our knowledge, their properties or possible role in intrinsic healing have not been studied in detail.

1.4.8

Other tissues

In the periosteum, the cambium layer contains multi-potent progenitor cell population that readily differentiates into chondrogenic lineage (reviewed by O'Driscoll and Fitzsimmons 2001). The advantage of this differentiation has been used for a long time in surgical repair procedures covering cartilage lesions by periosteal flaps (Jaroma and RitsilaÈ 1987; O'Driscoll 1998). Although the method shows short-term benefit, the repair tissue degenerates in the follow-up and at present periosteal transplantation is not a recommended method for cartilage repair (Hoikka et al. 1990; Hunziker 2002). Basic research shows still progress. A recent study in a goat model with human periosteumderived progenitor cells showed highly clonogenic cells with differentiation potential into chondrogenic, osteogenic, adipogenic, and myogenic lineages (De Bari et al. 2006). In another study, a simple isolation technique for skeletal tissue repair purposes of periosteal multi-potential MSCs was described (Choi et al. 2008).

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Regenerative medicine and biomaterials in connective tissue repair

Intra-articular infrapatellar adipose tissue, i.e., Hoffa's fat pad was shown to contain highly clonogenic, multi-potential MSCs that were capable of maintaining chondrogenesis for a long time (Wickham et al. 2003; English et al. 2007). Also, the infrapatellar fat pad contained pericyte marker 3G5-positive clonogenic cells that expressed MSC markers with enhanced chondrogenic differentiation after expansion in presence of FGF-2 (Khan et al. 2008). As reviewed by El Tamer and Reis (2009) and Tapp et al. (2009), MSCs isolated from adipose tissue are multi-potent, but they differ from MSCs derived from other mesenchymal tissues in that they endogenously express lower levels of BMP2, -4, and -6 in comparison to bone marrow. Their chondrogenic potential can be significantly increased in vitro by supplementing the cultures with exogenous BMP in addition to TGF (Hennig et al. 2007). Skeletal muscle tissue contains two stem cell populations with a possible MSC character: satellite cells and muscle-derived stem cells. Satellite cells have been considered as precursor cells rather than stem cells and have been shown so far to have osteogenic properties (Hashimoto et al. 2008; Sun et al. 2008). Muscle-derived stem cells likely locate in the connective tissue regions of skeletal muscle or in the capillaries surrounding myofibers. Great variation has been shown to exist between different populations greatly depending on the isolation methods (O'Brien et al. 2002). Animal muscle-derived stem cells have been shown to differentiate at least into osteblasts, myofibroblasts, chondrocytes and hematopoietic lineages, in addition to myoblasts (Cao and Huard 2004; Usas and Huard 2007; El Tamer and Reis 2009; Kubo et al. 2009). A myoendothelial cell population expressing myogenic and endothelial markers was recently isolated from human muscle with potential to differentiate into myogenic, osteogenic, and chondrogenic lineages (Zheng et al. 2007).

1.5

Conclusions and future trends

In this chapter we have described the macromolecular structure of the knee joint, organization of the tissues, their cellular and extracellular matrix composition and interrelationships with the biomechanical properties of the tissues. The major emphasis has been AC. Also we have reviewed the current knowledge of the presence of MSCs and progenitor cells in human joint tissues and briefly discussed their possible input in the joint homeostasis and endogenous repair potential. Inhomogeneous structure, anisotropic and nonlinear mechanical properties are characteristic of joint tissues. In functional adaptation, the structure, composition, and properties of the joint tissues are conditioned to withstand the loads which they are exposed to. In healthy joints, this will lead to optimized joint function. In the knee joint, structure, composition, and mechanical properties of AC show significant topographical variation. The topographical variation in mechanical properties has been revealed also by in vivo indentation

The structure and regenerative capacity of synovial joint tissues

27

measurements (Lyyra et al. 1999). Compression-tension non-linearity of AC is a further indication of tissue adaptation to the mechanical environment. Changes in the mechanical properties of joint tissues may indicate early pathology. In OA, equilibrium stiffness of cartilage can typically decrease by 50%. Minor degenerative changes in matrix, based on the scoring the histological integrity of the cartilage, have been shown to lead to extensive impairments in mechanical competence of AC (Laasanen et al. 2003). This has led to development of mechanical instrumentation for early in vivo diagnostics of cartilage degeneration. Compared with classical diagnostic techniques, these methods may provide minimally invasive ways for more sensitive diagnostics. Some techniques, e.g. quantitative ultrasound, may provide a method for simultaneous diagnostics of both AC and subchondral bone (Saarakkala et al. 2006). Potentially, small changes in tissue structure and composition may manifest themselves as more significant changes in tissue mechanical properties. This is an idea behind the concept of functional imaging (Julkunen et al. 2008). A combination of quantitative non-invasive imaging, such as magnetic resonance imaging (MRI), combined with the realistic theoretical model of joint function, helps to diagnose early tissue pathology and may enable functional analysis and prediction of the development of tissue properties in the future. This would be highly useful, for example, when assessing the outcome of cartilage repair operations. All joint tissues contain intrinsic repair potential in the form of resident tissue specific multi-potent progenitor cell populations. The presence of MSCs in the synovial fluid but also in the other tissues may have important function in the homeostasis of the joint, and particularly during traumatic or inflammatory conditions. The influence of stem cells in the tissues and synovial fluid may not be restricted to the direct effect during tissue remodeling or repair but they may also act as trophic mediators to assist these processes by secreting bioactive factors such as cytokines or factors that suppress immune recognition mechanisms (Caplan and Dennis 2006; Caplan 2009). Differences in the cell surface antigen presentation, gene expression profiles, in their effectivity to expand and self-renew in the culture, and potential to differentiate into different mesenchymal tissues are seen between MSCs derived from intra-articular and extra-articular tissues. There are differences also within the cell populations or single cell-derived clones from the same tissues, indicating the heterogeneous nature of these progenitor populations. MSC populations derived from intra-articular tissue are more homogenous than those from extra-articular sources, particularly from bone marrow. Synovial MSCs are superior over the others in their chondrogenic differentiation potential but they appear to favor development of fibrocartilagenous phenotype under conditions studied so far. Unfortunately, spontaneous repair by intrinsic MSCs seldom results in a formation of biomechanically adequate repair tissue. MSCs present in synovial fluid are increased in ligament injury and align with injured

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Regenerative medicine and biomaterials in connective tissue repair

tissue for regeneration, but perhaps unfavourable biomechanical and other factors prevent full healing (Strand et al. 2005; Morito et al. 2008). Further, chondrogenic progenitors are present in adult AC. This questions the prevailing opinion that the essential cause for poor repair capacity of AC is the shortage of intrinsic chondrogenic cells. It remains a challenge for future regenerative medicine to learn how to trigger intrinsic repair potentials of joint tissues, especially that of MSCs by taking advantage of the progenitor cell migration through tissues and homing to the repair site (Chamberlain et al. 2007; Koelling et al. 2009) and to induce synthesis of hyaline cartilage, instead of fibrocartilage.

1.6

Sources of further information and advice

1.6.1

Useful links

· Gray's Anatomy http://education.yahoo.com/reference/gray/subjects/ · Wheeless' Textbook of Orthopaedics: http://www.wheelessonline.com/ · Stem cell links http://stemcells.nih.gov/info/basics http://learn.genetics.utah.edu/content/tech/stemcells/

1.6.2

Books

Bronner F II and Farach-Carson M C (2007), Topics in Bone Biology, London, Springer. Cowin S C and Doty S B (2007), Tissue Mechanics, New York, Springer. JoÂzsa L G and Kannus P (1997), Human Tendons, Anatomy, Physiology, and Pathology, Champaign, IL, Human Kinetics. Nordin M and Frankel V H, (2001), Basic Biomechanics of the Musculoskeletal System, 3rd ed., Philadelphia, Lippincott Williams & Wilkins.

1.6.3

Book chapters

Bartl R and Frisch B (2009), `Biology of bone' in Bartl R and Frisch B, Osteoporosis, Diagnosis, Prevention, Therapy, Springer, Berlin, 7±28. Sandell L, HeinegaÊrd D and Hering T M (2007), `Cell biology, biochemistry, and molecular biology of articular cartilage in osteoarthritis' in Moskowitz R W, Altman R D, Hochberg M C, Buckwalter J A and Goldberg V M, Osteoarthritis. Diagnosis and medical/surgical management, 4th ed., Philadelphia, Lippincott Williams & Wilkins, 73±106. Zamorani M P and Valle M (2007), `Bone and joint' in Bianchi S, Martinoli C, Medical Radiology. Ultrasound of the Musculoskeletal System. Berlin, Heidelberg, New York, Springer, 137±85.

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1.7

29

References

Abdallah B M and Kassem M, `Human mesenchymal stem cells: from basic biology to clinical applications', Gene Ther, 2008, 15, 109±16. Abdallah B M and Kassem M, `The use of mesenchymal (skeletal) stem cells for treatment of degenerative diseases: current status and future perspectives', J Cell Physiol, 2009, 218, 9±12. Review. Aigner T and StoÈve J, `Collagens ± major component of the physiological cartilage matrix, major target of cartilage degeneration, major tool in cartilage repair', Adv Drug Deliv Rev, 2003, 55, 1569±93. Review. Alsalameh S, Amin R, Gemba T and Lotz M, `Identification of mesenchymal progenitor cells in normal and osteoarthrititc human articular cartilage', Arthritis Rheum, 2004, 50, 1522±32. Archer C W, Dowthwaite G P and Francis-West P, `Development of synovial joints', Birth Defects Res C Embryo Today, 2003, 69, 144±55. Review. Arokoski J P A, Jurvelin J, VaÈaÈtaÈinen U and Helminen H J, `Normal and pathological adaptation of articular cartilage to joint loading', Scand J Med Sci Sports, 2000, 10, 186±98. Review. Arthur A, Zannettino A and Gronthos S, `The therapeutic applications of multipotential mesenchymal/stromal stem cells in skeletal tissue repair', J Cell Physiol, 2009, 218, 237±45. Review. Ateshian G A, `The role of interstitial fluid pressurization in articular cartilage lubrication', J Biomechanics, 2009, 42, 1163±76. Barbero A, Ploegert S, Heberer M and Martin I, `Plasticity of clonal populations of dedifferentiated adult human articular chondrocytes', Arthritis Rheum, 2003, 48, 1315±25. Barry F P and Murphy J M, `Mesenchymal stem cells: clinical applications and biological characterization', Int J Biochem Cell Biol, 2004, 36, 568±84. Review. Bartl R and Frisch B (2009) `Biology of bone' in Bartl R and Frisch B, Osteoporosis, Diagnosis, Prevention, Therapy, Springer, Berlin, 7±28. Bastiaansen-Jenniskens Y M, Koevoet W, de Bart AC, van der Linden J C, Zuurmond A M, Weinans H, Verhaar J A, van Osch G J and Degroot J, `Contribution of collagen network features to functional properties of engineered cartilage', Osteoarthritis Cartilage, 2008, 16, 359±66. Benjamin M, Toumi H, Ralphs J R, Bydder G, Best T M and Milz S, `Where tendons and ligaments meet bone: attachment sites (``entheses'') in relation to exercise and/or mechanical load', J Anat, 2006, 208, 471±90. Bhosale A M and Richardson J B, `Articular cartilage: structure, injuries and review of management', Br Med Bull, 2008, 87, 77±95. Review. Bi Y, Ehirchiou D, Kilts T M, Inkson C A, Embree M C, Sonoyama W, Li L, Leet A I, Seo B M, Zhang L, Shi S and Young M F, `Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche', Nat Med, 2007, 13, 1219±27. Blom A B, van der Kraan P M and van den Berg W B, `Cytokine targeting in osteoarthritis', Curr Drug Targets, 2007, 8, 283±92. Review. Boschetti F, Pennati G, Gervaso F, Peretti G M and Dubini G, `Biomechanical properties of human articular cartilage under compressive loads', Biorheology, 2004, 41, 159± 66. Broom N D, `The altered biomechanical state of human femoral head osteoarthritic articular cartilage', Arthritis Rheum, 1984, 27, 1028±39.

30

Regenerative medicine and biomaterials in connective tissue repair

Burr D B, `Anatomy and physiology of the mineralized tissues: role in the pathogenesis of osteoarthrosis', Osteoarthritis Cartilage, 2004, 12 Suppl A, S20±30. Review. Burr D B and Radin E L, `Microfractures and microcracks in subchondral bone: are they relevant to osteoarthrosis?', Rheum Dis Clin North Am, 2003, 29, 675±85. Review. Cao B and Huard J, `Muscle-derived stem cells', Cell Cycle, 2004, 3, 104±7. Review. Caplan A I, `Adult mesenchymal stem cells for tissue engineering versus regenerative medicine', J Cell Physiol, 2007, 213, 341±7. Review. Caplan A I, `Why are MSCs therapeutic? New data: new insight', J Pathol, 2009, 217, 318±24. Review. Caplan A I and Dennis J E, `Mesenchymal stem cells as trophic mediators', J Cell Biochem, 2006, 98, 1076±84. Review. Chamberlain G, Fox J, Ashton B and Middleton J, `Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing', Stem Cells, 2007, 25, 2739±49. Review. Chen F H and Tuan R S, `Mesenchymal stem cells in arthritic diseases', Arthritis Res Ther, 2008, 10, 223. Review. Cheng M T, Yang H W, Chen T H and Lee O K, `Isolation and characterization of multipotent stem cells from human cruciate ligaments', Cell Prolif, 2009, 42, 448± 60. Chiquet-Ehrismann R and Tucker R P, `Connective tissues: signalling by tenascins' Int J Biochem Cell Biol, 2004, 36, 1085±9. Review. Choi Y S, Noh S E, Lim S M, Lee C W, Kim C S, Im M W, Lee M H and Kim D I, `Multipotency and growth characteristic of periosteum-derived progenitor cells for chondrogenic, osteogenic, and adipogenic differentiation', Biotechnol Lett, 2008, 30, 593±601. Cowin S C and Doty S B (2007a), `Cartilage', in Cowin S C and Doty S B, Tissue Mechanics, New York, Springer, 471±505. Cowin S C and Doty S B (2007b), `Bone', in Cowin S C and Doty S B, Tissue Mechanics, New York, Springer, 341±84. Cowin S C and Doty S B (2007c), `Tendon and ligaments', in Cowin S C and Doty S B, Tissue Mechanics, New York, Springer, 559±94. da Silva Meirelles L, Chagastelles P C and Nardi N B, `Mesenchymal stem cells reside in virtually all post-natal organs and tissues', J Cell Sci, 2006, 119, 2204±13. De Bari C, Dell'Accio F, Tylzanowski P and Luyten F P, `Multipotent mesenchymal stem cells from adult human synovial membrane', Arthritis Rheum, 2001, 44, 1928±42. De Bari C, Dell'Accio F, Vanlauwe J, Eyckmans J, Khan I M, Archer C W, Jones E A, McGonagle D, Mitsiadis T A, Pitzalis C and Luyten F P, `Mesenchymal multipotency of adult human periosteal cells demonstrated by single-cell lineage analysis', Arthritis Rheum, 2006, 54, 1209±21. de Mos M, Koevoet W J, Jahr H, Verstegen M M, Heijboer M P, Kops N, van Leeuwen J P, Weinans H, Verhaar J A and van Osch G J, `Intrinsic differentiation potential of adolescent human tendon tissue: an in-vitro cell differentiation study', BMC Musculoskelet Disord, 2007, 8, 16. D'Lima D D, Hashimoto S, Chen P C, Colwell C W and Lotz M K, `Human chondrocyte apoptosis in response to mechanical injury', Osteoarthritis Cartilage, 2001, 9, 712± 19. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D J and Horwitz E `Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement', Cytotherapy, 2006, 8, 315±17.

The structure and regenerative capacity of synovial joint tissues

31

Dowthwaite G P, Bishop J C, Redman S N, Khan I M, Rooney P, Evans D J, Haughton L, Bayram Z, Boyer S, Thomson B, Wolfe M S and Archer C W, `The surface of articular cartilage contains a progenitor cell population', J Cell Sci, 2004, 117, 889±97. Duthon V B, Barea C, Abrassart S, Fasel J H, Fritschy D and MeÂneÂtrey J, `Anatomy of the anterior cruciate ligament', Knee Surg Sports Traumatol Arthrosc, 2006, 14, 204±13. Review. Elsaid K A, Jay G D and Chichester C O, `Reduced expression and proteolytic susceptibility of lubricin/superficial zone protein may explain early elevation in the coefficient of friction in the joints of rats with antigen-induced arthritis', Arthritis Rheum, 2007, 56, 108±16. El Tamer M K and Reis R L, `Progenitor and stem cells for bone and cartilage regeneration', J Tissue Eng Regen Med, 2009, 3, 327±37. Review. English A, Jones E A, Corscadden D, Henshaw K, Chapman T, Emery P and McGonagle D, `A comparative assessment of cartilage and joint fat pad as a potential source of cells for autologous therapy development in knee osteoarthritis', Rheumatology, 2007, 46, 1676±83. Eyre D, `Collagen of articular cartilage', Arthritis Res, 2002, 4, 30±5. Review. Fam H, Bryant J T and Kontopoulou M, `Rheological properties of synovial fluid', Biorheology, 2007, 44, 59±74. Fan J, Varshney R R, Ren L, Cai D and Wang D A, `Synovium-derived mesenchymal stem cells: a new cell source for musculoskeletal regeneration', Tissue Eng Part B Rev, 2009, 15, 75±86. Review. Fickert S, Fiedler J and Brenner R E, `Identification of subpopulations with characteristics of mesenchymal progenitor cells from human osteoarthritic cartilage using triple staining for cell surface markers', Arthritis Res Ther, 2004, 6, R422±32. FitzGerald O and Bresnihan B, `Synovial membrane cellularity and vascularity', Ann Rheum Dis, 1995, 54, 511±15. Review. Gentili C and Cancedda R, `Cartilage and bone extracellular matrix', Curr Pharm Des, 2009, 15, 1334±48. Review. Ghosh P and Taylor T K, `The knee joint meniscus. A fibrocartilage of some distinction', Clin Orthop Relat Res, 1987, 224, 52±63. Review. Goldring M B and Marcu K B, `Cartilage homeostasis in health and rheumatic diseases', Arthritis Res Ther, 2009, 11, 224. Goldring M B, Otero M, Tsuchimochi K, Ijiri K and Li Y, `Defining the roles of inflammatory and anabolic cytokines in cartilage metabolism', Ann Rheum Dis, 2008, 67 Suppl 3, iii75±82. Review. Gregory C A, YloÈstalo J and Prockop D J, `Adult bone marrow stem/progenitor cells (MSCs) are preconditioned by microenvironmental ``niches'' in culture: a two-stage hypothesis for regulation of MSC fate', Sci STKE, 2005, pe37. Review. Grogan S P, Miyaki S, Asahara H, D'Lima D D and Lotz M K, `Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis', Arthritis Res Ther, 2009, 11, R85. Hashimoto N, Kiyono T, Wada M R, Umeda R, Goto Y, Nonaka I, Shimizu S, Yasumoto S and Inagawa-Ogashiwa M, `Osteogenic properties of human myogenic progenitor cells', Mech Dev, 2008, 125, 257±69. Hayes A J, Tudor D, Nowell M A, Caterson B and Hughes C E, `Chondroitin sulfate sulfation motifs as putative biomarkers for isolation of articular cartilage progenitor cells', J Histochem Cytochem, 2008, 56, 125±38. Hayes W C, Keer L M, Herrmann G and Mockros L F, `A mathematical analysis for indentation tests of articular cartilage', J Biomech, 1972, 5, 541±51.

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Regenerative medicine and biomaterials in connective tissue repair

Haynesworth S E, Baber M A and Caplan A I, `Cytokine expression by human marrowderived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1 alpha', J Cell Physiol, 1996, 166, 585±92. Helminen H J, Hyttinen M, Lammi M J, Arokoski J P A, LapvetelaÈinen T, Jurvelin J, Kiviranta I and Tammi M I, `Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life: a hypothesis', J Bone Miner Metab, 2000, 18, 245±57. Hennig T, Lorenz H, Thiel A, Goetzke K, Dickhut A, Geiger F and Richter W, `Reduced chondrogenic potential of adipose tissue derived stromal cells correlates with an altered TGFbeta receptor and BMP profile and is overcome by BMP-6', J Cell Physiol, 2007, 211, 682±91. Hiraoka K, Grogan S, Olee T and Lotz M, `Mesenchymal progenitor cells in adult human articular cartilage', Biorheology, 2006, 43, 447±54. Hoikka V E, Jaroma H J and RitsilaÈ V A, `Reconstruction of the patellar articulation with periosteal grafts. 4-year follow-up of 13 cases', Acta Orthop Scand, 1990, 61, 36±9. Horwitz E M, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini F C, Deans R J, Krause D S and Keating A, `Clarification of the nomenclature for MSC, The International Society for Cellular Therapy position statement', Cytotherapy, 2005, 7, 393±5. Huang C Y, Mow V C and Ateshian G A, `The role of flow-independent viscoelasticity in the biphasic tensile and compressive responses of articular cartilage', J Biomech Eng, 2001, 123, 410±17. Huang T F, Chen Y T, Yang T H, Chen L L, Chiou S H, Tsai T H, Tsai C C, Chen M H, Ma H L and Hung S C, `Isolation and characterization of mesenchymal stromal cells from human anterior cruciate ligament', Cytotherapy, 2008, 10, 806±14. Hunziker E B, `Articular cartilage repair, basic science and clinical progress. A review of the current status and prospects', Osteoarthritis Cartilage, 2002, 10, 432±63. Hyttinen M M, Arokoski J P, Parkkinen J J, Lammi M J, LapvetelaÈinen T, Mauranen K, KiraÂly K, Tammi M I and Helminen H J, `Age matters: collagen birefringence of superficial articular cartilage is increased in young guinea-pigs but decreased in older animals after identical physiological type of joint loading', Osteoarthritis Cartilage, 2001, 9, 694±701. Imhof H, Sulzbacher I, Grampp S, Czerny C, Youssefzadeh S and Kainberger F, `Subchondral bone and cartilage disease: a rediscovered functional unit', Invest Radiol, 2000, 35, 581±8. Ingram K R, Wann A K, Angel C K, Coleman P J and Levick J R, `Cyclic movement stimulates hyaluronan secretion into the synovial cavity of rabbit joints', J Physiol, 2008, 586, 1462. Iwanaga T, Shikichi M, Kitamura H, Yanase H and Nozawa-Inoue K, `Morphology and functional roles of synoviocytes in the joint', Arch Histol Cytol, 2000, 63, 17±31. Jaroma H J and RitsilaÈ V A, `Reconstruction of patellar cartilage defects with free periosteal grafts. An experimental study' Scand J Plast Reconstr Surg, 1987, 21, 175±81. Johnstone B, Hering T M, Caplan A I, Goldberg V M and Yoo J U, `In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells', Exp Cell Res, 1998, 238, 265±72. Jones D L and Wagers A J, `No place like home: anatomy and function of the stem cell niche', Nat Rev Mol Cell Biol, 2008, 9, 11±21. Review. Jones E and McGonagle D, `Human bone marrow mesenchymal stem cells in vivo',

The structure and regenerative capacity of synovial joint tissues

33

Rheumatology, 2008, 47(2), 126±31. Review. Jones E A, English A, Henshaw K, Kinsey S E, Markham A F, Emery P and McGonagle D, `Enumeration and phenotypic characterization of synovial fluid multipotential mesenchymal progenitor cells in inflammatory and degenerative arthritis', Arthritis Rheum, 2004, 50, 817±27. Jones E A, Crawford A, English A, Henshaw K, Mundy J, Corscadden D, Chapman T, Emery P, Hatton P and McGonagle D, `Synovial fluid mesenchymal stem cells in health and early osteoarthritis: detection and functional evaluation at the single-cell level', Arthritis Rheum, 2008, 58, 1731±40. Julkunen P, Korhonen R K, Nissi M J and Jurvelin J S, `Mechanical characterization of articular cartilage by combining magnetic resonance imaging and finite-element analysis: a potential functional imaging technique', Phys Med Biol, 2008, 53, 2425± 38. Jurvelin J, Kiviranta I, SaÈaÈmaÈnen A-M, Tammi M and Helminen H J, `Indentation stiffness of young canine knee articular cartilage ± influence of strenuous joint loading', J Biomech, 1990, 23, 1239±46. Kannus P, `Structure of the tendon connective tissue', Scand J Med Sci Sports, 2000, 10, 312±20. Review. Karlsson C, Stenhamre H, Sandstedt J and Lindahl A, `Neither Notch1 expression nor cellular size correlate with mesenchymal stem cell properties of adult articular chondrocytes', Cells Tissues Organs, 2008, 187, 275±85. Karlsson C, Thornemo M, Henriksson H B and Lindahl A, `Identification of a stem cell niche in the zone of Ranvier within the knee joint', J Anat, 2009, 215, 355±63. Katta J, Jin Z, Ingham E and Fisher J, `Biotribology of articular cartilage ± a review of the recent advances', Med Eng Phys, 2008, 30, 1349±63. Keating A, `Mesenchymal stromal cells', Curr Opin Hematol, 2006, 13, 419±25. Review. Kempson G E, Muir H, Pollard C and Tuke M, `The tensile properties of the cartilage of human femoral condyles related to the content of collagen and glycosaminoglycans', Biochim Biophys Acta, 1973, 297, 456±72. Ker R F, `Mechanics of tendons, from an engineering perspective', Int J Fatique, 2007, 29, 1001±9. Review. Khan I M, Redman S N, Williams R, Dowthwaite G P, Oldfield S F and Archer C W, `The development of synovial joints', Curr Top Dev Biol 2007, 79, 1±36. Review. Khan I M, Williams R and Archer C W, `One flew over the progenitor's nest: migratory cells find a home in osteoarthritic cartilage', Cell Stem Cell, 2009, 4, 282±4. Preview. Khan W S, Tew S R, Adesida A B and Hardingham T E, `Human infrapatellar fat padderived stem cells express the pericyte marker 3G5 and show enhanced chondrogenesis after expansion in fibroblast growth factor-2', Arthritis Res Ther, 2008, 10, R74. Knudson C B and Knudson W, `Cartilage proteoglycans', Semin Cell Dev Biol, 2001, 12, 69±78. Review. Knutsen G, Drogset J O, Engebretsen L, Grùntvedt T, Isaksen V, Ludvigsen T C, Roberts S, Solheim E, Strand T and Johansen O, `A randomized trial comparing autologous chondrocyte implantation with microfracture. Findings at five years', J Bone Joint Surg Am, 2007, 89, 2105±12. Koelling S, Kruegel J, Irmer M, Path J R, Sadowski B, Miro X and Miosge N (2009) `Migratory chondrogenic progenitor cells from repair tissue during the later stages of human osteoarthritis', Cell Stem Cell, 4, 324±335. Koga H, Muneta T, Nagase T, Nimura A, Ju Y J, Mochizuki T and Sekiya I, `Comparison

34

Regenerative medicine and biomaterials in connective tissue repair

of mesenchymal tissues-derived stem cells for in vivo chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit', Cell Tissue Res, 2008, 333, 207±15. Korhonen R K, Laasanen M S, ToÈyraÈs J, Lappalainen R, Helminen H J and Jurvelin J S, `Fibril reinforced poroelastic model predicts specifically mechanical behavior of normal, proteoglycan depleted and collagen degraded articular cartilage', J Biomech, 2003, 36, 1373±9. Kouri J B, JimeÂnez S A, Quintero M and Chico A, `Ultrastructural study of chondrocytes from fibrillated and non-fibrillated human osteoarthritic cartilage', Osteoarthritis Cartilage, 1996, 4, 111±25. Kubo S, Cooper G M, Matsumoto T, Phillippi J A, Corsi K A, Usas A, Li G, Fu F H and Huard J, `Blocking vascular endothelial growth factor with soluble Flt-1 improves the chondrogenic potential of mouse skeletal muscle-derived stem cells', Arthritis Rheum, 2009, 60, 155±65. Kuster M S and Wood G A, Stachowiak G W and GaÈchter A, `Joint load considerations in total knee replacement', J Bone Joint Surg Br, 1997, 79, 109±13. Laasanen M S, ToÈyraÈs J, Vasara A I, Hyttinen M M, Saarakkala S, Hirvonen J, Jurvelin J S and Kiviranta I, `Mechano-acoustic diagnosis of cartilage degeneration and repair', J Bone Joint Surg Am, 2003, 85-A Suppl 2, 78±84. Lai W M, Hou J S and Mow V C, `A triphasic theory for the swelling and deformation behaviors of articular cartilage', J Biomech Eng, 1991, 113, 245±58. Levick J R (1987), `Synovial fluid and trans-synovial flow in stationary and moving joints', in Helminen H, Kiviranta I, Tammi M, Saamanen A-M, Paukonen K and Jurvelin J, Joint Loading, Biology and Health of Articular Structures, Bristol, Wright & Sons, 149±86. Lutzky V, Hannawi S and Thomas R, `Cells of the synovium in rheumatoid arthritis. Dendritic cells', Arthritis Res Ther, 2007, 9, 219. Review. Lyyra T, Kiviranta I, VaÈaÈtaÈinen U, Helminen H J and Jurvelin J S, `In vivo characterization of indentation stiffness of articular cartilage in the normal human knee', Biomed Mater Res, 1999, 48, 482±7. Mackie E J, Ahmed Y A, Tatarczuch L, Chen K S and Mirams M, `Endochondral ossification: how cartilage is converted into bone in the developing skeleton', Int J Biochem Cell Biol, 2008, 40, 46±62. Review. Magnuson P B, 'Joint debridement: Surgical treatment of degenerative arthritis', Surg Gynecol Obstet, 1941, 73, 1±4. Majumdar M K, Thiede M A, Mosca J D, Moorman M and Gerson S L, `Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells', J Cell Physiol, 1998, 176, 57±66. Maroudas A, Palla G and Gilav E, `Racemization of aspartic acid in human articular cartilage', Connect Tissue Res, 1992, 28, 161±9. Melrose J, Smith S, Cake M, Read R and Whitelock J, `Comparative spatial and temporal localisation of perlecan, aggrecan and type I, II and IV collagen in the ovine meniscus: an ageing study', Histochem Cell Biol, 2005, 124, 225±35. Melrose J, Hayes A J, Whitelock J M and Little C B, `Perlecan, the `jack of all trades' proteoglycan of cartilaginous weight-bearing connective tissues', Bioessays, 2008, 30, 457±69. Review. Mente P L and Lewis J L, `Elastic modulus of calcified cartilage is an order of magnitude less than that of subchondral bone', J Orthop Res, 1994, 12, 637±47. Mithoefer K, McAdams T, Williams R J, Kreuz P C and Mandelbaum B R, `Clinical efficacy of the microfracture technique for articular cartilage repair in the knee, an

The structure and regenerative capacity of synovial joint tissues

35

evidence-based systematic analysis', Am J Sports Med, 2009, 37, 2053±63. Mochizuki T, Muneta T, Sakaguchi Y, Nimura A, Yokoyama A, Koga H and Sekiya I, `Higher chondrogenic potential of fibrous synovium- and adipose synoviumderived cells compared with subcutaneous fat-derived cells, distinguishing properties of mesenchymal stem cells in humans', Arthritis Rheum, 2006, 54, 843±53. Morales T I, `Chondrocyte moves: clever strategies?', Osteoarthritis Cartilage, 2007, 15, 861±71. Review. Morito T, Muneta T, Hara K, Ju Y-J, Mochizuki T, Makino H, Umezawa A and Sekiya I, `Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans', Rheumatology, 2008, 47, 1137±43. Mow V C, Kuei S C, Lai W M and Armstrong C G, `Biphasic creep and stress relaxation of articular cartilage in compression? Theory and experiments', J Biomech Eng, 1980, 102, 73±84. Nisell R, `Mechanics of the knee. A study of joint and muscle load with clinical applications', Acta Orthop Scand Suppl, 1985, 216, 1±42. Nordin M and Frankel V H (1989) Basic Biomechanics of the Musculoskeletal System, 2nd ed., Philadelphia, Lea & Febiger. NoÈth U, Steinert A F and Tuan R S, `Technology insight: adult mesenchymal stem cells for osteoarthritis therapy', Nat Clin Pract Rheumatol, 2008, 4, 371±80. O'Brien K, Muskiewicz K and Gussoni E, `Recent advances in and therapeutic potential of muscle-derived stem cells', J Cell Biochem, 2002, 38 Suppl, 80±7. Review. O'Driscoll S W, `The healing and regeneration of articular cartilage' J Bone Joint Surg, 1998, 80-A, 1795±812. O'Driscoll S W and Fitzsimmons J S, `The role of periosteum in cartilage repair', Clin Orthop Relat Res, 2001, 391 Suppl, S190±207. Review. Oettmeier R, Arokoski J, Roth A J, Abendroth K, Jurvelin J, Tammi M, Kiviranta I and Helminen H J, `Subchondral bone and articular cartilage responses to long distance running training (40 km per day) in the beagle knee joint', Eur J Exp Musculoskel Res, 1992, 1, 145±54. Petersen W and Tillmann B, `Collagenous fibril texture of the human knee joint menisci', Anat Embryol (Berl), 1998, 197, 317±24. Petersen W and Zantop T, `Anatomy of the anterior cruciate ligament with regard to its two bundles' Clin Orthop Relat Res, 2007, 454, 35±47. Review. Phinney D G and Prockop D J, `Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair ± current views', Stem Cells, 2007, 25, 2896±902. Pittenger M F, Mackay A M, Beck SC, Jaiswal R K, Douglas R, Mosca J D, Moorman M A, Simonetti D W, Craig S and Marshak D R, `Multilineage potential of adult human mesenchymal stem cells', Science, 1999, 284, 143±7. Poole C A, `Articular cartilage chondrons: form, function and failure' J Anat, 1997, 191, 1±13. Review. Pridie K H, `A method of resurfacing osteoarthritic knee joints', J Bone Joint Surg 1959, 41B, 618±19. Prockop D J, `Repair of tissues by adult stem/progenitor cells (MSCs): controversies, myths, and changing paradigms', Mol Ther, 2009, 17, 939±46. Qu C (2007), `Articular cartilage proteoglycan biosynthesis and sulfation', Ph.D. Thesis. Kuopio University Publications D. Medical Sciences, 419 (http://www.uku.fi/ vaitokset/2007/index.shtml). Radin E L and Rose R M, `Role of subchondral bone in the initiation and progression of cartilage damage', Clin Orthop Relat Res, 1986, 213, 34±40.

36

Regenerative medicine and biomaterials in connective tissue repair

Rhee D K, Marcelino J, Al-Mayouf S, Schelling D K, Bartels C F, Cui Y, Laxer R, Goldbach-Mansky R and Warman M L, `Consequences of disease-causing mutations on lubricin protein synthesis, secretion, and post-translational processing', J Biol Chem, 2005, 280, 31325±32. Riley G, `Medscape. Tendinopathy ± from basic science to treatment', Nat Clin Pract Rheumatol, 2008, 4, 82±9. Review. Roughley P J, `Articular cartilage and changes in arthritis: noncollagenous proteins and proteoglycans in the extracellular matrix of cartilage', Arthritis Res, 2001, 3, 342±7. Review. Roughley P J, `The structure and function of cartilage proteoglycans', Eur Cell Mater, 2006, 12, 92±101. Review. Rumian A P, Wallace A L and Birch H L, `Tendons and ligaments are anatomically distinct but overlap in molecular and morphological features ± a comparative study in an ovine model', J Orthop Res, 2007, 25, 458±64. SaÈaÈmaÈnen A-M, Vasara A, Lammi M J and Kiviranta I, [`Cell transplants and biomaterials in the surgical repair of cartilage lesions', Finnish]. Duodecim, 2008, 124, 1910±18. Review. Saarakkala S (2007), `Pre-clinical ultrasound diagnostics of articular cartilage and subchondral bone', Ph.D. Thesis, Kuopio University Publications C. Natural and Environmental Sciences, 205 (http://www.uku.fi/vaitokset/2007/index.shtml). Saarakkala S, Laasanen M S, Jurvelin J S and ToÈyraÈs J, `Quantitative ultrasound imaging detects degenerative changes in articular cartilage surface and subchondral bone', Phys Med Biol, 2006, 51, 5333±46. Sakaguchi Y, Sekiya I, Uagishita K and Muneta T, `Comparison of human stem cells derived from various mesenchymal tissues. Superiority of synovium as a cell source', Arthritis Rheum, 2005, 52, 2521±9. Salingcarnboriboon R, Yoshitake H, Tsuji K, Obinata M, Amagasa T, Nifuji A and Noda M, `Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property', Exp Cell Res, 2003, 287, 289±300. Schaefer L and Iozzo R V, `Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction', J Biol Chem, 2008, 283, 21305±9. Review. Schinagl R M, Gurskis D, Chen A C and Sah R L, `Depth-dependent confined compression modulus of full-thickness bovine articular cartilage', J Orthop Res, 1997, 15, 499±506. Segawa Y, Muneta T, Makino H, Nimura A, Mochizuki T, Ju Y-J, Ezura Y, Umezawa A and Sekiya I, `Mesenchymal stem cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression profiles', J Orhop Res, 2009, 27, 435±41. Selmani Z, Naji A, Gaiffe E, Obert L, Tiberghien P, Rouas-Freiss N, Carosella E D and Deschaseaux F, `HLA-G is a crucial immunosuppressive molecule secreted by adult human mesenchymal stem cells', Transplantation, 2009, 87, 9 Suppl, S62±6. Setton L A, Guilak F, Hsu E W and Vail T P, `Biomechanical factors in tissue engineered meniscal repair', Clin Orthop Relat Res, 1999a, 367 Suppl, S254±72. Review. Setton L A, Elliott D M and Mow V C, `Altered mechanics of cartilage with osteoarthritis: human osteoarthritis and an experimental model of joint degeneration', Osteoarthritis Cartilage, 1999b, 7, 2±14. Review. Shakibaei M, Csaki C and Mobasheri A, `Diverse roles of integrin receptors in articular cartilage', Adv Anat Embryol Cell Biol, 2008, 197, 1±60. Review. Siegel G, SchaÈfer R and Dazzi F, `The immunosuppressive properties of mesenchymal stem cells', Transplantation, 2009, 87, 9 Suppl, S45±9.

The structure and regenerative capacity of synovial joint tissues

37

Soulhat J, Buschmann M D and Shirazi-Adl A, `A fibril-network-reinforced biphasic model of cartilage in unconfined compression', J Biomech Eng, 1999, 121, 340±7. Steadman J R, Rodkey W G and Briggs K K, `Microfracture to treat full thickness chondral defects, surgical technique, rehabilitation, and outcomes', J Knee Surg, 2002, 15, 170±6. Strand T, Mùlster A, Hordvik M and Krukhaug Y, `Long-term follow-up after primary repair of the anterior cruciate ligament: clinical and radiological evaluation 15±23 years postoperatively', Arch Orthop Trauma Surg, 2005, 125, 217±21. Sun S, Wang Z and Hao Y, `Osterix overexpression enhances osteoblast differentiation of muscle satellite cells in vitro', Int J Oral Maxillofac Surg, 2008, 37, 350±6. Sutton S, Clutterbuck A, Harris P, Gent T, Freeman S, Foster N, Barrett-Jolley R and Mobasheri A, `The contribution of the synovium, synovial derived inflammatory cytokines and neuropeptides to the pathogenesis of osteoarthritis', Vet J, 2009, 179, 10±24. Review. Sweigart M A and Athanasiou K A, `Toward tissue engineering of the knee meniscus' Tissue Eng, 2001, 7, 111±29. Review. Tallheden T, Brittberg M, Peterson L and Lindahl A, `Human articular chondrocytes ± plasticity and differentiation potential', Cells Tissues Organs, 2006, 184, 55±67. Review. Tammi M, Paukkonen K, Kiviranta I, Jurvelin J, SaÈaÈmaÈnen A-M and Helminen H J (1987), `Joint loading-induced alterations in articular cartilage', in: Helminen HJ, Kiviranta I, Tammi M, SaÈaÈmaÈnen A-M, Paukkonen K and Jurvelin J, Joint Loading ± Biology and Health of Articular Structures, Bristol, John Wright and Sons Ltd, 50±66. Tapp H, Hanley E N Jr, Patt J C and Gruber H E, `Adipose-derived stem cells: characterization and current application in orthopaedic tissue repair', Exp Biol Med, 2009, 234, 1±9. Review. Thambyah A, Goh J C and De S D, `Contact stresses in the knee joint in deep flexion', Med Eng Phys, 2005, 27, 329±35. Thornemo M, Tallheden T, Sjogren Jansson E, Larsson A, Lovstedt K, Nannmark U, Brittberg M and Lindahl A, `Clonal populations of chondrocytes with progenitor properties identified within human articular cartilage', Cells Tissues Organs, 2005, 180, 141±50. Torzilli P A, Deng X-H and Ramcharan M, `Effect of compressive strain on cell viability in statically loaded articular cartilage', Biomechan Model Mechanobiol, 2006, 5, 123±32. Unsworth A, Dowson D and Wright V, `Some new evidence on human joint lubrication', Ann Rheum Dis, 1975, 34, 277±85. Usas A and Huard J, `Muscle-derived stem cells for tissue engineering and regenerative therapy', Biomaterials, 2007, 28, 5401±6. Review. Valiyaveettil M, Mort J S and McDevitt C A, `The concentration, gene expression, and spatial distribution of aggrecan in canine articular cartilage, meniscus, and anterior and posterior cruciate ligaments: a new molecular distinction between hyaline cartilage and fibrocartilage in the knee joint', Connect Tissue Res, 2005, 46, 83±91. van der Kraan P M, Buma P, van Kuppevelt T and van den Berg W B, `Interaction of chondrocytes, extracellular matrix and growth factors: relevance for articular cartilage tissue engineering', Osteoarthritis Cartilage, 2002, 10, 631±7. Review. Vandenabeele F, De Bari C, Moreels M, Lambrichts I, Dell'Accio F, Lippens P L and Luyten F P, `Morphological and immunocytochemical characterization of cultured fibroblast-like cells derived from adult human synovial membrane', Arch Histol Cytol, 2003, 66, 145±53.

38

Regenerative medicine and biomaterials in connective tissue repair

Verdonk P C, Forsyth R G, Wang J, Almqvist K F, Verdonk R, Veys E M and Verbruggen G, `Characterisation of human knee meniscus cell phenotype', Osteoarthritis Cartilage, 2005, 13, 548±60. Verzijl N, DeGroot J, Thorpe S R, Bank R A, Shaw J N, Lyons T J, Bijlsma J W, Lafeber F P, Baynes J W and TeKoppele J M, `Effect of collagen turnover on the accumulation of advanced glycation end products', J Biol Chem, 2000, 275, 39027±31. Walker M R, Patel K K and Stappenbeck T S, `The stem cell niche', J Pathol, 2009, 217, 169±80. Review. Wann A K, Ingram K R, Coleman P J, McHale N and Levick J R, `Mechanosensitive hyaluronan secretion: stimulus±response curves and role of transcription± translation±translocation in rabbit joints', Exp Physiol, 2009, 94, 350±61. Wickham M Q, Erickson G R, Gimble J M, Vail T P and Guilak F, `Multipotent stromal cells derived from the infrapatellar fat pad of the knee', Clin Orthop Relat Res, 2003, 412, 196±212. Williams J T, Southerland S S, Souza J, Calcutt A F and Cartledge R G, `Cells isolated from adult human skeletal muscle capable of differentiating into multiple mesodermal phenotypes', Am Surg, 1999, 65, 22±6. Wilson W, van Donkelaar C C, van Rietbergen B, Ito K and Huiskes R, `Stresses in the local collagen network of articular cartilage: a poroviscoelastic fibril-reinforced finite element study', J Biomech, 2004, 37, 357±66. Wolff J (1892), Das Gesetz der Transformation der Knochen, Berlin, Hirschwald. Yoon J H and Halper J, `Tendon proteoglycans: biochemistry and function', J Musculoskelet Neuronal Interact, 2005, 5, 22±34. Review. Zhang G, Young B B, Ezura Y, Favata M, Soslowsky L J, Chakravarti S and Birk D E, `Development of tendon structure and function: regulation of collagen fibrillogenesis', J Musculoskelet Neuronal Interact, 2005, 5, 5±21. Review. Zheng B, Cao B, Crisan M, Sun B, Li G, Logar A, Yap S, Pollett J B, Drowley L, Cassino T, Gharaibeh B, Deasy BM, Huard J and PeÂault B, `Prospective identification of myogenic endothelial cells in human skeletal muscle', Nat Biotechnol, 2007, 25, 1025±34.

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The myofibroblast in connective tissue repair and regeneration B . H I N Z , University of Toronto, Canada

Abstract: Myofibroblasts contribute to the normal healing of connective tissues such as skin, bone and cartilage and assure tissue integrity by forming a mechanically resisting scar after injury of heart, lung, liver and kidney. Excessive extracellular matrix (ECM) secreting and contractile activities of the myofibroblast result in the development of fibrosis that dramatically impedes normal tissue function. Just like many other cell types, mesenchymal stem cells which are implanted to regenerate tissue are prone to this transformation and are in danger of generating scar instead of functional tissue. This chapter will define the characteristics of the myofibroblast with the ultimate aim of controlling its activity in tissue repair and regeneration. Keywords: -smooth muscle actin, fibrosis, stroma reaction to tumour, transforming growth factor beta (TGF 1), wound healing.

2.1

Introduction

When severely damaged tissues cannot be regenerated by the routine repair mechanism of the body or when physiological healing is imperfect, then regenerative medicine and tissue engineering are considered. Most cell therapeutic applications involve the isolation of autologous regenerative cells that are then expanded in culture and re-implanted with the aim of restoring organ function. Depending on the type and the structure of tissue to repair, different cell delivery strategies have been developed, ranging from direct injection into the damaged tissue (site-directed delivery), infusion by the intravenous and left ventricular pathways (systemic delivery), and delivery in various scaffolds (Caplan, 2007; Clark et al., 2007; Giordano et al., 2007; Karp and Langer, 2007; Lutolf and Hubbell, 2005; Pittenger and Martin, 2004; Robertson et al., 2008; Sands and Mooney, 2007; Segers and Lee, 2008; Weaver and Garry, 2008). In addition to choosing the appropriate delivery strategy, the success of regenerative medicine strongly depends on selecting the right cell type for implantation. The perfect regenerative cell must be able to replicate in culture for rapid cell population expansion and must exhibit (or develop) phenotypic features that are suitable to restore the function of the organ to repair. The `right cell' is not necessarily one that exhibits the required features in the preparative cell culture but the one that

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develops and more importantly keeps these features in the body implant or graft and that attains/retains these features in the body. This is the basic dilemma of stem cell therapy: pluripotency is wanted because it enables stem cells to finally attain the characteristics of cells that construct the normal organ. However, when delivered for organ regeneration, pluripotent cells do not experience the normal organ but the hostile diseased environment which can drive their differentiation toward unwanted and even destructive phenotypes. If regenerative strategies fail, the outcome can be teratoma formation (implanted cells continue to proliferate and become tumorigenic) (Hentze et al., 2007; Rice and Scolding, 2008) or tissue contracture and deformation (implanted cells become fibrogenic). To better understand the challenges that regenerative cells face after engrafting injured organs we have to consider the body's inherent reparative machinery and the cells involved in physiological tissue repair. When organs and tissues are damaged such as after cardiac infarct, skin wounding or bone fracture the inherent repair mechanisms of our body have to fulfil two urgent tasks: (1) establishing tissue haemostasis, fighting inflammation and discarding debris which is carried out by immune and inflammatory cells and (2) providing mechanical tissue coherence by forming a scar which is the task of fibroblasts and so-called myofibroblasts (Baudino et al., 2006; Brown et al., 2005; Desmouliere et al., 2005; Gurtner et al., 2008; Werner and Grose, 2003). The myofibroblast is the most prominent cell phenotype, generated to populate and to repair injured tissues by secreting extracellular matrix (ECM) and organizing this ECM in a contractile process (Desmouliere et al., 2005; Hinz et al., 2007; Tomasek et al., 2002). Myofibroblasts severely impair organ function when their contractile and ECM protein secretory activities become deregulated as is the case in virtually all fibrotic diseases. After briefly summarizing the characteristic features and functions of the myofibroblast I will ask, and at least partly answer, the question of the myofibroblast origin. It becomes increasingly clear that myofibroblasts can arise from a plethora of precursor cells predominantly of mesenchymal but also from ectodermal origin (Hinz et al., 2007). It appears that damaged tissues can recruit myofibroblast precursors from several sources to satisfy the temporarily high demand of cells with tissue remodelling and repair activity. Most clinical approaches implanting differentiated or pluripotent stem cells or introducing biomaterials to engineer damaged and fibrotic tissue have to cope with the special cellular, chemical and mechanical environment created by the myofibroblast. Worse, regenerative cells themselves are at high risk of attaining the fibrogenic myofibroblast character being subjected to the `bad' neighbourhood of the scar. As a consequence, they can switch sides and become the enemies of regenerative medicine and tissue engineering by creating fibrotic scar tissue as opposed to the aim of restoring organ function. To prevent this development it is crucial to understand the general molecular pathways regulating evolution and function of the myofibroblast.

The myofibroblast in connective tissue repair and regeneration

2.2

41

Myofibroblasts: humble tissue construction workers

One has to bear in mind that the aim of the body response to injury, in contrast to regenerative medicine and tissue engineering, is to provide rapid repair even at the expense of losing tissue functionality. Indeed, a considerable level of scarring and fibrosis is required to preserve the mechanical stability of an injured organ against rupture. I will here concentrate on the processes that are involved in providing this mechanical stability and shed light on the major cell phenotype involved, the myofibroblast. The forces leading to remodelling and contraction of damaged tissues are generated within the wounded tissue itself. In the early 1970s Gabbiani and coworkers (1971) identified specialized fibroblasts as the active component in dermal wound contraction, which were named myofibroblasts to account for their ultrastructural similarity to smooth muscle cells (SMC) (Gabbiani et al., 1971). From a historical perspective it is important to point out that at the time of their discovery, the definition of `myofibroblast' was exclusively based on the co-existence of mesenchymal morphological features including a developed endoplasmic reticulum as well as SMC features such as actin filament bundles and contractile activity. Specific molecular markers had only been defined about ten years later. Further morphological features of the myofibroblast are high ECM synthesizing activity, development of cell-to-cell and cell-to-matrix adhesions (fibronexus), and secretion of growth factors (for reviews see Eyden, 2008; Hinz, 2007). Over the last three decades myofibroblasts have been found in a variety of physiological and pathological situations that are characterized by enhanced remodelling and tension production. Myofibroblasts can be of very heterogeneous origins as summarized in Section 2.4; however their development follows a well-established sequence of events. De novo myofibroblast differentiation in response to tissue injury is initiated by changes in the composition, organization and the mechanical properties of the ECM (Hinz and Gabbiani, 2003) and by various cytokines that are released by inflammatory and resident cells (Gurtner et al., 2008; Werner and Grose, 2003). The progress of myofibroblast differentiation can be separated into two main phases that are each characterized by specific cytoskeletal characteristics (Fig. 2.1). First, to re-populate cell-denuded damaged tissue, myofibroblast precursor cells acquire contractile bundles. These in vivo stress fibres generate sufficient forces to pull the cells forward during the migration process and to pre-remodel the ECM (Hinz et al., 2001b). To discriminate such activated and low contractile cells from quiescent fibroblastic cells which are devoid of any contractile features, the term `proto-myofibroblast' was proposed (Tomasek et al., 2002). It has to be noted that in standard culture most fibroblastic cells attain this phenotype by developing stress fibres composed of cytoplasmic actins on the rigid culture plastic surface that represents a considerable mechanical stimulus.

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2.1 The myofibroblast differentiation spectrum. Co-immunostaining for Factin (phalloidin, all lower image parts) and for -SMA (all upper image parts) discriminates between fibroblasts (left), proto- (middle) and differentiated myofibroblasts (right column). All three phenotypes occur sequentially in the granulation tissue of healing rat open wounds (Hinz et al., 2001b), and during the maturation (stiffening) of mechanically restrained collagen gels (Hinz, 2006). Myofibroblasts cultured on silicone culture substrates (elastic modulus indicated in kPa) fully differentiate on stiff, are proto-myofibroblasts on medium stiff and fibroblasts on very soft polymers. Similar control over myofibroblast differentiation is achieved by growing them on microcontactprinted (CP) arrays of very small (1 m length), medium long (4 m) and super-sized (20 m) adhesion islets (Goffin et al., 2006).

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Mechanical stress is also prerequisite for the second stage of myofibroblast differentiation hallmarked by de novo expression of the actin isoform -smooth muscle actin (-SMA) and its incorporation into pre-existing stress fibres (Clement et al., 2005; Goffin et al., 2006; Tomasek et al., 2002). Expression of -SMA discriminates differentiated myofibroblasts from proto-myofibroblasts (Fig. 2.1). Indeed, neo-expression of -SMA by fibroblastic cells is the most widely used criterion to define the differentiated myofibroblast and to diagnose myofibroblast-related diseases. Besides serving as molecular marker, incorporation of -SMA into stress fibres significantly augments the contractile activity of fibroblastic cells and hallmarks the contraction phase of connective tissue remodelling (Hinz et al., 2001a). As elaborated in greater detail in Section 2.6, expression of -SMA is precisely controlled by the joint action of growth factors like transforming growth factor beta 1 (TGF 1), of specialized ECM proteins such as the extra domain A (ED-A) containing fibronectin (FN) splice variant ED-A FN and of the mechanical microenvironment (Tomasek et al., 2002). Under physiological conditions the contractile and secretory activities of myofibroblasts are terminated when the tissue is sufficiently remodelled and repaired. Under most circumstances `sufficient repair' signifies that the damaged tissue regains mechanical coherence but does not necessarily mean the restoration of functionality. At this point -SMA expression becomes down-regulated and myofibroblasts disappear by massive apoptosis, leaving the mature scar behind (Desmouliere et al., 1995). However, in excessive repair myofibroblast activity persists and results in tissue deformation by contracture. This is particularly evident in hypertrophic scars such as those developing after burns (Atiyeh et al., 2005), in scleroderma (Strehlow and Korn, 1998; Varga and Abraham, 2007) and in the palmar fibromatosis of Dupuytren's disease (Tomasek et al., 1999). Myofibroblast-generated contractures are also characteristic for fibrosis affecting vital organs such as liver (Desmouliere et al., 2003; Gressner and Weiskirchen, 2006), heart (Baudino et al., 2006; Brown et al., 2005; Virag and Murry, 2003), lung (Chiappara et al., 2001; Phan, 2002; Thannickal et al., 2004) and kidney (Lan, 2003). Cells with myofibroblastic phenotype further contribute to the development of atheromatous plaques after blood vessel injury (Bochaton-Piallat and Gabbiani, 2006). A number of different biomaterials have been shown to activate macrophages, which in turn contribute to the generation of myofibroblast by producing TGF 1 (Anderson et al., 2008; Li et al., 2007a). Myofibroblasts are further considered to be key elements in creating circumferential tissue constrictions that form around solid body implants (Comut et al., 2000; Suska et al., 2008) and contract silicone breast implants (Coleman et al., 1993; Rudolph et al., 1978; Siggelkow et al., 2003). Finally, myofibroblasts play a role in the process called stroma reaction to epithelial tumours which promotes cancer progression by creating a stimulating microenvironment for the transformed cells (Bhowmick and Moses, 2005; De Wever and Mareel, 2003; Desmouliere et al., 2004). This tumour-

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promoting feature has raised the concept that myofibroblasts as one subpopulation of tumour-associated fibroblasts represent important targets of anticancer treatments (Albini and Sporn, 2007; Mueller and Fusenig, 2004). Given the diversity of organs that can host myofibroblasts it is intuitive to ask where these cells come from ± often with the aim of preventing their formation and destructive accumulation.

2.3

Know thy enemy: a quick guide to identify the myofibroblast

The original and most basic myofibroblast definition describes a cell of mesenchymal character joining fibroblastic and SMC features, which exhibits pronounced actin±myosin filament bundles and contractile activity. Cells missing these features cannot be functional myofibroblasts. On the other hand, no molecular marker has been described that unmistakably identifies a cell as being myofibroblast. One way to approach the problem of its identification is to consider that myofibroblasts are rarely present in normal tissue and predominantly arise in pathological and physiological repair processes. Under most circumstances, myofibroblasts differentiate from other cell types by de novo expressing stress fibres and -SMA (the exception of SMC will be discussed below) and can retain features of their precursors (Section 2.4). A warrant of typical proteins expressed by differentiated myofibroblasts and characteristic morphological features has been summarized elsewhere (Eyden, 2007, 2008; Hinz, 2007; Schurch et al., 2007); here I want to shed light from a slightly different angle: against which cell type do you want to discriminate the myofibroblast? Distinction from epithelial cells is straightforward by assessing expression of mesenchymal markers that are not expressed in epithelium, in particular the cytoskeletal protein vimentin. Generally, -SMA can be used as discriminator with the exception of myoepithelial cells that are also -SMA-positive. Myoepithelium however is negative for vimentin and in contrast to myofibroblasts expresses keratins, E-cadherin and desmoplakin (Bissell and Radisky, 2001; Lazard et al., 1993; Savera and Zarbo, 2004). Another special case is transformed epithelium that can lose epithelial characteristics and acquires mesenchymal features during epithelial-to-mesenchymal transition (EMT) (Kalluri and Zeisberg, 2006). The fraction of these cancer-derived cells that further differentiate to express -SMA are functionally and morphologically myofibroblasts. Once one has defined the criteria for the fibroblast (which is a daunting task in itself) the detection of -SMA-positive stress fibres suffices to distinguish fibroblasts from differentiated myofibroblasts. The same criterion can apply to sort myofibroblasts from other normal connective tissue cells, including chondrocytes and osteoblasts (Spector, 2001) as well as endothelial cells that all express vimentin. In addition, differentiated myofibroblasts in vivo and in

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vitro can de novo express the cell±cell contact protein OB-cadherin that has not yet been reported on the surface of -SMA-negative fibroblasts (Hinz et al., 2004). In strict terms and according to the original definition of the myofibroblast, the development of -SMA-negative contractile stress fibres alone is a hallmark of myofibroblast differentiation because normal fibroblasts are noncontractile (Gabbiani et al., 1971). If both phenotypes co-exist in vivo, such as during dermal wound healing (Hinz et al., 2001b) and in lung alveolar septa in the context of lung fibrosis (Kapanci et al., 1990), `proto-myofibroblast' (SMA-negative stress fibres) and `differentiated myofibroblast' (-SMA-positive stress fibres) are the appropriate terms (Tomasek et al., 2002). In standard cell culture, in which fibroblasts inevitably form stress fibres, `myofibroblast' generally describes only the -SMA expressing cells. It is tedious to discriminate between myofibroblasts and SMC, for which SMA obviously fails to make the distinction (Fujimoto and Singer, 1987). In normal adult tissue, SMC express a number of late differentiation markers that are usually not in the repertoire of the myofibroblast, including smooth muscle myosin heavy chain (Benzonana et al., 1988), h-caldesmon (Eyden, 2007, 2008), and smoothelin (van der Loop et al., 1996). The muscle intermediate filament protein desmin is an often reliable and widely used exclusion criterion but can be expressed in myofibroblasts in some particular conditions (Hinz et al., 2001b; Skalli et al., 1986). Moreover, SMC normal vessels tissue do not only express desmin but also (or only) vimentin; both intermediate filament proteins are therefore not reliable markers for one or the other cell type (Frank and Warren, 1981; Gabbiani et al., 1981). Equally problematic is the differentiation between myofibroblasts and pericytes if only molecular markers are considered. Depending on their tissue location pericytes can express vimentin and desmin, are -SMA positive and smooth muscle myosin negative (Armulik et al., 2005; Eyden, 2007; Hughes, 2008). However, pericytes are characterized by their close interaction with endothelial cells and lack of contractile features in normal tissues (Eyden, 2007). Discriminating myofibroblasts and SMC as well as pericytes becomes practically impossible in conditions of smooth muscle injury and in cell culture, in particular if the provenance of the cells is unclear. For example, remodelling of injured arteries is thought to be predominantly driven by SMC from the media, de-differentiating into myofibroblasts but the contribution from adventitial fibroblasts to the myofibroblast population has also been suggested (BochatonPiallat and Gabbiani, 2006; Hao et al., 2006; Sartore et al., 2001; Zalewski et al., 2002). Similar uncertainty exists in explant and digestion cultures of tissue containing both connective tissue and smooth muscle. As during arterial remodelling, SMC here lose their late differentiation markers desmin, smooth muscle myosin and smoothelin and acquire a myofibroblastic and synthetic phenotype (Benzonana et al., 1988; Christen et al., 2001; Larson et al., 1984). On the other hand, gene expression profiling supported by protein biochemistry

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demonstrated that some of the late SMC markers are expressed in fibroblasts after treatment with TGF 1, the most potent myofibroblast inducer (Chambers et al., 2003; Malmstrom et al., 2004). Hence, considering the expression profile of cytoskeletal proteins, the differentiated myofibroblast appears to exist in a continuous spectrum between fibroblasts and SMC. On this background, it seems appropriate to regard the myofibroblast as a phenotype rather than a cell type. Ultimately, one will have to determine what is more important: what a particular cell represents at the time of being assessed or where it came from.

2.4

Origins of the myofibroblast

2.4.1

Local precursors of the myofibroblast

Myofibroblasts are recruited from a variety of precursor cells whose nature depends on the injured tissue and the microenvironment (Hinz et al., 2007) (Fig. 2.2). In many organs, locally residing fibroblasts are considered to be the major source of -SMA positive myofibroblasts in response to injury and development of fibrosis, such as in the skin (Gabbiani, 2003; Hinz, 2007), in fibrotic reactions to body implants (Ariyan et al., 1978; Suska et al., 2008), in liver (Li et al., 2007b; Ramadori and Saile, 2004), kidney (Desmouliere et al., 2003; Qi et al., 2006), and in the stroma reaction to epithelial tumours (De Wever and Mareel, 2002; Desmouliere et al., 2004). EMT is another mechanism to generate fibroblasts and myofibroblasts from epithelial and endothelial precursors in tumour development (Kalluri and Zeisberg, 2006; Thiery, 2002), as well as in kidney fibrosis (Iwano et al., 2002; Kalluri and Neilson, 2003) and possibly lung fibrosis (Chilosi et al., 2003; Kim et al., 2006). EMT has further been demonstrated to contribute to fibrosis of heart (Zeisberg et al., 2007a) and liver (Zeisberg et al., 2007b). In fibrotic liver, hepatic stellate cells are another important source for myofibroblasts (Bataller and Brenner, 2005; Friedman, 2004b; Guyot et al., 2006). De-differentiation of SMC contributes to the generation of myofibroblasts in atheromatous plaques (Bochaton-Piallat and Gabbiani, 2006; Hao et al., 2006). In systemic sclerosis, vessel repair and dermal scarring, pericytes have been suggested to attain contractile myofibroblast features (Rajkumar et al., 2006; Sundberg et al., 1996). Although the repair processes of injured brain exhibit many specific features compared with other organs, astrocytes seem to develop a myofibroblastic phenotype in the glial scar (Moreels et al., 2008; Silver and Miller, 2004).

2.4.2

The circulating fibrocyte, another myofibroblast precursor ± or not?

Another source for reparative fibroblasts that has attracted great interest over the last decade is the so-called fibrocyte, a bone-marrow (BM) derived circulating

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2.2 Myofibroblast precursor cells. Locally residing fibroblasts appear to be the main myofibroblast progenitors in most tissues. In the liver, myofibroblasts are additionally recruited from hepatic stellate cells (HSC). In tumours and in fibrotic liver, lung, and kidney, differentiated myofibroblasts can arise from epithelial- and endothelial-to-mesenchymal transition (EMT). During atheromatous plaque evolution, myofibroblasts arise from de-differentiating smooth muscle cells (SMC). The relative contribution of bone marrow-derived circulating fibrocytes to the formation of differentiated myofibroblasts is unclear at present. Finally, MSC have been shown to acquire the differentiated myofibroblast phenotype in vitro and in vivo.

cell that is recruited to sites of organ injury, inflammation and fibrosis (Abe et al., 2001) (Fig. 2.2). Fibrocytes have been first characterized by the coexpression of fibroblast markers collagen types I and II and fibronectin, of monocyte markers CD13 (aminopeptidase N) and CD11b (M integrin), and haematopoietic progenitor markers CD34, CD45 (protein tyrosine phosphatase receptor type C) and CD105 (endoglin) (Bucala et al., 1994); a more complete characterization has recently been reviewed (Bellini and Mattoli, 2007; Metz, 2003). Early works estimated that fibrocytes make up 0.1±0.5% of the nonerythrocytic cell population circulating in the peripheral blood (Bucala et al., 1994), whereas later studies suggested that fibrocytes do not circulate in their mature form but differentiate from mononuclear precursor cells (Bellini and Mattoli, 2007; Gordon and Taylor, 2005). Mononuclear precursors of the fibrocyte express the CC chemokine receptor (CCR) CCR2 that is implicated in their recruitment to different tissues (Gordon and Taylor, 2005; Moore et al.,

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2005) and that CCR2 becomes down-regulated during fibrocyte maturation at the tissue site (Abe et al., 2001). It is believed that the fibrocyte is a mandatory differentiation stage before attaining the fibroblastic phenotype in the target tissue (Bellini and Mattoli, 2007; Mattoli et al., 2009) (Fig. 2.2). In culture, fibrocyte-to-myofibroblast differentiation can occurs spontaneously to a certain extent and is inducible with endothelin-1 and TGF 1 (Hong et al., 2007; Schmidt et al., 2003). Up-regulation of myofibroblast markers including -SMA is correlated with down-regulation of the haematopoietic markers CD34 and CD45 (Schmidt et al., 2003). In an animal model of wound healing, loss of CD34 and CD45 fibrocyte markers occurs within hours of fibrocyte recruitment/maturation in inflamed tissue and renders it difficult to determine whether fibrocytes as such are able to differentiate into the myofibroblast in vivo or whether they preferentially localize to sites of myofibroblast accumulation (Mori et al., 2005; Quan et al., 2006). Leukocytespecific protein-1 (LSP-1) has been identified as another fibrocyte marker (Yang et al., 2002). Fibrocyte-derived fibroblastic cells were shown to express LSP-1 up to two months, when CD45 expression is mostly lost (Phillips et al., 2004; Wu et al., 2007b). It remains to be shown whether LSP-1 positive cells can co-express SMA or whether they play a supportive role. Indeed, fibrocytes were shown to promote myofibroblast differentiation from resident fibroblast during the healing of burns by secreting specific growth factors (Wang et al., 2007). To solve the question of whether fibrocytes can differentiate into -SMApositive myofibroblasts, a number of studies used irradiated wild-type mice, engrafted with BM obtained from GFP-expressing transgenic mice or from sexmismatched animals. BM-derived cells were associated in varying numbers with myofibroblast-containing lesions in animals subjected to fibrotic stimuli in different organs (Andersson-Sjoland et al., 2008; Forbes et al., 2004; Haudek et al., 2006; Ishii et al., 2005; Kisseleva et al., 2006; Phillips et al., 2004; Sakai et al., 2006; Schmidt et al., 2003; Wada et al., 2007), during vascular remodelling (Frid et al., 2006; Varcoe et al., 2006), in the context of tumour development (Direkze et al., 2004; Ishii et al., 2005), in lung fibrosis (Moeller et al., 2009), in chronic asthma (Wang et al., 2008) and following dermal wounding (Direkze et al., 2003; Fathke et al., 2004; Mori et al., 2005). On the basis of the used markers it is also possible that fibrocytes are involved in cardiac repair and pathogenesis of atherosclerosis (Fujita et al., 2007; Sata et al., 2002). One study has demonstrated that CD13/collagen I-positive fibrocytes isolated form wound granulation tissue contain BM-derived cells as well as cell populations expressing CD34, CD45 and -SMA; however, these markers were assessed separately (Mori et al., 2005). Because most studies did not simultaneously evaluate BM origin, the fibrocyte character and -SMA expression, fibrocyteto-myofibroblast differentiation in vivo is still a matter of debate (Hinz, 2007). Indeed, some studies appear to rule out that BM-derived fibrocyte progenitors contribute to myofibroblast formation in liver (Kisseleva et al., 2006) and lung

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fibrosis (Hashimoto et al., 2004); in the latter, myofibroblast differentiation from fibrocytes was inducible by TGF 1 in culture. Fibrocytes are not the only cell types that originate from BM precursors to possibly giving rise to myofibroblasts. BM contains a small fraction of -SMA expressing cells that were considered as being myofibroblasts (Peled et al., 1991; Schmitt-Graff et al., 1989). Using a transgenic mouse that expresses GFP under the control of the -SMA promoter it was suggested that these cells are part of the stromal compartment of the BM with the capacity to circulate in the peripheral blood. These cells were not of hematopoietic character; however, fibrocyte (precursor) marker expression has not been assessed (Yokota et al., 2006). Another possibility is that these BM-derived cells represent a circulating fraction of mesenchymal stem cells (MSC) (Roufosse et al., 2004). Because a number of animal experiments and clinical trials have demonstrated the potential of MSCs to treat human diseases as a cell therapy and in tissue engineering, this cell type will be discussed in greater detail below.

2.5

Mesenchymal stem cells (MSC) and the myofibroblast phenotype: regeneration, repair or risk?

2.5.1

The regenerative potential of MSCs

For regenerative medicine, the population of BM-derived multipotent mesenchymal stromal cells is of major interest (Bianco et al., 2001; He et al., 2007). In culture these cells acquire a fibroblastic character which has been defined as colony-forming unit fibroblast (CFU-F) (Bianco et al., 2001; Friedenstein et al., 1970). The clonogenic cells among the CFU-Fs which exhibit self-renewal and multilineage differentiation character are generally referred to as BM-MSCs. It has to be noted that the stem cell identity of BM-MSC, i.e. the potential for selfrenewal, multilineage differentiation and reconstitution of functional tissue in vivo (Verfaillie, 2002), is not always consequently tested in studies that report to work with this cell type. For this reason, some authors prefer to use the term `mesenchymal precursor cell' instead (Roufosse et al., 2004). BM-MSC currently serve as the major source for experimental and clinical purposes (Caplan, 1991; Chamberlain et al., 2007; Giordano et al., 2007; Pittenger et al., 1999; Pittenger and Martin, 2004; Segers et al., 2006). Other MSC sources are umbilical cord blood (Erices et al., 2000; Lee et al., 2004), adipose tissue (Aust et al., 2004; Zuk et al., 2001), pancreas (Seeberger et al., 2006), pleural cavity (Metcalf, 1972), muscle and brain (Jiang et al., 2002), connective tissue of dermis and skeletal muscle (Young et al., 2001), exfoliated deciduous teeth, and the eye conjunctiva (Nadri et al., 2008). As stated above, MSC have also been suggested to circulate in the peripheral blood but this population is very difficult to identify and/or purify and their contribution to

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tissue regeneration is debated (Fernandez et al., 1997; Orlic et al., 2001b; Roufosse et al., 2004). It is believed that circulating MSC are capable of specifically targeting and entering sites of tissue injury (Fox et al., 2007). MSC in culture are generally identified by the expression of specific cell surface proteins such as CD105, CD90 (Thy1), CD44 (HA receptor), CD73 (SH3 or SH4), CD166 (ALCAM), CD29 ( 1 integrin), CD106 (VCAM) and Stro-1 (Minguell et al., 2001; Pittenger et al., 1999; Simmons and Torok-Storb, 1991). Hence, MSC are different from fibrocytes by being negative for monocyte surface proteins (CD13, CD11b) and haematopoietic markers (CD34, CD45) (He et al., 2007; Pittenger et al., 1999). MSC can differentiate into a variety of cell types that are of potential use for regenerative medicine and tissue engineering (Caplan, 2007); these include chondrocytes and osteocytes for cartilage and bone reconstitution (Bruder et al., 1998; Horwitz et al., 1999; Kadiyala et al., 1997; Noel et al., 2002; Pittenger et al., 1999), myoblasts for skeletal muscle repair (Ferrari et al., 1998), hepatocytes for liver regeneration (Petersen et al., 1999), cardiomyocytes and SMC to repair the cardiovascular system and to promote neo-vessel formation (Chen et al., 2004; Giordano et al., 2007; Orlic et al., 2001a; Psaltis et al., 2008; Rafii and Lyden, 2003; Strauer et al., 2002), and even neuronal cells to treat neurological disorders (Mezey et al., 2000; Picinich et al., 2007). MSC give rise to fibroblasts that can regenerate soft connective tissue (Pittenger and Marshak, 2001; Young et al., 1998) and tendon (Butler et al., 2008) and that are potential candidates for supporting skin repair after wounding (Wu et al., 2007b). In the context of wound healing, MSC-to-epidermal cell differentiation seems to be possible (Deng et al., 2005; Nakagawa et al., 2005; Sasaki et al., 2008; Wu et al., 2007a). In addition to directly regenerating tissue and organs, MSC can exhibit properties that modulate the body-inherent repair processes. Directly injected BM-MSC contribute to skin regeneration after wounding and supernatants from these cells stimulate tube formation of vascular endothelial cells and recruitment of macrophages to the wound, suggesting positive paracrine effects (Chen et al., 2008; Wu et al., 2007a). In co-culture, paracrine actions of BM-MSC stimulate proliferation and differentiation of skin epidermal cells (Aoki et al., 2004). Moreover, MSC are able to migrate to sites of damaged tissues where their immunosuppressive and immunomodulatory properties improve the tissue transplantation success (Le Blanc and Pittenger, 2005; Uccelli et al., 2008). Finally, MSC are immunologically immature and do not elicit inflammatory responses, which extends their possible use to gene delivery (Chamberlain et al., 2007; Pereboeva et al., 2003).

2.5.2

MSC-to-myofibroblast differentiation

Many of the potential therapeutic applications that have been proposed for MSC imply their engraftment into fibrotic tissue. In chronic fibrosis the high ECM

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secreting and contractile activities of endogenous myofibroblasts destroy the functional architecture of the remaining healthy tissue. Even if progression of fibrosis could be prevented, no efficient treatment exists to reverse this destruction and to reconstruct normal tissue. This is particularly evident in cardiac fibrosis following infarct and in pulmonary fibrosis (Gauldie et al., 2007; Phan, 2002; Wynn, 2008). MSC therapy has been proposed to regenerate fibrotic tissue (Ortiz et al., 2003) but it is presently unclear whether transplantation of undifferentiated MSCs can possibly fulfil this task. A number of recent findings rather suggest that the diseased microenvironment will drive regenerative MSC into fibrogenic myofibroblasts that will further distort the present ECM. What is the evidence for MSC-to-myofibroblast differentiation? One of the defining features of MSC in cell culture is their well-spread morphology and the formation of contractile stress fibres (Pittenger et al., 1999), which fulfils the criteria for the proto-myofibroblast phenotype (Tomasek et al., 2002). A number of studies have further reported spontaneous development of the differentiated myofibroblast phenotype in cultured BM-MSC, evidenced by de novo expression of -SMA and up-regulated contractile activity (Bonanno et al., 1994; Cai et al., 2001; Kinner et al., 2002b; Peled et al., 1991) (Fig. 2.2). The percentage of BM-MSC spontaneously expressing -SMA is gradually increasing with long-term culture (Charbord et al., 1985; Galmiche et al., 1993; Yokota et al., 2006). Moreover, MSC from different origins have been shown differentiate into myofibroblasts in response to stimuli that are known inducers of -SMA in fibroblastic cells. Proteomic profiling of BM-MSC revealed a myofibroblast differentiation program upon treatment with TGF 1 (Wang et al., 2004). Similarly, MSC derived from human adipose tissue de novo express -SMA upon treatment with lysophosphatic acid and with bradykinin in TGF 1 and Smad2/3 dependent manner (Jeon et al., 2008; Kim et al., 2008). Treatment with basic fibroblast growth factor (FGF-2) (Hankemeier et al., 2005) as well as application of cyclic mechanical strain (Kobayashi et al., 2004) induced -SMA expression in cultured BM-MSC and MSC differentiate into myofibroblasts in chondrogenic conditions (Hung et al., 2006). In co-culture with PDGF-Bactivated fibroblasts, BM-MSC are specifically recruited and differentiate into myofibroblasts in a process involving FGF-2 and CXCL5 (Nedeau et al., 2008). Application of mechanical stimuli drives MSC along a myogenic lineage, including expression of -SMA and other markers of the myofibroblast (Park et al., 2007). It has to be noted that not all -SMA-positive MSC in culture are necessarily myofibroblasts but may represent presumptive stages of differentiation into SMCs or pericytes (Bianco et al., 2001; Charbord et al., 1990). This distinction, however, is difficult to make because SMC attain the myofibroblast phenotype in culture (Bochaton-Piallat et al., 1992) and myofibroblasts can extend to the very far spectrum of SMC differentiation upon treatment with TGF 1 (Chambers et al., 2003).

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The threat of MSC-to-myofibroblast differentiation for regenerative medicine

What is known about the significance of MSC-to-myofibroblast differentiation in vivo? Recent studies evaluating the interaction between MSC and epithelial tumour cells in vivo and in vitro indicate that acquisition of the myofibroblast phenotype by MSC can reduce the success of the envisaged therapy and can even amplify the disease. Different groups have shown that systemically transplanted MSC target to the stroma environment of epithelial tumours (Hall et al., 2007; Hung et al., 2005; Menon et al., 2007; Studeny et al., 2004). This specific homing together with the immuno-inactivity of MSC was suggested to be useful for the tumour-specific delivery of anti-cancer drugs, cytokines and viruses (Komarova et al., 2006; Studeny et al., 2002) and to suppress tumour development as such (Khakoo et al., 2006). On the other hand, the tumour appears to activate MSC-to-myofibroblast differentiation in a fashion similar to carcinomaassociated fibroblasts (Mishra et al., 2009). BM-MSC cultured in tumourconditioned medium were shown to differentiate into -SMA-positive myofibroblasts (Mishra et al., 2008). MSC that have become activated by mildly invasive human breast carcinoma cells in culture enhance the metastatic potential of the cancer cells when injected subcutaneously; this effect is mediated in a feedback paracrine loop (Karnoub et al., 2007). Another detrimental effect of MSC-to-myofibroblast differentiation has been observed for organs that are sought to be regenerated with MSC therapy. Schneider and coworkers have tested the stability of the MSC character in a three-dimensional culture model of air-exposed dermal equivalent, similar to those used in skin tissue engineering (Schneider et al., 2008). In these keratinocyte-promoting growth conditions MSC did not trans-differentiate along the anticipated epithelial lineage but instead developed into fibrogenic myofibroblasts that contracted the ECM. Similarly, the high contractile activity of myofibroblastic MSC has been shown to deform scaffold matrices that are used to deliver MSC for musculoskeletal tissue engineering purposes (Kinner et al., 2002b). Excessive myofibroblast activity has further been demonstrated to have detrimental effects for the healing of these tissues (Premdas et al., 2001) although the in vivo function of myofibroblastic MSC in these conditions has not been tested yet. Yan and collaborators analysed the success of mouse BM-MSC transplantation as a treatment of lung injury induced by irradiation (Yan et al., 2007). MSC injected into the injured lung immediately after irradiation differentiated into functional lung epithelial and endothelial cells. In contrast, MSC injected 2 months after irradiation when fibrosis has developed, cells appeared as -SMApositive `myofibrocytes' that were involved in fibrosis progression (Yan et al., 2007). Similarly, injection of MSC into mice was shown to improve the outcome of acute renal injury, presumably by restoring the glomerular basement

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membrane but not of the interstitial fibrosis following chronic renal disease (Ninichuk et al., 2006). Likewise, in a model of acute liver injury and fibrosis in immunodeficient mice, systemically transplanted human BM-MSC were shown to engraft in high numbers in the damaged liver. However, the wanted differentiation into hepatocytes occurred only at very low rates whereas most cells of human origin developed a myofibroblastic phenotype (di Bonzo et al., 2008). It appears likely that these cells will rather contribute to fibrosis than to regenerate the liver. Concomitantly, MSC reduced development of liver fibrosis when systemically transfused immediately after inducing fibrosis but not when delivered after 1 week of fibrosis development (Fang et al., 2004). Another major application of MSC is the potential repair of infarcted heart which already entered advanced clinical trials with variable success rates (Chamberlain et al., 2007; Giordano et al., 2007; Laflamme and Murry, 2005; Pittenger and Martin, 2004; Prockop and Olson, 2007; Segers et al., 2006; Shake et al., 2002). It has been acknowledged that the hostile environment of the infarcted heart, including ischaemia, inflammation and fibrosis is one important factor that reduces the success of endogenously and exogenously promoted heart regeneration (Segers and Lee, 2008). Fibrosis has been identified as a strong physical barrier for the entry of regenerative cells in zebrafish heart regeneration (Poss et al., 2002) and the fibrotic environment was shown to modulate regenerative cells in unwanted ways. BM-derived stem cells injected into the myocardial scar after infarct can lead to myocardial calcifications (Breitbach et al., 2007). On the other hand, MSC delivered to acutely infarcted heart were shown to attenuate development of fibrosis, presumably by exerting `trophic' paracrine effects that reduce myofibroblast differentiation of cardiac fibroblasts (Caplan and Dennis, 2006; Nagaya et al., 2005; Ohnishi et al., 2007). This effect could underlie the observation that injection of MSC early after cardiac infarct reduces the stiffness of the scar which should reduce its physical barrier function; softer environment is expected to reduce myofibroblast differentiation as discussed further below (Berry et al., 2006). In summary, is it conceivable that the timing of MSC transplantation is critical for the success of the regenerative therapy; when engrafting immediately after organ damage before the onset of fibrosis the local microenvironment will be very different from later fibrotic stages. In light of the fact that most patients are first seen by a clinician when lung and liver fibrosis has already progressed, transplanting MSC without additional measures into these organs appears a difficult and even dangerous strategy. This situation is different in the case of cardiac fibrosis following myocardial infarction because the onset of the damage is generally known.

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Possible benefits of MSC-to-myofibroblast differentiation for regenerative medicine

From the studies above it is amply clear that MSC have great potential to improve tissue regeneration when transplanted under the right conditions but can become detrimental when differentiating into fibrogenic cells. The question remains whether MSC-to-myofibroblast differentiation can exert beneficial actions and actually support tissue reconstruction. In a rabbit model of corneal wound healing, systemically transplanted BM-MSC were shown to home to the injured site and to improve corneal wound healing by differentiating into myofibroblasts (Ye et al., 2006). Similarly, GFP-expressing BM-MSC topically applied to open rat wounds differentiate into granulation tissue myofibroblasts and accelerated the wound healing progress (Yamaguchi et al., 2005). In a mouse skin wound model, intravenously injected BM-MSC were shown to recruit to the wound site and to accelerate wound healing by differentiating into different cell types, including -SMA-positive cells (Sasaki et al., 2008). Because rodents do not develop hypertrophic scars it is difficult to predict if such a treatment will also improve the quality of human skin wound healing. In conditions of experimental colitis, BM-derived MSC were shown to contribute to gut repair by replenishing the population of pericryptal myofibroblasts (Brittan et al., 2002, 2005). MSC-to-myofibroblast differentiation may also play a beneficial role for the repair of certain musculoskeletal tissues. Differentiated chondrocytes and osteoblasts, the cells involved in physiological cartilage and bone repair, pass over a controlled myofibroblast-stage which supports the healing process by priming the specific fibrous tissue architecture (Kinner et al., 2002a; Spector, 2001; Wang et al., 2000). Chondrocytes expressing -SMA populate the superficial layer of articular cartilage which is suggested to protect cartilage from damage (Kim and Spector, 2000). A similar role in preserving cartilage-like structure has been demonstrated for MSC subjected to chondrogenic differentiation in culture (Hung et al., 2006). MSC grown in chondrogenic culture in the presence of TGF 1 generated cartilagelike pellets with an annular surface that was populated by -SMA-positive cells. In contrast, removal of TGF 1 and consequent loss of the myofibroblast phenotype resulted in the loss of the structural integrity of the pellets (Hung et al., 2006). The question of whether the MSC-to-myofibroblast differentiation is beneficial or detrimental will depend on the specific repair demands in a given tissue and on the nature of the tissue itself. It appears that acute repair of connective tissue can be supported by MSC-derived myofibroblasts whereas myofibroblastic MSC will not directly contribute to the regeneration of other organs. In either case endogenous control over myofibroblast development is desired which requires profound knowledge of the control mechanisms and factors involved.

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What drives myofibroblast differentiation?

To therapeutically counteract organ dysfunction caused by myofibroblasts it is crucial to understand the general molecular pathways regulating their evolution and function. I will here concentrate on the mechanisms that regulate myofibroblasts independently from a specific organ, disease or myofibroblast origin.

2.6.1

Mechanical stress regulates myofibroblast development

It emerges as a common theme that mechanical stress is one of the most potent factors controlling myofibroblast fate and development from different precursor cells (Fig. 2.1). After injury of connective tissues, mechanical stress results from the partial or total loss of the mechano-protective ECM architecture and the residing cells are directly exposed to the stress. To resist the mechanical load arising during tissue repair and remodelling processes and to prevent tissue rupture myofibroblast precursors develop tension on their own by building up mechano-resistant stress fibres. The ultimate goal of myofibroblast activity is to restore the mechanical integrity of the tissue by secreting and organizing new ECM, a process that is precisely controlled by mechanical feedback signals from the ECM, such as ECM stiffness. What is a `stiff' ECM in a physiological and pathological sense and how does ECM stiffness develop during tissue repair and remodelling? The stiffness of the provisional ECM laid down after acute wounding is low, with an elastic modulus (the physical unit of compliance) of ~100±1000 Pa. Fibroblastic cells subjected to similar mechanical conditions in vitro by growth on soft two-dimensional polyacrylamide gels do not develop stress fibres (Discher et al., 2005; Tamariz and Grinnell, 2002; Yeung et al., 2005) (Fig. 2.1). Hence, acquisition of the proto-myofibroblast phenotype and consequently of the differentiated myofibroblast is suppressed on soft substrates. In contrast, SMA-negative stress fibres start to develop on increasingly stiff twodimensional culture substrates exhibiting an elastic modulus of 3000±6000 Pa (Yeung et al., 2005). The threshold ECM stiffness for occurrence of -SMA in stress fibres ranges around 20 000 Pa as demonstrated for myofibroblasts cultured on silicone surfaces with tunable stiffness (Goffin et al., 2006) (Fig. 2.1). A comparable ECM stiffness of ~15 000 Pa activates hepatic stellate cells into -SMA-positive myofibroblasts in the appropriate growth conditions (Wells, 2005). The synthetic polyacrylamide and silicone substrates used in the studies above provide stable mechanical growth conditions; in contrast, reparative cells in vivo change and stiffen their own mechanical microenvironment. This situation can partly be reproduced in vitro using three-dimensional collagen gels with different stiffness and under different mechanical constraints (Grinnell, 2003). In mechanically unrestrained and/or newly polymerized collagen gels fibro-

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blastic cells attain a stellate-like morphology and do not develop contractile features (Grinnell et al., 2003; Hinz, 2006). Resulting from the cell remodelling activity ECM stiffness increases over time which is reflected by the de novo development of stress fibres within hours of remodelling of anchored gels (Fig. 2.1) (Hinz, 2006). Exertion of cellular force in this phase is appreciated from the increasing ECM organization level in mechanically restrained (anchored) gels and from gel size reduction in unloaded (free floating) gels (Grinnell, 2000). Full myofibroblast differentiation, i.e. expression of -SMA, requires an even higher level of collagen gel stiffness that builds up with increasing culture time. Importantly, even the strongly pro-fibrotic cytokine TGF 1 is not effective to promote myofibroblast differentiation in soft substrate conditions (Arora et al., 1999; Goffin et al., 2006) (Fig. 2.1). A similar sequence of events that is linked with increasing ECM stiffness and stress has been demonstrated for in vivo healing of full thickness dermal wounds. Fibroblasts populating very early wound granulation tissue (1±3 days postwounding) are devoid of stress fibres (Hinz et al., 2001b) but develop actin filament bundles in 5±6-day-old wound granulation tissue (Hinz et al., 2001b). Expression of -SMA and occurrence of differentiated myofibroblasts is not observed before day 8 in experimental rat wounds (Darby et al., 1990; Hinz et al., 2001b), when ECM stiffness reaches values around 30 000 Pa (Goffin et al., 2006) (Fig. 2.1). Preventing wound closure by mechanically splinting the edges of experimental wounds accelerates expression of -SMA compared with normally healing wounds; stress release by removing the splint leads to reduced -SMA expression (Hinz et al., 2001b). In other fibrotic tissues and in granulation tissue toward the end of wound healing ECM stiffness values of greater than 50 000 Pa have been measured (Goffin et al., 2006). Increased stiffness of a damaged organ is not necessarily associated with scar formation but can precede and promote fibrosis as shown after liver injury (Georges et al., 2007). The mechanisms and intracellular signalling pathways through which tension controls -SMA transcription appear to involve Rho/Rho-associated kinase and have been reviewed elsewhere (Wang et al., 2006; Zhao et al., 2007). In addition to being regulated on the expression level, -SMA is considered as mechanosensitive protein. Reducing stress fibre tension by reducing substrate stiffness and/or by inhibiting intracellular contraction first results in the selective removal of -SMA from persisting stress fibres (Goffin et al., 2006) (Fig. 2.1). The fact that -SMA only localizes to stress fibres under significant mechanical load is believed to provide a mechanism for rapidly controlling myofibroblast contractile function (Goffin et al., 2006; Hinz, 2006). This cellular control mechanism provides a possible target to therapeutically counteract myofibroblast function and progression in excessive tissue contractures as elaborated in Section 2.7.

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Latent TGF 1: pro-fibrotic cytokine with mechanosensory function

TGF 1 is the most potent myofibroblast-inducing factor and one of the strongest pro-fibrotic cytokines presently known. TGF 1 exerts its pro-fibrotic activities by mediating the inflammatory response, causing excessive ECM production, increasing the synthesis of tissue inhibitors of metallo-proteinases (TIMPs), decreasing protease synthesis and finally by inducing myofibroblast differentiation (Desmouliere et al., 1993; Grainger, 2007; Hinz, 2007; Leask and Abraham, 2004; Ruiz-Ortega et al., 2007; Taipale et al., 1998). The fact that cancer cells become insensitive to the growth-arresting action of TGF together with its proangiogenic effects and its potential to inducing EMT of cancer cells allocates TGF 1 also a central role in tumour development (Bierie and Moses, 2006; Pardali and Moustakas, 2007; Siegel and Massague, 2003). Because TGF 1 signalling also assures homeostasis of adult tissues by controlling proliferation of epithelial cells, endothelial cells, immune cells and fibroblasts (Feng and Derynck, 2005; ten Dijke and Arthur, 2007; Wakefield and Stuelten, 2007), global inhibition of TGF 1 is problematic as anti-fibrotic therapeutic strategy with many uncontrollable side-effects. On the other hand, the complex and diverse mechanisms leading to the activation of latent TGF 1 potentially provide the means for a cell-specific inhibition of TGF 1 action. Activation of latent TGF 1 requires its dissociation from the latency associated peptide (LAP) that is co-synthesized in complex with TGF 1 (Annes et al., 2003). The vast majority of cell types secrete TGF 1 as part of a large latent complex, consisting of TGF 1, LAP and the latent TGF 1 binding protein (LTBP-1) (Annes et al., 2003; Todorovic et al., 2005) (Fig. 2.3a). LTBP-1 is a member of the fibrillin family of ECM proteins that binds to several other ECM components, including fibrillin-1, FN, and vitronectin, thereby providing a reservoir of latent TGF 1 in the ECM (Annes et al., 2003; Todorovic et al., 2005). Activation of latent TGF 1 by its dissociation from LAP is promoted by various mechanisms which differ according to the cell type and the physiological context. Latent TGF 1 activation occurs upon proteolytic cleavage, by interaction with thrombospondin 1 and with the mannose-6-phosphate receptor (Annes et al., 2003; Jenkins, 2008) (Fig. 2.3b). Moreover integrins, as transmembrane components of cell-ECM adhesions, have been reported to play a major role in activating latent TGF 1 (Sheppard, 2005; Wipff and Hinz, 2008). Two principal mechanisms have been proposed and are experimentally supported how integrins can activate a growth factor. The first mechanism is sensitive to protease inhibitors and proposes integrins as common docking point for latent TGF 1 and its activating proteases. The second mechanism is independent from any proteolytic action and involves cell traction forces which are directly transmitted to the large latent complex via integrins (Jenkins, 2008; Sheppard, 2005; Wipff and Hinz, 2008) (Fig. 2.3c).

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2.3 Activation of latent TGF 1. (a) TGF 1 is secreted in a ECM-bound large latent complex (LLC), consisting of TGF 1 and the latency associated peptide (LAP) that form the small latent complex (SLC), and the latent TGF 1-binding protein (LTBP-1. (b) Integrin-independent proteolytic activation of latent TGF 1 occurs at specific sites that are sensitive to proteolytic digestion (scissors), leading to TGF 1 release. (c) Integrins v 6, v 5 and v 3 have been shown to activate latent TGF 1 independently from proteolytic activity. They all recognize the integrin binding sequence RGD of the LAP moiety in the LLC. It has been proposed that when the LLC is covalently bound to a mechanically resistant ECM, cell traction forces exerted to LAP will result in a deformation change of the latent complex that liberates active TGF 1 (from Wipff and Hinz, 2008).

First evidence that integrins can directly activate latent TGF 1 independently from any proteolytic activity was provided for the epithelial integrin v 6 (Annes et al., 2004; Jenkins et al., 2006; Munger et al., 1999). Functional knockout of this integrin produces a phenotype in mice that closely resembles that of a TGF 1 knockout (Huang et al., 1996; Shull et al., 1992). Concomitantly, the lungs of 6 knockout mice are protected from fibrosis (Munger et al., 1999). Later, the integrins v 5, v 8, a yet unidentified 1 integrin and possibly v 3 integrin have also been reported to participate in activating latent TGF 1 (Sheppard, 2005; Wipff and Hinz, 2008). All integrins that contribute to

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the activation of latent TGF 1 physically interact with the LAP portion of the large latent complex (Sheppard, 2005). Inhibiting the respective binding sequence in vitro abolishes latent TGF 1 activation in epithelial cells (Munger et al., 1999) and in myofibroblasts (Wipff et al., 2007); genetic deletion of this sequence in mice strongly resembles the TGF 1 knockout (Yang et al., 2007). The following model has been proposed for direct and integrin-mediated activation of latent TGF 1 by contractile myofibroblasts: (1) The integrin v 5 binds extracellularly to the LAP portion of the large latent complex and intracellularly to -SMA positive stress fibres. (2) The forces generated by contractile stress fibres pull on the latent complex via its integrin binding site. (3) Binding of LTBP-1 to the ECM provides mechanical resistance to the pulling which leads to opening of the complex and release/presentation of active TGF 1 (Fig. 2.3c). This model is supported by the findings that inhibition of integrin v 5 with function blocking antibodies diminishes latent TGF 1 activation by cultured myofibroblasts and reduces the fibrogenic character of fibroblastic cells (Asano et al., 2006). In addition, myofibroblasts activate latent TGF 1 as a function of their contractile activity (Wipff et al., 2007). Inducing myofibroblast contraction with thrombin, angiotensin-II and endothelin-1 increases latent TGF 1 activation; this effect depends on integrin binding to LAP (Wipff et al., 2007). Finally, to induce a putative conformational change in the latent TGF 1 complex by integrin-mediated pulling on the LAP, the ECM must provide mechanical resistance. Indeed, myofibroblasts only activate TGF 1 by integrinmediated contraction when cultured on substrates with a stiffness that corresponds to that of contracting fibrotic and granulation tissue but not when grown on substrates exhibiting the compliance of normal connective tissue (Wipff et al., 2007). This dependence of the availability of active TGF 1 on ECM stiffness would restrict generation and autocrine maintenance of the myofibroblast phenotype to a mechanical microenvironment that has been sufficiently pre-remodelled and stiffened for being efficiently contracted. Interfering with the integrins that are implicated in latent TGF 1 activation is one possible strategy to therapeutically counteract the harmful activity of active TGF 1 in a cell-specific manner, without impairing its beneficial effects on other cell types.

2.6.3

Other factors that modulate myofibroblast differentiation

Discussing all the intracellular signalling molecules, cytokines and ECM proteins that modulate myofibroblast differentiation and -SMA expression would by far exceed the scope of this chapter and the reader is referred to the respective literature (Hinz, 2007; Horowitz and Thannickal, 2006; Schurch et al., 2007; Wynn, 2007). Most differentiated myofibroblast-inducing factors act in synergy with TGF 1 signalling and are not effective in inducing -SMA on

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their own (Desmouliere et al., 1993; Ronnov-Jessen and Petersen, 1993). Factors that stimulate the differentiation of myofibroblasts from their precursors without involving paracrine effects from other cell types, include CTGF (connective tissue growth factor) (Shi-Wen et al., 2008), IL-6 (Gallucci et al., 2006), Fizz1 (found in inflammatory zone) (Liu et al., 2004), galectin-3 (Henderson et al., 2006), osteopontin (Lenga et al., 2008; Mori et al., 2008), endothelin-1 (Jain et al., 2007; Shi-Wen et al., 2007), angiotensin II (Guo et al., 2001; Mezzano et al., 2001; Rosenkranz, 2004; Uhal et al., 2007), thrombin (Bogatkevich et al., 2003), possibly semaphorin 7A (Kang et al., 2007), NGF (nerve growth factor) (Micera et al., 2001) and cleavage of the urokinase receptor (Bernstein et al., 2007). Myofibroblast differentiation is further promoted by cell adhesion to specific ECM proteins including collagen type VI (Naugle et al., 2006), tenascin-C (De Wever et al., 2004; Tamaoki et al., 2005) and most importantly ED-A FN (Serini et al., 1998). In addition, pathogen-associated molecular patterns, such as bacterial lipoproteins, DNA and double-stranded RNA can bind to receptors on the surface of fibroblastic cells (Akira and Takeda, 2004) and have been shown to activate myofibroblasts in the intestine (Otte et al., 2003). Another important stimulating factor for myofibroblast differentiation appears to be the production of reactive oxygen species by NADPH oxidases (NOX) in fibroblastic cells (Shen et al., 2006). NOX are transmembrane proteins that regulate intracellular redox signalling by reducing extracellular molecular oxygen to superoxide generating downstream reactive oxygen species (Bedard and Krause, 2007). The predominant NOX isoform in fibroblasts is NOX4, which has been shown to mediate TGF 1-induced conversion of cardiac fibroblasts into myofibroblasts (Cucoranu et al., 2005). The vast majority of factors that were shown to exert a myofibroblast-inducing effect have been tested on fibroblasts from different tissue origins. It remains to be shown whether all identified myofibroblast precursor cells similarly respond to these factors. This appears the case for TGF 1, being an accepted myofibroblast promoter in fibroblasts (Desmouliere et al., 1993; Ronnov-Jessen and Petersen, 1993), hepatic stellate cells (Gressner and Weiskirchen, 2006), astrocytes (Moreels et al., 2008), epithelial cells (Masszi et al., 2003; Willis et al., 2005), fibrocytes (Hong et al., 2007) and MSC (Wang et al., 2004).

2.7

Lessons to be learned from the myofibroblast for the effective use of mesenchymal stem cells (MSC)

To suppress and/or reduce the contribution of engrafted MSC to the development of fibrotic scar, MSC delivery to diseased organs could be combined with the delivery of agents that down-regulate myofibroblast development. The search for anti-fibrotic drugs is intense and tissue engineering as well as regenerative medicine will benefit from the discovery of new therapies against

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organ fibrosis (Brown et al., 2005; Friedman, 2004a; Gharaee-Kermani et al., 2007; Horowitz and Thannickal, 2006; Scotton and Chambers, 2007). At present, however, none of the countless factors that are implied in normal and pathological wound healing has been successfully targeted to significantly improve the tissue repair process in clinical applications. This is particularly true for therapies that imply growth factors which are subjected to rapid degradation in the aggressive wound environment and require precisely controlled timing of administration to achieve the wanted effects (Chan et al., 2006). Growth factors that antagonize myofibroblast development in culture and in animal models have been described, including IL-1 (Kanangat et al., 2006; Shephard et al., 2004), tumour necrosis factor- (TNF-) (Goldberg et al., 2007; Saika et al., 2006), TGF 3 (Shah et al., 1995) and interferon- (IFN- ) (Desmouliere et al., 1992). Using MSC as vehicle to deliver these growth factors specifically to fibrotic scars and simultaneously to regenerate the tissue can be one future strategy to improve tissue regeneration. However, much is left to be done before this utopian vision can be applied in clinics. It is unclear whether MSC respond to the above-mentioned factors in a similar fashion than fibroblasts and how the engrafted microenvironment will modulate their response. For instance, FGF-2 appears to support myofibroblast differentiation of MSC (Hankemeier et al., 2005; Nedeau et al., 2008) but antagonizes myofibroblast development of SMC, pericytes and fibroblasts (Cushing et al., 2008; Maltseva et al., 2001; Papetti et al., 2003). Another possibility to suppress development of the myofibroblast phenotype in MSC is to interfere with the mechanical feedback loop of high contractile activity and ECM stiffening that induces and maintains the fibrogenic cell character (Wipff et al., 2007) (see Section 2.6.1). This strategy has been proven successful to inhibit fibroblast-to-myofibroblast differentiation in different ways. First, reducing the stiffness of the microenvironment will lower the stress exerted on the MSC and possibly suppress its myofibroblast development. It is difficult to imagine how one could reduce the stiffness of a scar tissue in a controlled manner if MSC are to be delivered systemically. However, when using a scaffold delivery strategy the mechanical property of the biomaterial or synthetic material can be adjusted to control the fibrogenic behaviour of implanted MSC (Ghosh and Ingber, 2007). Second, if the mechanical properties of the microenvironment are not controllable, interfering with the mechanisms through which MSC perceive extracellular stress is another option. It has been shown that the level of substrate rigidity determines the size and molecular composition of cell±matrix focal adhesions that perceive and communicate extracellular mechanical signals to the cytoskeleton, leading to specific gene expression (Bershadsky et al., 2003; Ingber, 2003). By artificially reducing the adhesion area available for cell attachment using microcontact printing on rigid surfaces it is possible to `simulate' a soft environment for myofibroblasts. As a consequence, these cells lose -SMA expression in stress fibres and contractile

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capacity similar to growth of soft substrates (Goffin et al., 2006). Third, the level of myofibroblast intracellular tension can be specifically and directly reduced by targeting -SMA positive stress fibres using a competitive peptide strategy. Inhibiting myofibroblast contraction by long-term administration of the so-called SMA fusion peptide results in decreased collagen production and finally disappearance of the myofibroblast (Clement et al., 2005; Hinz et al., 2002). Whereas the latter option has not yet been tested for MSC, modulating substrate stiffness and cell adhesion area have already been shown to influence MSC differentiation into different lineages. Engler and collaborators could demonstrate that growth on differently compliant polymer surfaces induces early lineage differentiation of MSC (Engler et al., 2006). In otherwise identical culture conditions, adjusting ECM stiffness to the stiffness of their natural body environment guides MSC along the according differentiation pathway. On `brain-soft' gels with elastic modulus of ~1000 Pa, MSC express early neurogenic marker, growth on `muscle-stiff' substrates of ~11 000 Pa induces early myogenic factors and `bone-stiff' substrates of >34 000 Pa induces osteogenic differentiation (Engler et al., 2006). Similar observations were made by dictating the size of the surface area available for MSC adhesion. MSC are commitment to the adipocyte lineage when grown on small adhesion islands or into osteoblasts when plated on large islands; this mechanical restriction was even capable to override the effect of specific differentiating growth factors added to the medium (McBeath et al., 2004). Myofibroblastic differentiation of MSC on soft and micropatterned substrates has not been assessed in the appropriate fibrogenic conditions but the clear mechano-responsiveness of MSC suggests an effect of substrate stiffness also on the development of this phenotype.

2.8

Conclusions and future trends

Most studies that search to understand how myofibroblasts develop and how their activity is controlled are motivated by the desire to fight organ fibrosis, one of the major causes of death in Western countries. I have here evaluated the threat and/or potential that this cell phenotype represents for regenerative medicine with a specific focus on MSC-to-myofibroblast transition. It remains to be shown whether and when the myofibroblastic MSC is our friend or our enemy; in any case knowing your enemy raises the chances of success of tissue regeneration. This spirit is expressed in a very loose translation of the wellknown statement made by Sun Tzu in `The Art of War': `So it is said that if you know your enemies and know yourself, you will fight without danger in battles. If you only know yourself, but not your opponent, you may win or may lose. If you know neither yourself nor your enemy, you will always endanger yourself.'

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References

Abe, R., S.C. Donnelly, T. Peng, R. Bucala, and C.N. Metz. 2001. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol. 166: 7556±62. Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nat Rev Immunol. 4: 499± 511. Albini, A., and M.B. Sporn. 2007. The tumour microenvironment as a target for chemoprevention. Nat Rev Cancer. 7: 139±47. Anderson, J.M., A. Rodriguez, and D.T. Chang. 2008. Foreign body reaction to biomaterials. Semin Immunol. 20: 86±100. Andersson-Sjoland, A., C.G. de Alba, K. Nihlberg, C. Becerril, R. Ramirez, A. Pardo, G. Westergren-Thorsson, and M. Selman. 2008. Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis. Int J Biochem Cell Biol. 40: 2129± 40. Annes, J.P., J.S. Munger, and D.B. Rifkin. 2003. Making sense of latent TGFbeta activation. J Cell Sci. 116: 217±24. Annes, J.P., Y. Chen, J.S. Munger, and D.B. Rifkin. 2004. Integrin V 6-mediated activation of latent TGF- requires the latent TGF- binding protein-1. J Cell Biol. 165: 723±34. Aoki, S., S. Toda, T. Ando, and H. Sugihara. 2004. Bone marrow stromal cells, preadipocytes, and dermal fibroblasts promote epidermal regeneration in their distinctive fashions. Mol Biol Cell. 15: 4647±57. Ariyan, S., R. Enriquez, and T.J. Krizek. 1978. Wound contraction and fibrocontractive disorders. Arch Surg. 113: 1034±46. Armulik, A., A. Abramsson, and C. Betsholtz. 2005. Endothelial/pericyte interactions. Circ Res. 97: 512±23. Arora, P.D., N. Narani, and C.A. McCulloch. 1999. The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts. Am. J. Pathol. 154: 871±82. Asano, Y., H. Ihn, K. Yamane, M. Jinnin, and K. Tamaki. 2006. Increased expression of integrin alphavbeta5 induces the myofibroblastic differentiation of dermal fibroblasts. Am J Pathol. 168: 499±510. Atiyeh, B.S., M. Costagliola, and S.N. Hayek. 2005. Keloid or hypertrophic scar: the controversy: review of the literature. Ann Plast Surg. 54: 676±80. Aust, L., B. Devlin, S.J. Foster, Y.D. Halvorsen, K. Hicok, T. du Laney, A. Sen, G.D. Willingmyre, and J.M. Gimble. 2004. Yield of human adipose-derived adult stem cells from liposuction aspirates. Cytotherapy. 6: 7±14. Bataller, R., and D.A. Brenner. 2005. Liver fibrosis. J Clin Invest. 115: 209±18. Baudino, T.A., W. Carver, W. Giles, and T.K. Borg. 2006. Cardiac fibroblasts: friend or foe? Am J Physiol Heart Circ Physiol. 291: H1015±26. Bedard, K., and K.H. Krause. 2007. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 87: 245±313. Bellini, A., and S. Mattoli. 2007. The role of the fibrocyte, a bone marrow-derived mesenchymal progenitor, in reactive and reparative fibroses. Lab Invest. 87: 858± 70. Benzonana, G., O. Skalli, and G. Gabbiani. 1988. Correlation between the distribution of smooth muscle or non muscle myosins and alpha-smooth muscle actin in normal and pathological soft tissues. Cell Motil Cytoskeleton. 11: 260±74. Bernstein, A.M., S.S. Twining, D.J. Warejcka, E. Tall, and S.K. Masur. 2007. Urokinase

64

Regenerative medicine and biomaterials in connective tissue repair

receptor cleavage: a crucial step in fibroblast-to-myofibroblast differentiation. Mol Biol Cell. 18: 2716±27. Berry, M.F., A.J. Engler, Y.J. Woo, T.J. Pirolli, L.T. Bish, V. Jayasankar, K.J. Morine, T.J. Gardner, D.E. Discher, and H.L. Sweeney. 2006. Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance. Am J Physiol Heart Circ Physiol. 290: H2196±203. Bershadsky, A.D., N.Q. Balaban, and B. Geiger. 2003. Adhesion-dependent cell mechanosensitivity. Annu Rev Cell Dev Biol. 19: 677±95. Bhowmick, N.A., and H.L. Moses. 2005. Tumor±stroma interactions. Curr Opin Genet Dev. 15: 97±101. Bianco, P., M. Riminucci, S. Gronthos, and P.G. Robey. 2001. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells. 19: 180±92. Bierie, B., and H.L. Moses. 2006. Tumour microenvironment: TGF : the molecular Jekyll and Hyde of cancer. Nat Rev Cancer. 6: 506±20. Bissell, M.J., and D. Radisky. 2001. Putting tumours in context. Nat Rev Cancer. 1: 46± 54. Bochaton-Piallat, M.L., and G. Gabbiani. 2006. Smooth muscle cell: a key cell for plaque vulnerability regulation? Circ Res. 98: 448±9. Bochaton-Piallat, M.L., F. Gabbiani, P. Ropraz, and G. Gabbiani. 1992. Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features. Differentiation. 49: 175±85. Bogatkevich, G.S., E. Tourkina, C.S. Abrams, R.A. Harley, R.M. Silver, and A. Ludwicka-Bradley. 2003. Contractile activity and smooth muscle alpha-actin organization in thrombin-induced human lung myofibroblasts. Am J Physiol Lung Cell Mol Physiol. 285: L334±43. Bonanno, E., L. Ercoli, P. Missori, G. Rocchi, and L.G. Spagnoli. 1994. Homogeneous stromal cell population from normal human adult bone marrow expressing alphasmooth muscle actin filaments. Lab Invest. 71: 308±15. Breitbach, M., T. Bostani, W. Roell, Y. Xia, O. Dewald, J.M. Nygren, J.W. Fries, K. Tiemann, H. Bohlen, J. Hescheler, A. Welz, W. Bloch, S.E. Jacobsen, and B.K. Fleischmann. 2007. Potential risks of bone marrow cell transplantation into infarcted hearts. Blood. 110: 1362±9. Brittan, M., T. Hunt, R. Jeffery, R. Poulsom, S.J. Forbes, K. Hodivala-Dilke, J. Goldman, M.R. Alison, and N.A. Wright. 2002. Bone marrow derivation of pericryptal myofibroblasts in the mouse and human small intestine and colon. Gut. 50: 752±7. Brittan, M., V. Chance, G. Elia, R. Poulsom, M.R. Alison, T.T. MacDonald, and N.A. Wright. 2005. A regenerative role for bone marrow following experimental colitis: contribution to neovasculogenesis and myofibroblasts. Gastroenterology. 128: 1984±95. Brown, R.D., S.K. Ambler, M.D. Mitchell, and C.S. Long. 2005. The cardiac fibroblast: therapeutic target in myocardial remodeling and failure. Annu Rev Pharmacol Toxicol. 45: 657±87. Bruder, S.P., A.A. Kurth, M. Shea, W.C. Hayes, N. Jaiswal, and S. Kadiyala. 1998. Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells. J Orthop Res. 16: 155±62. Bucala, R., L.A. Spiegel, J. Chesney, M. Hogan, and A. Cerami. 1994. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med. 1: 71±81. Butler, D.L., N. Juncosa-Melvin, G.P. Boivin, M.T. Galloway, J.T. Shearn, C. Gooch, and H. Awad. 2008. Functional tissue engineering for tendon repair: a multidisciplinary

The myofibroblast in connective tissue repair and regeneration

65

strategy using mesenchymal stem cells, bioscaffolds, and mechanical stimulation. J Orthop Res. 26: 1±9. Cai, D., R. Marty-Roix, H.P. Hsu, and M. Spector. 2001. Lapine and canine bone marrow stromal cells contain smooth muscle actin and contract a collagen± glycosaminoglycan matrix. Tissue Eng. 7: 829±41. Caplan, A.I. 1991. Mesenchymal stem cells. J Orthop Res. 9: 641±50. Caplan, A.I. 2007. Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol. 213: 341±7. Caplan, A.I., and J.E. Dennis. 2006. Mesenchymal stem cells as trophic mediators. J Cell Biochem. 98: 1076±84. Chamberlain, G., J. Fox, B. Ashton, and J. Middleton. 2007. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells. 25: 2739±49. Chambers, R.C., P. Leoni, N. Kaminski, G.J. Laurent, and R.A. Heller. 2003. Global expression profiling of fibroblast responses to transforming growth factor-beta1 reveals the induction of inhibitor of differentiation-1 and provides evidence of smooth muscle cell phenotypic switching. Am J Pathol. 162: 533±46. Chan, R.K., P.H. Liu, G. Pietramaggiori, S.I. Ibrahim, H.B. Hechtman, and D.P. Orgill. 2006. Effect of recombinant platelet-derived growth factor (Regranex) on wound closure in genetically diabetic mice. J Burn Care Res. 27: 202±5. Charbord, P., A.M. Gown, A. Keating, and J.W. Singer. 1985. CGA-7 and HHF, two monoclonal antibodies that recognize muscle actin and react with adherent cells in human long-term bone marrow cultures. Blood. 66: 1138±42. Charbord, P., H. Lerat, I. Newton, E. Tamayo, A.M. Gown, J.W. Singer, and P. Herve. 1990. The cytoskeleton of stromal cells from human bone marrow cultures resembles that of cultured smooth muscle cells. Exp Hematol. 18: 276±82. Chen, L., E.E. Tredget, P.Y. Wu, and Y. Wu. 2008. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS ONE. 3: e1886. Chen, S.L., W.W. Fang, F. Ye, Y.H. Liu, J. Qian, S.J. Shan, J.J. Zhang, R.Z. Chunhua, L.M. Liao, S. Lin, and J.P. Sun. 2004. Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol. 94: 92±5. Chiappara, G., R. Gagliardo, A. Siena, M.R. Bonsignore, J. Bousquet, G. Bonsignore, and A.M. Vignola. 2001. Airway remodelling in the pathogenesis of asthma. Curr Opin Allergy Clin Immunol. 1: 85±93. Chilosi, M., V. Poletti, A. Zamo, M. Lestani, L. Montagna, P. Piccoli, S. Pedron, M. Bertaso, A. Scarpa, B. Murer, A. Cancellieri, R. Maestro, G. Semenzato, and C. Doglioni. 2003. Aberrant Wnt/beta-catenin pathway activation in idiopathic pulmonary fibrosis. Am J Pathol. 162: 1495±502. Christen, T., V. Verin, M. Bochaton-Piallat, Y. Popowski, F. Ramaekers, P. Debruyne, E. Camenzind, G. van Eys, and G. Gabbiani. 2001. Mechanisms of neointima formation and remodeling in the porcine coronary artery. Circulation. 103: 882±8. Clark, R.A., K. Ghosh, and M.G. Tonnesen. 2007. Tissue engineering for cutaneous wounds. J Invest Dermatol. 127: 1018±29. Clement, S., B. Hinz, V. Dugina, G. Gabbiani, and C. Chaponnier. 2005. The N-terminal Ac-EEED sequence plays a role in -smooth-muscle actin incorporation into stress fibers. J Cell Sci. 118: 1395±404. Coleman, D.J., D.T. Sharpe, I.L. Naylor, C.L. Chander, and S.E. Cross. 1993. The role of the contractile fibroblast in the capsules around tissue expanders and implants. Br J

66

Regenerative medicine and biomaterials in connective tissue repair

Plast Surg. 46: 547±56. Comut, A.A., S. Shortkroff, X. Zhang, and M. Spector. 2000. Association of fibroblast orientation around titanium in vitro with expression of a muscle actin. Biomaterials. 21: 1887±96. Cucoranu, I., R. Clempus, A. Dikalova, P.J. Phelan, S. Ariyan, S. Dikalov, and D. Sorescu. 2005. NAD(P)H oxidase 4 mediates transforming growth factor-beta1induced differentiation of cardiac fibroblasts into myofibroblasts. Circ Res. 97: 900±7. Cushing, M.C., P.D. Mariner, J.T. Liao, E.A. Sims, and K.S. Anseth. 2008. Fibroblast growth factor represses Smad-mediated myofibroblast activation in aortic valvular interstitial cells. Faseb J. 22: 1769±77. Darby, I., O. Skalli, and G. Gabbiani. 1990. Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab. Invest. 63: 21±9. De Wever, O., and M. Mareel. 2002. Role of myofibroblasts at the invasion front. Biol Chem. 383: 55±67. De Wever, O., and M. Mareel. 2003. Role of tissue stroma in cancer cell invasion. J Pathol. 200: 429±47. De Wever, O., Q.D. Nguyen, L. Van Hoorde, M. Bracke, E. Bruyneel, C. Gespach, and M. Mareel. 2004. Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon cancer cells through RhoA and Rac. Faseb J. 18: 1016±18. Deng, W., Q. Han, L. Liao, C. Li, W. Ge, Z. Zhao, S. You, H. Deng, F. Murad, and R.C. Zhao. 2005. Engrafted bone marrow-derived flk-(1+) mesenchymal stem cells regenerate skin tissue. Tissue Eng. 11: 110±19. Desmouliere, A., L. Rubbia-Brandt, A. Abdiu, T. Walz, A. Macieira-Coelho, and G. Gabbiani. 1992. Alpha-smooth muscle actin is expressed in a subpopulation of cultured and cloned fibroblasts and is modulated by gamma-interferon. Exp. Cell Res. 201: 64±73. Desmouliere, A., A. Geinoz, F. Gabbiani, and G. Gabbiani. 1993. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J. Cell Biol. 122: 103±11. Desmouliere, A., M. Redard, I. Darby, and G. Gabbiani. 1995. Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Am. J. Pathol. 146: 56±66. Desmouliere, A., I.A. Darby, and G. Gabbiani. 2003. Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis. Lab Invest. 83: 1689±707. Desmouliere, A., C. Guyot, and G. Gabbiani. 2004. The stroma reaction myofibroblast: a key player in the control of tumor cell behavior. Int J Dev Biol. 48: 509±17. Desmouliere, A., C. Chaponnier, and G. Gabbiani. 2005. Tissue repair, contraction, and the myofibroblast. Wound Repair Regen. 13: 7±12. di Bonzo, L.V., I. Ferrero, C. Cravanzola, K. Mareschi, D. Rustichell, E. Novo, F. Sanavio, S. Cannito, E. Zamara, M. Bertero, A. Davit, S. Francica, F. Novelli, S. Colombatto, F. Fagioli, and M. Parola. 2008. Human mesenchymal stem cells as a two-edged sword in hepatic regenerative medicine: engraftment and hepatocyte differentiation versus profibrogenic potential. Gut. 57: 223±31. Direkze, N.C., S.J. Forbes, M. Brittan, T. Hunt, R. Jeffery, S.L. Preston, R. Poulsom, K. Hodivala-Dilke, M.R. Alison, and N.A. Wright. 2003. Multiple organ engraftment

The myofibroblast in connective tissue repair and regeneration

67

by bone-marrow-derived myofibroblasts and fibroblasts in bone-marrowtransplanted mice. Stem Cells. 21: 514±20. Direkze, N.C., K. Hodivala-Dilke, R. Jeffery, T. Hunt, R. Poulsom, D. Oukrif, M.R. Alison, and N.A. Wright. 2004. Bone marrow contribution to tumor-associated myofibroblasts and fibroblasts. Cancer Res. 64: 8492±5. Discher, D.E., P. Janmey, and Y.L. Wang. 2005. Tissue cells feel and respond to the stiffness of their substrate. Science. 310: 1139±43. Engler, A.J., S. Sen, H.L. Sweeney, and D.E. Discher. 2006. Matrix elasticity directs stem cell lineage specification. Cell. 126: 677±89. Erices, A., P. Conget, and J.J. Minguell. 2000. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol. 109: 235±42. Eyden, B. 2007. The myofibroblast: a study of normal, reactive and neoplastic tissues, with an emphasis on ultrastructure. J Submicrosc Cytol Pathol. 1: 7±166. Eyden, B. 2008. The myofibroblast: phenotypic characterization as a prerequisite to understanding its functions in translational medicine. J Cell Mol Med. 12: 22±37. Fang, B., M. Shi, L. Liao, S. Yang, Y. Liu, and R.C. Zhao. 2004. Systemic infusion of FLK1(+) mesenchymal stem cells ameliorate carbon tetrachloride-induced liver fibrosis in mice. Transplantation. 78: 83±8. Fathke, C., L. Wilson, J. Hutter, V. Kapoor, A. Smith, A. Hocking, and F. Isik. 2004. Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair. Stem Cells. 22: 812±22. Feng, X.H., and R. Derynck. 2005. Specificity and versatility in TGF-signaling through Smads. Annu Rev Cell Dev Biol. 21: 659±93. Fernandez, M., V. Simon, G. Herrera, C. Cao, H. Del Favero, and J.J. Minguell. 1997. Detection of stromal cells in peripheral blood progenitor cell collections from breast cancer patients. Bone Marrow Transplant. 20: 265±71. Ferrari, G., G. Cusella-De Angelis, M. Coletta, E. Paolucci, A. Stornaiuolo, G. Cossu, and F. Mavilio. 1998. Muscle regeneration by bone marrow-derived myogenic progenitors. Science. 279: 1528±30. Forbes, S.J., F.P. Russo, V. Rey, P. Burra, M. Rugge, N.A. Wright, and M.R. Alison. 2004. A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis. Gastroenterology. 126: 955±63. Fox, J.M., G. Chamberlain, B.A. Ashton, and J. Middleton. 2007. Recent advances into the understanding of mesenchymal stem cell trafficking. Br J Haematol. 137: 491± 502. Frank, E.D., and L. Warren. 1981. Aortic smooth muscle cells contain vimentin instead of desmin. Proc Natl Acad Sci USA. 78: 3020±4. Frid, M.G., J.A. Brunetti, D.L. Burke, T.C. Carpenter, N.J. Davie, J.T. Reeves, M.T. Roedersheimer, N. van Rooijen, and K.R. Stenmark. 2006. Hypoxia-induced pulmonary vascular remodeling requires recruitment of circulating mesenchymal precursors of a monocyte/macrophage lineage. Am J Pathol. 168: 659±69. Friedenstein, A.J., R.K. Chailakhjan, and K.S. Lalykina. 1970. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 3: 393±403. Friedman, S.L. 2004a. Mechanisms of disease: mechanisms of hepatic fibrosis and therapeutic implications. Nat Clin Pract Gastroenterol Hepatol. 1: 98±105. Friedman, S.L. 2004b. Stellate cells: a moving target in hepatic fibrogenesis. Hepatology. 40: 1041±3. Fujimoto, T., and S.J. Singer. 1987. Immunocytochemical studies of desmin and vimentin in pericapillary cells of chicken. J Histochem Cytochem. 35: 1105±15.

68

Regenerative medicine and biomaterials in connective tissue repair

Fujita, J., M. Mori, H. Kawada, Y. Ieda, M. Tsuma, Y. Matsuzaki, H. Kawaguchi, T. Yagi, S. Yuasa, J. Endo, T. Hotta, S. Ogawa, H. Okano, R. Yozu, K. Ando, and K. Fukuda. 2007. Administration of granulocyte colony-stimulating factor after myocardial infarction enhances the recruitment of hematopoietic stem cell-derived myofibroblasts and contributes to cardiac repair. Stem Cells. 25: 2750±9. Gabbiani, G. 2003. The myofibroblast in wound healing and fibrocontractive diseases. J Pathol. 200: 500±3. Gabbiani, G., G.B. Ryan, and G. Majno. 1971. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia. 27: 549±50. Gabbiani, G., E. Schmid, S. Winter, C. Chaponnier, C. de Ckhastonay, J. Vandekerckhove, K. Weber, and W.W. Franke. 1981. Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific alpha-type actin. Proc Natl Acad Sci USA. 78: 298±302. Gallucci, R.M., E.G. Lee, and J.J. Tomasek. 2006. IL-6 modulates alpha-smooth muscle actin expression in dermal fibroblasts from IL-6-deficient mice. J Invest Dermatol. 126: 561±8. Galmiche, M.C., V.E. Koteliansky, J. Briere, P. Herve, and P. Charbord. 1993. Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway. Blood. 82: 66±76. Gauldie, J., P. Bonniaud, P. Sime, K. Ask, and M. Kolb. 2007. TGF-beta, Smad3 and the process of progressive fibrosis. Biochem Soc Trans. 35: 661±4. Georges, P.C., J.J. Hui, Z. Gombos, M.E. McCormick, A.Y. Wang, M. Uemura, R. Mick, P.A. Janmey, E.E. Furth, and R.G. Wells. 2007. Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis. Am J Physiol Gastrointest Liver Physiol. 293: G1147±54. Gharaee-Kermani, M., B. Hu, V.J. Thannickal, S.H. Phan, and M.R. Gyetko. 2007. Current and emerging drugs for idiopathic pulmonary fibrosis. Expert Opin Emerg Drugs. 12: 627±46. Ghosh, K., and D.E. Ingber. 2007. Micromechanical control of cell and tissue development: implications for tissue engineering. Adv Drug Deliv Rev. 59: 1306± 18. Giordano, A., U. Galderisi, and I.R. Marino. 2007. From the laboratory bench to the patient's bedside: an update on clinical trials with mesenchymal stem cells. J Cell Physiol. 211: 27±35. Goffin, J.M., P. Pittet, G. Csucs, J.W. Lussi, J.J. Meister, and B. Hinz. 2006. Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers. J Cell Biol. 172: 259±68. Goldberg, M.T., Y.P. Han, C. Yan, M.C. Shaw, and W.L. Garner. 2007. TNF-alpha suppresses alpha-smooth muscle actin expression in human dermal fibroblasts: an implication for abnormal wound healing. J Invest Dermatol. 127: 2645±55. Gordon, S., and P.R. Taylor. 2005. Monocyte and macrophage heterogeneity. Nat Rev Immunol. 5: 953±64. Grainger, D.J. 2007. TGF-beta and atherosclerosis in man. Cardiovasc Res. 74: 213±22. Gressner, A.M., and R. Weiskirchen. 2006. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med. 10: 76±99. Grinnell, F. 2000. Fibroblast-collagen-matrix contraction: growth-factor signalling and mechanical loading. Trends Cell Biol. 10: 362±5.

The myofibroblast in connective tissue repair and regeneration

69

Grinnell, F. 2003. Fibroblast biology in three-dimensional collagen matrices. Trends Cell Biol. 13: 264±9. Grinnell, F., C.H. Ho, E. Tamariz, D.J. Lee, and G. Skuta. 2003. Dendritic fibroblasts in three-dimensional collagen matrices. Mol Biol Cell. 14: 384±95. Guo, G., J. Morrissey, R. McCracken, T. Tolley, H. Liapis, and S. Klahr. 2001. Contributions of angiotensin II and tumor necrosis factor-alpha to the development of renal fibrosis. Am J Physiol Renal Physiol. 280: F777±85. Gurtner, G.C., S. Werner, Y. Barrandon, and M.T. Longaker. 2008. Wound repair and regeneration. Nature. 453: 314±21. Guyot, C., S. Lepreux, C. Combe, E. Doudnikoff, P. Bioulac-Sage, C. Balabaud, and A. Desmouliere. 2006. Hepatic fibrosis and cirrhosis: the (myo)fibroblastic cell subpopulations involved. Int J Biochem Cell Biol. 38: 135±51. Hall, B., J. Dembinski, A.K. Sasser, M. Studeny, M. Andreeff, and F. Marini. 2007. Mesenchymal stem cells in cancer: tumor-associated fibroblasts and cell-based delivery vehicles. Int J Hematol. 86: 8±16. Hankemeier, S., M. Keus, J. Zeichen, M. Jagodzinski, T. Barkhausen, U. Bosch, C. Krettek, and M. Van Griensven. 2005. Modulation of proliferation and differentiation of human bone marrow stromal cells by fibroblast growth factor 2: potential implications for tissue engineering of tendons and ligaments. Tissue Eng. 11: 41±9. Hao, H., G. Gabbiani, E. Camenzind, M. Bacchetta, R. Virmani, and M.L. BochatonPiallat. 2006. Phenotypic modulation of intima and media smooth muscle cells in fatal cases of coronary artery lesion. Arterioscler Thromb Vasc Biol. 26: 326±32. Hashimoto, N., H. Jin, T. Liu, S.W. Chensue, and S.H. Phan. 2004. Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest. 113: 243±52. Haudek, S.B., Y. Xia, P. Huebener, J.M. Lee, S. Carlson, J.R. Crawford, D. Pilling, R.H. Gomer, J. Trial, N.G. Frangogiannis, and M.L. Entman. 2006. Bone marrowderived fibroblast precursors mediate ischemic cardiomyopathy in mice. Proc Natl Acad Sci USA. 103: 18284±9. He, Q., C. Wan, and G. Li. 2007. Concise review: multipotent mesenchymal stromal cells in blood. Stem Cells. 25: 69±77. Henderson, N.C., A.C. Mackinnon, S.L. Farnworth, F. Poirier, F.P. Russo, J.P. Iredale, C. Haslett, K.J. Simpson, and T. Sethi. 2006. Galectin-3 regulates myofibroblast activation and hepatic fibrosis. Proc Natl Acad Sci USA. 103: 5060±5. Hentze, H., R. Graichen, and A. Colman. 2007. Cell therapy and the safety of embryonic stem cell-derived grafts. Trends Biotechnol. 25: 24±32. Hinz, B. 2006. Masters and servants of the force: The role of matrix adhesions in myofibroblast force perception and transmission. Eur J Cell Biol. 85: 175±81. Hinz, B. 2007. Formation and function of the myofibroblast during tissue repair. J Invest Dermatol. 127: 526±37. Hinz, B., and G. Gabbiani. 2003. Mechanisms of force generation and transmission by myofibroblasts. Curr Opin Biotechnol. 14: 538±46. Hinz, B., G. Celetta, J.J. Tomasek, G. Gabbiani, and C. Chaponnier. 2001a. Alphasmooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell. 12: 2730±41. Hinz, B., D. Mastrangelo, C.E. Iselin, C. Chaponnier, and G. Gabbiani. 2001b. Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation. Am J Pathol. 159: 1009±20. Hinz, B., G. Gabbiani, and C. Chaponnier. 2002. The NH2-terminal peptide of alphasmooth muscle actin inhibits force generation by the myofibroblast in vitro and in

70

Regenerative medicine and biomaterials in connective tissue repair

vivo. J Cell Biol. 157: 657±63. Hinz, B., P. Pittet, J. Smith-Clerc, C. Chaponnier, and J.J. Meister. 2004. Myofibroblast development is characterized by specific cell±cell adherens junctions. Mol Biol Cell. 15: 4310±20. Hinz, B., S.H. Phan, V.J. Thannickal, A. Galli, M.L. Bochaton-Piallat, and G. Gabbiani. 2007. The myofibroblast: one function, multiple origins. Am J Pathol. 170: 1807± 16. Hong, K.M., J.A. Belperio, M.P. Keane, M.D. Burdick, and R.M. Strieter. 2007. Differentiation of human circulating fibrocytes as mediated by transforming growth factor-beta and peroxisome proliferator-activated receptor gamma. J Biol Chem. 282: 22910±20. Horowitz, J.C., and V.J. Thannickal. 2006. Idiopathic pulmonary fibrosis: new concepts in pathogenesis and implications for drug therapy. Treat Respir Med. 5: 325±42. Horwitz, E.M., D.J. Prockop, L.A. Fitzpatrick, W.W. Koo, P.L. Gordon, M. Neel, M. Sussman, P. Orchard, J.C. Marx, R.E. Pyeritz, and M.K. Brenner. 1999. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med. 5: 309±13. Huang, X.Z., J.F. Wu, D. Cass, D.J. Erle, D. Corry, S.G. Young, R.V. Farese, Jr, and D. Sheppard. 1996. Inactivation of the integrin beta 6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lung and skin. J Cell Biol. 133: 921±8. Hughes, C.C. 2008. Endothelial±stromal interactions in angiogenesis. Curr Opin Hematol. 15: 204±9. Hung, S.C., W.P. Deng, W.K. Yang, R.S. Liu, C.C. Lee, T.C. Su, R.J. Lin, D.M. Yang, C.W. Chang, W.H. Chen, H.J. Wei, and J.G. Gelovani. 2005. Mesenchymal stem cell targeting of microscopic tumors and tumor stroma development monitored by noninvasive in vivo positron emission tomography imaging. Clin Cancer Res. 11: 7749±56. Hung, S.C., P.Y. Kuo, C.F. Chang, T.H. Chen, and L.L. Ho. 2006. Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells. Cell Tissue Res. 324: 457±66. Ingber, D.E. 2003. Tensegrity II. How structural networks influence cellular information processing networks. J Cell Sci. 116: 1397±408. Ishii, G., T. Sangai, K. Sugiyama, T. Ito, T. Hasebe, Y. Endoh, J. Magae, and A. Ochiai. 2005. In vivo characterization of bone marrow-derived fibroblasts recruited into fibrotic lesions. Stem Cells. 23: 699±706. Iwano, M., D. Plieth, T.M. Danoff, C. Xue, H. Okada, and E.G. Neilson. 2002. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest. 110: 341±50. Jain, R., P.W. Shaul, Z. Borok, and B.C. Willis. 2007. Endothelin-1 induces alveolar epithelial-mesenchymal transition through endothelin type A receptor-mediated production of TGF-beta1. Am J Respir Cell Mol Biol. 37: 38±47. Jenkins, G. 2008. The role of proteases in transforming growth factor-beta activation. Int J Biochem Cell Biol. 40: 1068±78. Jenkins, R.G., X. Su, G. Su, C.J. Scotton, E. Camerer, G.J. Laurent, G.E. Davis, R.C. Chambers, M.A. Matthay, and D. Sheppard. 2006. Ligation of protease-activated receptor 1 enhances alpha(v)beta6 integrin-dependent TGF-beta activation and promotes acute lung injury. J Clin Invest. 116: 1606±14. Jeon, E.S., H.J. Moon, M.J. Lee, H.Y. Song, Y.M. Kim, M. Cho, D.S. Suh, M.S. Yoon, C.L. Chang, J.S. Jung, and J.H. Kim. 2008. Cancer-derived lysophosphatidic acid

The myofibroblast in connective tissue repair and regeneration

71

stimulates differentiation of human mesenchymal stem cells to myofibroblast-like cells. Stem Cells. 26: 789±97. Jiang, Y., B. Vaessen, T. Lenvik, M. Blackstad, M. Reyes, and C.M. Verfaillie. 2002. Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp Hematol. 30: 896±904. Kadiyala, S., R.G. Young, M.A. Thiede, and S.P. Bruder. 1997. Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant. 6: 125±34. Kalluri, R., and E.G. Neilson. 2003. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest. 112: 1776±84. Kalluri, R., and M. Zeisberg. 2006. Fibroblasts in cancer. Nat Rev Cancer. 6: 392±401. Kanangat, S., A.E. Postlethwaite, G.C. Higgins, and K.A. Hasty. 2006. Novel functions of intracellular IL-1ra in human dermal fibroblasts: implications in the pathogenesis of fibrosis. J Invest Dermatol. 126: 756±65. Kang, H.R., C.G. Lee, R.J. Homer, and J.A. Elias. 2007. Semaphorin 7A plays a critical role in TGF-beta1-induced pulmonary fibrosis. J Exp Med. 204: 1083±93. Kapanci, Y., S. Burgan, G.G. Pietra, B. Conne, and G. Gabbiani. 1990. Modulation of actin isoform expression in alveolar myofibroblasts (contractile interstitial cells) during pulmonary hypertension. Am. J. Pathol. 136: 881±9. Karnoub, A.E., A.B. Dash, A.P. Vo, A. Sullivan, M.W. Brooks, G.W. Bell, A.L. Richardson, K. Polyak, R. Tubo, and R.A. Weinberg. 2007. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. 449: 557±63. Karp, J.M., and R. Langer. 2007. Development and therapeutic applications of advanced biomaterials. Curr Opin Biotechnol. 18: 454±9. Khakoo, A.Y., S. Pati, S.A. Anderson, W. Reid, M.F. Elshal, Rovira, II, A.T. Nguyen, D. Malide, C.A. Combs, G. Hall, J. Zhang, M. Raffeld, T.B. Rogers, W. StetlerStevenson, J.A. Frank, M. Reitz, and T. Finkel. 2006. Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi's sarcoma. J Exp Med. 203: 1235±47. Kim, A.C., and M. Spector. 2000. Distribution of chondrocytes containing alpha-smooth muscle actin in human articular cartilage. J Orthop Res. 18: 749±55. Kim, K.K., M.C. Kugler, P.J. Wolters, L. Robillard, M.G. Galvez, A.N. Brumwell, D. Sheppard, and H.A. Chapman. 2006. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci USA. 103: 13180±5. Kim, Y.M., E.S. Jeon, M.R. Kim, J.S. Lee, and J.H. Kim. 2008. Bradykinin-induced expression of alpha-smooth muscle actin in human mesenchymal stem cells. Cell Signal. 20: 1882±9. Kinner, B., L.C. Gerstenfeld, T.A. Einhorn, and M. Spector. 2002a. Expression of smooth muscle actin in connective tissue cells participating in fracture healing in a murine model. Bone. 30: 738±45. Kinner, B., J.M. Zaleskas, and M. Spector. 2002b. Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells. Exp Cell Res. 278: 72±83. Kisseleva, T., H. Uchinami, N. Feirt, O. Quintana-Bustamante, J.C. Segovia, R.F. Schwabe, and D.A. Brenner. 2006. Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis. J Hepatol. 45: 429±38. Kobayashi, N., T. Yasu, H. Ueba, M. Sata, S. Hashimoto, M. Kuroki, M. Saito, and M. Kawakami. 2004. Mechanical stress promotes the expression of smooth muscle-like properties in marrow stromal cells. Exp Hematol. 32: 1238±45.

72

Regenerative medicine and biomaterials in connective tissue repair

Komarova, S., Y. Kawakami, M.A. Stoff-Khalili, D.T. Curiel, and L. Pereboeva. 2006. Mesenchymal progenitor cells as cellular vehicles for delivery of oncolytic adenoviruses. Mol Cancer Ther. 5: 755±66. Laflamme, M.A., and C.E. Murry. 2005. Regenerating the heart. Nat Biotechnol. 23: 845± 56. Lan, H.Y. 2003. Tubular epithelial-myofibroblast transdifferentiation mechanisms in proximal tubule cells. Curr Opin Nephrol Hypertens. 12: 25±9. Larson, D.M., K. Fujiwara, R.W. Alexander, and M.A. Gimbrone, Jr. 1984. Myosin in cultured vascular smooth muscle cells: immunofluorescence and immunochemical studies of alterations in antigenic expression. J Cell Biol. 99: 1582±9. Lazard, D., X. Sastre, M.G. Frid, M.A. Glukhova, J.P. Thiery, and V.E. Koteliansky. 1993. Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue. Proc. Natl. Acad. Sci. USA. 90: 999±1003. Le Blanc, K., and M. Pittenger. 2005. Mesenchymal stem cells: progress toward promise. Cytotherapy. 7: 36±45. Leask, A., and D.J. Abraham. 2004. TGF-beta signaling and the fibrotic response. Faseb J. 18: 816±27. Lee, O.K., T.K. Kuo, W.M. Chen, K.D. Lee, S.L. Hsieh, and T.H. Chen. 2004. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood. 103: 1669±75. Lenga, Y., A. Koh, A.S. Perera, C.A. McCulloch, J. Sodek, and R. Zohar. 2008. Osteopontin expression is required for myofibroblast differentiation. Circ Res. 102: 319±27. Li, A.G., M.J. Quinn, Y. Siddiqui, M.D. Wood, I.F. Federiuk, H.M. Duman, and W.K. Ward. 2007a. Elevation of transforming growth factor beta (TGFbeta) and its downstream mediators in subcutaneous foreign body capsule tissue. J Biomed Mater Res A. 82: 498±508. Li, Z., J.A. Dranoff, E.P. Chan, M. Uemura, J. Sevigny, and R.G. Wells. 2007b. Transforming growth factor-beta and substrate stiffness regulate portal fibroblast activation in culture. Hepatology. 46: 1246±56. Liu, T., S.M. Dhanasekaran, H. Jin, B. Hu, S.A. Tomlins, A.M. Chinnaiyan, and S.H. Phan. 2004. FIZZ1 stimulation of myofibroblast differentiation. Am J Pathol. 164: 1315±26. Lutolf, M.P., and J.A. Hubbell. 2005. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol. 23: 47±55. Malmstrom, J., H. Lindberg, C. Lindberg, C. Bratt, E. Wieslander, E.L. Delander, B. Sarnstrand, J.S. Burns, P. Mose-Larsen, S. Fey, and G. Marko-Varga. 2004. Transforming growth factor-beta 1 specifically induce proteins involved in the myofibroblast contractile apparatus. Mol Cell Proteomics. 3: 466±77. Maltseva, O., P. Folger, D. Zekaria, S. Petridou, and S.K. Masur. 2001. Fibroblast growth factor reversal of the corneal myofibroblast phenotype. Invest Ophthalmol Vis Sci. 42: 2490±5. Masszi, A., C. Di Ciano, G. Sirokmany, W.T. Arthur, O.D. Rotstein, J. Wang, C.A. McCulloch, L. Rosivall, I. Mucsi, and A. Kapus. 2003. Central role for Rho in TGFbeta1-induced alpha-smooth muscle actin expression during epithelialmesenchymal transition. Am J Physiol Renal Physiol. 284: F911±24. Mattoli, S., A. Bellini, and M. Schmidt. 2009. The role of a human hematopoietic mesenchymal progenitor in wound healing and fibrotic diseases and implications

The myofibroblast in connective tissue repair and regeneration

73

for therapy. Curr Stem Cell Res Ther. e-pub ahead of print. McBeath, R., D.M. Pirone, C.M. Nelson, K. Bhadriraju, and C.S. Chen. 2004. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell. 6: 483±95. Menon, L.G., S. Picinich, R. Koneru, H. Gao, S.Y. Lin, M. Koneru, P. Mayer-Kuckuk, J. Glod, and D. Banerjee. 2007. Differential gene expression associated with migration of mesenchymal stem cells to conditioned medium from tumor cells or bone marrow cells. Stem Cells. 25: 520±8. Metcalf, D. 1972. New type of colony-forming cell located in the pleural cavity. Nat New Biol. 238: 253±4. Metz, C.N. 2003. Fibrocytes: a unique cell population implicated in wound healing. Cell Mol Life Sci. 60: 1342±50. Mezey, E., K.J. Chandross, G. Harta, R.A. Maki, and S.R. McKercher. 2000. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science. 290: 1779±82. Mezzano, S.A., M. Ruiz-Ortega, and J. Egido. 2001. Angiotensin II and renal fibrosis. Hypertension. 38: 635±8. Micera, A., E. Vigneti, D. Pickholtz, R. Reich, O. Pappo, S. Bonini, F.X. Maquart, L. Aloe, and F. Levi-Schaffer. 2001. Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair. Proc Natl Acad Sci USA. 98: 6162±7. Minguell, J.J., A. Erices, and P. Conget. 2001. Mesenchymal stem cells. Exp Biol Med (Maywood). 226: 507±20. Mishra, P.J., P.J. Mishra, R. Humeniuk, D.J. Medina, G. Alexe, J.P. Mesirov, S. Ganesan, J.W. Glod, and D. Banerjee. 2008. Carcinoma-associated fibroblast-like differentiation of human mesenchymal stem cells. Cancer Res. 68: 4331±9. Mishra, P.J., J.W. Glod, and D. Banerjee. 2009. Mesenchymal stem cells: flip side of the coin. Cancer Res. 69: 1255±8. Moeller, A., S.E. Gilpin, K. Ask, G. Cox, D. Cook, J. Gauldie, P.J. Margetts, L. Farkas, J. Dobranowski, C. Boylan, P.M. O'Byrne, R.M. Strieter, and M. Kolb. 2009. Circulating fibrocytes are an indicator of poor prognosis in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 179: 588±94. Moore, B.B., J.E. Kolodsick, V.J. Thannickal, K. Cooke, T.A. Moore, C. Hogaboam, C.A. Wilke, and G.B. Toews. 2005. CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury. Am J Pathol. 166: 675±84. Moreels, M., F. Vandenabeele, D. Dumont, J. Robben, and I. Lambrichts. 2008. Alphasmooth muscle actin (alpha-SMA) and nestin expression in reactive astrocytes in multiple sclerosis lesions: potential regulatory role of transforming growth factorbeta 1 (TGF-beta1). Neuropathol Appl Neurobiol. 34: 532±46. Mori, L., A. Bellini, M.A. Stacey, M. Schmidt, and S. Mattoli. 2005. Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow. Exp Cell Res. 304: 81±90. Mori, R., T.J. Shaw, and P. Martin. 2008. Molecular mechanisms linking wound inflammation and fibrosis: knockdown of osteopontin leads to rapid repair and reduced scarring. J Exp Med. 205: 43±51. Mueller, M.M., and N.E. Fusenig. 2004. Friends or foes ± bipolar effects of the tumour stroma in cancer. Nat Rev Cancer. 4: 839±49. Munger, J.S., X. Huang, H. Kawakatsu, M.J. Griffiths, S.L. Dalton, J. Wu, J.F. Pittet, N. Kaminski, C. Garat, M.A. Matthay, D.B. Rifkin, and D. Sheppard. 1999. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for

74

Regenerative medicine and biomaterials in connective tissue repair

regulating pulmonary inflammation and fibrosis. Cell. 96: 319±28. Nadri, S., M. Soleimani, J. Kiani, A. Atashi, and R. Izadpanah. 2008. Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells. Differentiation. 76: 223±31. Nagaya, N., K. Kangawa, T. Itoh, T. Iwase, S. Murakami, Y. Miyahara, T. Fujii, M. Uematsu, H. Ohgushi, M. Yamagishi, T. Tokudome, H. Mori, K. Miyatake, and S. Kitamura. 2005. Transplantation of mesenchymal stem cells improves cardiac function in a rat model of dilated cardiomyopathy. Circulation. 112: 1128±35. Nakagawa, H., S. Akita, M. Fukui, T. Fujii, and K. Akino. 2005. Human mesenchymal stem cells successfully improve skin-substitute wound healing. Br J Dermatol. 153: 29±36. Naugle, J.E., E.R. Olson, X. Zhang, S.E. Mase, C.F. Pilati, M.B. Maron, H.G. Folkesson, W.I. Horne, K.J. Doane, and J.G. Meszaros. 2006. Type VI collagen induces cardiac myofibroblast differentiation: implications for postinfarction remodeling. Am J Physiol Heart Circ Physiol. 290: H323±30. Nedeau, A.E., R.J. Bauer, K. Gallagher, H. Chen, Z.J. Liu, and O.C. Velazquez. 2008. A CXCL5- and bFGF-dependent effect of PDGF-B-activated fibroblasts in promoting trafficking and differentiation of bone marrow-derived mesenchymal stem cells. Exp Cell Res. 314: 2176±86. Ninichuk, V., O. Gross, S. Segerer, R. Hoffmann, E. Radomska, A. Buchstaller, R. Huss, N. Akis, D. Schlondorff, and H.J. Anders. 2006. Multipotent mesenchymal stem cells reduce interstitial fibrosis but do not delay progression of chronic kidney disease in collagen4A3-deficient mice. Kidney Int. 70: 121±9. Noel, D., F. Djouad, and C. Jorgense. 2002. Regenerative medicine through mesenchymal stem cells for bone and cartilage repair. Curr Opin Investig Drugs. 3: 1000±4. Ohnishi, S., H. Sumiyoshi, S. Kitamura, and N. Nagaya. 2007. Mesenchymal stem cells attenuate cardiac fibroblast proliferation and collagen synthesis through paracrine actions. FEBS Lett. 581: 3961±6. Orlic, D., J. Kajstura, S. Chimenti, I. Jakoniuk, S.M. Anderson, B. Li, J. Pickel, R. McKay, B. Nadal-Ginard, D.M. Bodine, A. Leri, and P. Anversa. 2001a. Bone marrow cells regenerate infarcted myocardium. Nature. 410: 701±5. Orlic, D., J. Kajstura, S. Chimenti, F. Limana, I. Jakoniuk, F. Quaini, B. Nadal-Ginard, D.M. Bodine, A. Leri, and P. Anversa. 2001b. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci USA. 98: 10344±9. Ortiz, L.A., F. Gambelli, C. McBride, D. Gaupp, M. Baddoo, N. Kaminski, and D.G. Phinney. 2003. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci USA. 100: 8407±11. Otte, J.M., I.M. Rosenberg, and D.K. Podolsky. 2003. Intestinal myofibroblasts in innate immune responses of the intestine. Gastroenterology. 124: 1866±78. Papetti, M., J. Shujath, K.N. Riley, and I.M. Herman. 2003. FGF-2 antagonizes the TGFbeta1-mediated induction of pericyte alpha-smooth muscle actin expression: a role for myf-5 and Smad-mediated signaling pathways. Invest Ophthalmol Vis Sci. 44: 4994±5005. Pardali, K., and A. Moustakas. 2007. Actions of TGF-beta as tumor suppressor and prometastatic factor in human cancer. Biochim Biophys Acta. 1775: 21±62. Park, J.S., N.F. Huang, K.T. Kurpinski, S. Patel, S. Hsu, and S. Li. 2007. Mechanobiology of mesenchymal stem cells and their use in cardiovascular repair. Front Biosci. 12: 5098±116.

The myofibroblast in connective tissue repair and regeneration

75

Peled, A., D. Zipori, O. Abramsky, H. Ovadia, and E. Shezen. 1991. Expression of alphasmooth muscle actin in murine bone marrow stromal cells. Blood. 78: 304±9. Pereboeva, L., S. Komarova, G. Mikheeva, V. Krasnykh, and D.T. Curiel. 2003. Approaches to utilize mesenchymal progenitor cells as cellular vehicles. Stem Cells. 21: 389±404. Petersen, B.E., W.C. Bowen, K.D. Patrene, W.M. Mars, A.K. Sullivan, N. Murase, S.S. Boggs, J.S. Greenberger, and J.P. Goff. 1999. Bone marrow as a potential source of hepatic oval cells. Science. 284: 1168±70. Phan, S.H. 2002. The myofibroblast in pulmonary fibrosis. Chest. 122: 286S±9S. Phillips, R.J., M.D. Burdick, K. Hong, M.A. Lutz, L.A. Murray, Y.Y. Xue, J.A. Belperio, M.P. Keane, and R.M. Strieter. 2004. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest. 114: 438±46. Picinich, S.C., P.J. Mishra, J. Glod, and D. Banerjee. 2007. The therapeutic potential of mesenchymal stem cells. Cell- and tissue-based therapy. Expert Opin Biol Ther. 7: 965±73. Pittenger, M.F., and D.R. Marshak. 2001. Mesenchymal stem cells of human adult bone marrow. In Marshak, D.R., Gardner, R.L., and Gottlieb, D. (eds), Stem Cell Biology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Pittenger, M.F., and B.J. Martin. 2004. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res. 95: 9±20. Pittenger, M.F., A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, and D.R. Marshak. 1999. Multilineage potential of adult human mesenchymal stem cells. Science. 284: 143±7. Poss, K.D., L.G. Wilson, and M.T. Keating. 2002. Heart regeneration in zebrafish. Science. 298: 2188±90. Premdas, J., J.B. Tang, J.P. Warner, M.M. Murray, and M. Spector. 2001. The presence of smooth muscle actin in fibroblasts in the torn human rotator cuff. J Orthop Res. 19: 221±8. Prockop, D.J., and S.D. Olson. 2007. Clinical trials with adult stem/progenitor cells for tissue repair: let's not overlook some essential precautions. Blood. 109: 3147±51. Psaltis, P.J., A.C. Zannettino, S.G. Worthley, and S. Gronthos. 2008. Concise review: mesenchymal stromal cells: potential for cardiovascular repair. Stem Cells. 26: 2201±10. Qi, W., X. Chen, P. Poronnik, and C.A. Pollock. 2006. The renal cortical fibroblast in renal tubulointerstitial fibrosis. Int J Biochem Cell Biol. 38: 1±5. Quan, T.E., S.E. Cowper, and R. Bucala. 2006. The role of circulating fibrocytes in fibrosis. Curr Rheumatol Rep. 8: 145±50. Rafii, S., and D. Lyden. 2003. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med. 9: 702±12. Rajkumar, V.S., X. Shiwen, M. Bostrom, P. Leoni, J. Muddle, M. Ivarsson, B. Gerdin, C.P. Denton, G. Bou-Gharios, C.M. Black, and D.J. Abraham. 2006. Plateletderived growth factor-beta receptor activation is essential for fibroblast and pericyte recruitment during cutaneous wound healing. Am J Pathol. 169: 2254±65. Ramadori, G., and B. Saile. 2004. Portal tract fibrogenesis in the liver. Lab Invest. 84: 153±9. Rice, C.M., and N.J. Scolding. 2008. Autologous bone marrow stem cells ± properties and advantages. J Neurol Sci. 265: 59±62. Robertson, M.J., P. Gip, and D.V. Schaffer. 2008. Neural stem cell engineering: directed differentiation of adult and embryonic stem cells into neurons. Front Biosci. 13: 21±50.

76

Regenerative medicine and biomaterials in connective tissue repair

Ronnov-Jessen, L., and O.W. Petersen. 1993. Induction of alpha-smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia. Lab. Invest. 68: 696± 707. Rosenkranz, S. 2004. TGF-beta1 and angiotensin networking in cardiac remodeling. Cardiovasc Res. 63: 423±32. Roufosse, C.A., N.C. Direkze, W.R. Otto, and N.A. Wright. 2004. Circulating mesenchymal stem cells. Int J Biochem Cell Biol. 36: 585±97. Rudolph, R., J. Abraham, T. Vecchione, S. Guber, and M. Woodward. 1978. Myofibroblasts and free silicon around breast implants. Plast Reconstr Surg. 62: 185±96. Ruiz-Ortega, M., J. Rodriguez-Vita, E. Sanchez-Lopez, G. Carvajal, and J. Egido. 2007. TGF-beta signaling in vascular fibrosis. Cardiovasc Res. 74: 196±206. Saika, S., K. Ikeda, O. Yamanaka, K.C. Flanders, Y. Okada, T. Miyamoto, A. Kitano, A. Ooshima, Y. Nakajima, Y. Ohnishi, and W.W. Kao. 2006. Loss of tumor necrosis factor alpha potentiates transforming growth factor beta-mediated pathogenic tissue response during wound healing. Am J Pathol. 168: 1848±60. Sakai, N., T. Wada, H. Yokoyama, M. Lipp, S. Ueha, K. Matsushima, and S. Kaneko. 2006. Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis. Proc Natl Acad Sci USA. 103: 14098±103. Sands, R.W., and D.J. Mooney. 2007. Polymers to direct cell fate by controlling the microenvironment. Curr Opin Biotechnol. 18: 448±53. Sartore, S., A. Chiavegato, E. Faggin, R. Franch, M. Puato, S. Ausoni, and P. Pauletto. 2001. Contribution of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent bystander to active participant. Circ Res. 89: 1111±21. Sasaki, M., R. Abe, Y. Fujita, S. Ando, D. Inokuma, and H. Shimizu. 2008. Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol. 180: 2581±7. Sata, M., A. Saiura, A. Kunisato, A. Tojo, S. Okada, T. Tokuhisa, H. Hirai, M. Makuuchi, Y. Hirata, and R. Nagai. 2002. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med. 8: 403±9. Savera, A.T., and R.J. Zarbo. 2004. Defining the role of myoepithelium in salivary gland neoplasia. Adv Anat Pathol. 11: 69±85. Schmidt, M., G. Sun, M.A. Stacey, L. Mori, and S. Mattoli. 2003. Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol. 171: 380±9. Schmitt-Graff, A., O. Skalli, and G. Gabbiani. 1989. Alpha-smooth muscle actin is expressed in a subset of bone marrow stromal cells in normal and pathological conditions. Virchows Arch B Cell Pathol Incl Mol Pathol. 57: 291±302. Schneider, R.K., S. Neuss, R. Stainforth, N. Laddach, M. Bovi, R. Knuechel, and A. Perez-Bouza. 2008. Three-dimensional epidermis-like growth of human mesenchymal stem cells on dermal equivalents: contribution to tissue organization by adaptation of myofibroblastic phenotype and function. Differentiation. 76: 156± 67. Schurch, W., T.A. Seemayer, B. Hinz, and G. Gabbiani. 2007. Myofibroblast. In Mills, S.E. (ed.), Histology for Pathologists. Lippincott-Williams & Wilkins Pub., Philadelphia, PA, 123±64. Scotton, C.J., and R.C. Chambers. 2007. Molecular targets in pulmonary fibrosis: the myofibroblast in focus. Chest. 132: 1311±21. Seeberger, K.L., J.M. Dufour, A.M. Shapiro, J.R. Lakey, R.V. Rajotte, and G.S. Korbutt. 2006. Expansion of mesenchymal stem cells from human pancreatic ductal

The myofibroblast in connective tissue repair and regeneration

77

epithelium. Lab Invest. 86: 141±53. Segers, V.F., and R.T. Lee. 2008. Stem-cell therapy for cardiac disease. Nature. 451: 937±42. Segers, V.F., I. Van Riet, L.J. Andries, K. Lemmens, M.J. Demolder, A.J. De Becker, M.M. Kockx, and G.W. De Keulenaer. 2006. Mesenchymal stem cell adhesion to cardiac microvascular endothelium: activators and mechanisms. Am J Physiol Heart Circ Physiol. 290: H1370±7. Serini, G., M.L. Bochaton-Piallat, P. Ropraz, A. Geinoz, L. Borsi, L. Zardi, and G. Gabbiani. 1998. The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1. J. Cell Biol. 142: 873± 81. Shah, M., D.M. Foreman, and M.W. Ferguson. 1995. Neutralisation 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 (Pt 3): 985±1002. Shake, J.G., P.J. Gruber, W.A. Baumgartner, G. Senechal, J. Meyers, J.M. Redmond, M.F. Pittenger, and B.J. Martin. 2002. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg. 73: 1919±25; discussion 1926. Shen, W.L., P.J. Gao, Z.Q. Che, K.D. Ji, M. Yin, C. Yan, B.C. Berk, and D.L. Zhu. 2006. NAD(P)H oxidase-derived reactive oxygen species regulate angiotensin-II induced adventitial fibroblast phenotypic differentiation. Biochem Biophys Res Commun. 339: 337±43. Shephard, P., G. Martin, S. Smola-Hess, G. Brunner, T. Krieg, and H. Smola. 2004. Myofibroblast differentiation is induced in keratinocyte-fibroblast co-cultures and is antagonistically regulated by endogenous transforming growth factor-beta and interleukin-1. Am J Pathol. 164: 2055±66. Sheppard, D. 2005. Integrin-mediated activation of latent transforming growth factor beta. Cancer Metastasis Rev. 24: 395±402. Shi-Wen, X., E.A. Renzoni, L. Kennedy, S. Howat, Y. Chen, J.D. Pearson, G. BouGharios, M.R. Dashwood, R.M. du Bois, C.M. Black, C.P. Denton, D.J. Abraham, and A. Leask. 2007. Endogenous endothelin-1 signaling contributes to type I collagen and CCN2 overexpression in fibrotic fibroblasts. Matrix Biol. 26: 625±32. Shi-Wen, X., A. Leask, and D. Abraham. 2008. Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis. Cytokine Growth Factor Rev. 19: 133±44. Shull, M.M., I. Ormsby, A.B. Kier, S. Pawlowski, R.J. Diebold, M. Yin, R. Allen, C. Sidman, G. Proetzel, D. Calvin, et al. 1992. Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature. 359: 693±9. Siegel, P.M., and J. Massague. 2003. Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer. 3: 807±21. Siggelkow, W., A. Faridi, K. Spiritus, U. Klinge, W. Rath, and B. Klosterhalfen. 2003. Histological analysis of silicone breast implant capsules and correlation with capsular contracture. Biomaterials. 24: 1101±9. Silver, J., and J.H. Miller. 2004. Regeneration beyond the glial scar. Nat Rev Neurosci. 5: 146±56. Simmons, P.J., and B. Torok-Storb. 1991. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood. 78: 55±62. Skalli, O., P. Ropraz, A. Trzeciak, G. Benzonana, D. Gillessen, and G. Gabbiani. 1986. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth

78

Regenerative medicine and biomaterials in connective tissue repair

muscle differentiation. J. Cell Biol. 103: 2787±96. Spector, M. 2001. Musculoskeletal connective tissue cells with muscle: expression of muscle actin in and contraction of fibroblasts, chondrocytes, and osteoblasts. Wound Repair Regen. 9: 11±18. Strauer, B.E., M. Brehm, T. Zeus, M. Kostering, A. Hernandez, R.V. Sorg, G. Kogler, and P. Wernet. 2002. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation. 106: 1913± 18. Strehlow, D., and J.H. Korn. 1998. Biology of the scleroderma fibroblast. Curr Opin Rheumatol. 10: 572±8. Studeny, M., F.C. Marini, R.E. Champlin, C. Zompetta, I.J. Fidler, and M. Andreeff. 2002. Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Res. 62: 3603±8. Studeny, M., F.C. Marini, J.L. Dembinski, C. Zompetta, M. Cabreira-Hansen, B.N. Bekele, R.E. Champlin, and M. Andreeff. 2004. Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst. 96: 1593±603. Sundberg, C., M. Ivarsson, B. Gerdin, and K. Rubin. 1996. Pericytes as collagenproducing cells in excessive dermal scarring. Lab Invest. 74: 452±66. Suska, F., L. Emanuelsson, A. Johansson, P. Tengvall, and P. Thomsen. 2008. Fibrous capsule formation around titanium and copper. J Biomed Mater Res A. 85: 888±96. Taipale, J., J. Saharinen, and J. Keski-Oja. 1998. Extracellular matrix-associated transforming growth factor-beta: role in cancer cell growth and invasion. Adv Cancer Res. 75: 87±134. Tamaoki, M., K. Imanaka-Yoshida, K. Yokoyama, T. Nishioka, H. Inada, M. Hiroe, T. Sakakura, and T. Yoshida. 2005. Tenascin-C regulates recruitment of myofibroblasts during tissue repair after myocardial injury. Am J Pathol. 167: 71±80. Tamariz, E., and F. Grinnell. 2002. Modulation of fibroblast morphology and adhesion during collagen matrix remodeling. Mol Biol Cell. 13: 3915±29. ten Dijke, P., and H.M. Arthur. 2007. Extracellular control of TGFbeta signalling in vascular development and disease. Nat Rev Mol Cell Biol. 8: 857±69. Thannickal, V.J., G.B. Toews, E.S. White, J.P. Lynch, 3rd, and F.J. Martinez. 2004. Mechanisms of pulmonary fibrosis. Annu Rev Med. 55: 395±417. Thiery, J.P. 2002. Epithelial±mesenchymal transitions in tumour progression. Nat Rev Cancer. 2: 442±54. Todorovic, V., V. Jurukovski, Y. Chen, L. Fontana, B. Dabovic, and D.B. Rifkin. 2005. Latent TGF-beta binding proteins. Int J Biochem Cell Biol. 37: 38±41. Tomasek, J.J., M.B. Vaughan, and C.J. Haaksma. 1999. Cellular structure and biology of Dupuytren's disease. Hand Clin. 15: 21±34. Tomasek, J.J., G. Gabbiani, B. Hinz, C. Chaponnier, and R.A. Brown. 2002. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol. 3: 349±63. Uccelli, A., L. Moretta, and V. Pistoia. 2008. Mesenchymal stem cells in health and disease. Nat Rev Immunol. 8: 726±36. Uhal, B.D., J.K. Kim, X. Li, and M. Molina-Molina. 2007. Angiotensin-TGF-beta 1 crosstalk in human idiopathic pulmonary fibrosis: autocrine mechanisms in myofibroblasts and macrophages. Curr Pharm Des. 13: 1247±56. van der Loop, F.T., G. Schaart, E.D. Timmer, F.C. Ramaekers, and G.J. van Eys. 1996. Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells. J Cell Biol. 134: 401±11.

The myofibroblast in connective tissue repair and regeneration

79

Varcoe, R.L., M. Mikhail, A.K. Guiffre, G. Pennings, M. Vicaretti, W.J. Hawthorne, J.P. Fletcher, and H.J. Medbury. 2006. The role of the fibrocyte in intimal hyperplasia. J Thromb Haemost. 4: 1125±33. Varga, J., and D. Abraham. 2007. Systemic sclerosis: a prototypic multisystem fibrotic disorder. J Clin Invest. 117: 557±67. Verfaillie, C.M. 2002. Adult stem cells: assessing the case for pluripotency. Trends Cell Biol. 12: 502±8. Virag, J.I., and C.E. Murry. 2003. Myofibroblast and endothelial cell proliferation during murine myocardial infarct repair. Am J Pathol. 163: 2433±40. Wada, T., N. Sakai, K. Matsushima, and S. Kaneko. 2007. Fibrocytes: a new insight into kidney fibrosis. Kidney Int. 72: 269±73. Wakefield, L.M., and C. Stuelten. 2007. Keeping order in the neighborhood: new roles for TGFbeta in maintaining epithelial homeostasis. Cancer Cell. 12: 293±5. Wang, C.H., C.D. Huang, H.C. Lin, K.Y. Lee, S.M. Lin, C.Y. Liu, K.H. Huang, Y.S. Ko, K.F. Chung, and H.P. Kuo. 2008. Increased circulating fibrocytes in asthma with chronic airflow obstruction. Am J Respir Crit Care Med. 178: 583±91. Wang, D., J.S. Park, J.S. Chu, A. Krakowski, K. Luo, D.J. Chen, and S. Li. 2004. Proteomic profiling of bone marrow mesenchymal stem cells upon transforming growth factor beta1 stimulation. J Biol Chem. 279: 43725±34. Wang, J., R. Zohar, and C.A. McCulloch. 2006. Multiple roles of alpha-smooth muscle actin in mechanotransduction. Exp Cell Res. 312: 205±14. Wang, J.F., H. Jiao, T.L. Stewart, H.A. Shankowsky, P.G. Scott, and E.E. Tredget. 2007. Fibrocytes from burn patients regulate the activities of fibroblasts. Wound Repair Regen. 15: 113±21. Wang, Q., H.A. Breinan, H.P. Hsu, and M. Spector. 2000. Healing of defects in canine articular cartilage: distribution of nonvascular alpha-smooth muscle actincontaining cells. Wound Repair Regen. 8: 145±58. Weaver, C.V., and D.J. Garry. 2008. Regenerative biology: a historical perspective and modern applications. Regen Med. 3: 63±82. Wells, R.G. 2005. The role of matrix stiffness in hepatic stellate cell activation and liver fibrosis. J Clin Gastroenterol. 39: S158±61. Werner, S., and R. Grose. 2003. Regulation of wound healing by growth factors and cytokines. Physiol Rev. 83: 835±70. Willis, B.C., J.M. Liebler, K. Luby-Phelps, A.G. Nicholson, E.D. Crandall, R.M. du Bois, and Z. Borok. 2005. Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1: potential role in idiopathic pulmonary fibrosis. Am J Pathol. 166: 1321±32. Wipff, P.J., and B. Hinz. 2008. Integrins and the activation of latent transforming growth factor beta1 ± an intimate relationship. Eur J Cell Biol. 87: 601±15. Wipff, P.J., D.B. Rifkin, J.J. Meister, and B. Hinz. 2007. Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix. J Cell Biol. 179: 1311±23. Wu, Y., L. Chen, P.G. Scott, and E.E. Tredget. 2007a. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells. 25: 2648±59. Wu, Y., J. Wang, P.G. Scott, and E.E. Tredget. 2007b. Bone marrow-derived stem cells in wound healing: a review. Wound Repair Regen. 15 Suppl 1: S18±26. Wynn, T.A. 2007. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest. 117: 524±9. Wynn, T.A. 2008. Cellular and molecular mechanisms of fibrosis. J Pathol. 214: 199±210. Yamaguchi, Y., T. Kubo, T. Murakami, M. Takahashi, Y. Hakamata, E. Kobayashi, S. Yoshida, K. Hosokawa, K. Yoshikawa, and S. Itami. 2005. Bone marrow cells

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differentiate into wound myofibroblasts and accelerate the healing of wounds with exposed bones when combined with an occlusive dressing. Br J Dermatol. 152: 616±22. Yan, X., Y. Liu, Q. Han, M. Jia, L. Liao, M. Qi, and R.C. Zhao. 2007. Injured microenvironment directly guides the differentiation of engrafted Flk-1(+) mesenchymal stem cell in lung. Exp Hematol. 35: 1466±75. Yang, L., P.G. Scott, J. Giuffre, H.A. Shankowsky, A. Ghahary, and E.E. Tredget. 2002. Peripheral blood fibrocytes from burn patients: identification and quantification of fibrocytes in adherent cells cultured from peripheral blood mononuclear cells. Lab Invest. 82: 1183±92. Yang, Z., Z. Mu, B. Dabovic, V. Jurukovski, D. Yu, J. Sung, X. Xiong, and J.S. Munger. 2007. Absence of integrin-mediated TGF 1 activation in vivo recapitulates the phenotype of TGF 1-null mice. J Cell Biol. 176: 787±93. Ye, J., K. Yao, and J.C. Kim. 2006. Mesenchymal stem cell transplantation in a rabbit corneal alkali burn model: engraftment and involvement in wound healing. Eye. 20: 482±90. Yeung, T., P.C. Georges, L.A. Flanagan, B. Marg, M. Ortiz, M. Funaki, N. Zahir, W. Ming, V. Weaver, and P.A. Janmey. 2005. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil Cytoskeleton. 60: 24± 34. Yokota, T., Y. Kawakami, Y. Nagai, J.X. Ma, J.Y. Tsai, P.W. Kincade, and S. Sato. 2006. Bone marrow lacks a transplantable progenitor for smooth muscle type alpha-actinexpressing cells. Stem Cells. 24: 13±22. Young, H.E., T.A. Steele, R.A. Bray, J. Hudson, J.A. Floyd, K. Hawkins, K. Thomas, T. Austin, C. Edwards, J. Cuzzourt, M. Duenzl, P.A. Lucas, and A.C. Black, Jr. 2001. Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec. 264: 51±62. Young, R.G., D.L. Butler, W. Weber, A.I. Caplan, S.L. Gordon, and D.J. Fink. 1998. Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair. J Orthop Res. 16: 406±13. Zalewski, A., Y. Shi, and A.G. Johnson. 2002. Diverse origin of intimal cells: smooth muscle cells, myofibroblasts, fibroblasts, and beyond? Circ Res. 91: 652±5. Zeisberg, E.M., O. Tarnavski, M. Zeisberg, A.L. Dorfman, J.R. McMullen, E. Gustafsson, A. Chandraker, X. Yuan, W.T. Pu, A.B. Roberts, E.G. Neilson, M.H. Sayegh, S. Izumo, and R. Kalluri. 2007a. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med. 13: 952±61. Zeisberg, M., C. Yang, M. Martino, M.B. Duncan, F. Rieder, H. Tanjore, and R. Kalluri. 2007b. Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition. J Biol Chem. 282: 23337±47. Zhao, X.H., C. Laschinger, P. Arora, K. Szaszi, A. Kapus, and C.A. McCulloch. 2007. Force activates smooth muscle alpha-actin promoter activity through the Rho signaling pathway. J Cell Sci. 120: 1801±9. Zuk, P.A., M. Zhu, H. Mizuno, J. Huang, J.W. Futrell, A.J. Katz, P. Benhaim, H.P. Lorenz, and M.H. Hedrick. 2001. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 7: 211±28.

3

The structure of articular cartilage E . B . H U N Z I K E R , University of Bern, Switzerland

Abstract: Mature articular cartilage has a highly anisotropic structure which is indispensable for its mechanical competence. Immature articular cartilage has an isotropic structure which reflects its dual function as an articulating layer and a superficial growth plate. In this chapter, the structures of immature and mature articular cartilage are described, and the physiological mechanism underlying the evolution of the former into the latter is discussed. Inter-species differences in articular cartilage structure, and structural-functional correlations in humans, are also addressed. Key words: tissue resorption, tissue substitution, inter-species differences, anisotropic structure, isotropic structure.

3.1

Introduction

The abutting ends of the long bones that constitute a synovial joint are mantled by a layer of articular cartilage, which mediates the transfer of loads and permits the frictionless movements of the skeletal elements. This primarily mechanical function of the mature tissue is reflected in its highly anisotropic organization into several morphologically distinct zones. However, during the early phase of postnatal development, the tissue manifests an isotropic structure, which likewise has its functional corollaries. At this time, the tissue acts not only as a layer that mediates frictionless joint movement, but also as a surface growth plate for the rapid elongation and modelling of the epiphyseal bone. At a later phase of postnatal development, the isotropic organization of the tissue is transformed into the anisotropic architecture that typifies the adult organism. This transformation is achieved by a process of tissue resorption and substitution. During the growth phase of postnatal development, stem cells within the superficial zone feed the proliferating pool within the transitional and upper radial ones. Within the lower radial zone, the cells hypertrophy, and their extracellular matrix then undergoes mineralization prior to resorption. Towards the end of this phase, as the organism approaches skeletal maturity, the proliferative, but not the metabolic, activity of the cells ceases: they continue to remodel their extracellular matrix. Adult human articular cartilage is characterized by an extremely low numerical density of cells, which constitute less than 2% of the tissue volume.

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The mechanical properties of the layer are conferred chiefly by the abundant extracellular matrix. The structure of adult articular cartilage and the degree of anisotropy differ greatly between mammalian species that are commonly used as experimental animal models, for example the rabbit, the goat and the miniature pig. Not only the overall height of the layer, but also the relative heights of each zone vary tremendously for a given joint. The numerical density of cells within each zone is also subject to great diversity, although the total number contained within a unit area of tissue beneath the joint surface is remarkably constant. The fibrillar and macromolecular organization of each zone is likewise similar in the different mammalian species. However, the temporal course of postnatal growth and maturation differs greatly between these.

3.2

General structure and function of articular cartilage

The synovial joints of mammals comprise the abutting ends of mostly long bones, each of which is covered with a layer of articular cartilage. The articular cartilage layer is continuous with the underlying subchondral bone. The bony heads are surrounded by the synovial membrane and the joint capsule. Also associated with the joint are ligaments, tendons and muscle. A synovial space intervenes between the abutting layers of articular cartilage. This space is filled with a fluid, which is secreted by the lining cells of the synovial membrane. The abutting layers of articular cartilage, together with the intervening synovial fluid, permit the practically frictionless movement of the two associated bones and the transfer of loads between them. These biomechanical functions are reflected in the structure of articular cartilage. In the light microscope, this layer is revealed to be highly anisotropic in structure. The cells are organized into distinct vertical columns and horizontal strata relative to the articular surface. The first horizontal layer, namely, that closest to the articular surface, is referred to as the superficial zone. It consists of cells with an ellipsoidal or spindle-like form, whose long axis runs parallel to the articular surface, and which exist singly. The cells are embedded within a meshwork of fine collagen fibrils and fibres, which likewise run parallel to the articular surface. The superficial zone generally makes up one-thirteenth of the total height of the articular cartilage layer. It gives way to the transitional zone, which contains cells with a more rounded profile. These cells exist singly or in pairs. The collagen fibres form hemispherical arcades, which are continuous with vertically-running counterparts in the deeper zones. The transitional zone makes up about two-thirteenths of the total height of the articular cartilage layer. The superficial and transitional zones, being relatively poor in proteoglycans, stain weakly with cationic dyes. The transitional zone gives way to the radial zone, which is subdivided into an upper and a lower portion, each of which makes up about five-thirteenths of the total height of the articular cartilage layer. This

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zone is characterized by a gradual and marked increase in the size of the cells, which, in form, assume the appearance of oblate spheroids, and which are grouped into chondrons containing up to four vertically stacked chondrocytes. The concentration of proteoglycans increases continuously between the upper and the lower radial zones. Both regions are rich in vertically oriented collagen fibres, which course between the vertical columns of cells and which increase in thickness with increasing depth. The thick fibres at the base of the lower radial zone penetrate the underlying and fairly thin layer of calcified cartilage, which gives way to the subchondral bone. The interface between the layer of calcified cartilage and the subchondral bone plate has a characteristically undulating profile, which reflects the interdigitation of the two tissues and assists in anchorage. The upper border of the calcified cartilage layer is sometimes referred to as the tidemark. Its prominence as a line in conventionally processed tissue sections is an artefact, which is generated by decalcification. In the absence of decalcification, the mineralization front is represented as a discrete boundary between the unmineralized and the mineralized cartilage compartments (Hunziker et al., 1997). The anisotropic structure of mature articular cartilage has been described in detail by Benninghoff (1925). It is apparent in all synovial joints, irrespective of their size or of the animal species (Fig. 3.1). The maintenance of this highly anisotropic structure is indispensable for the functionality of the skeletal system as a whole (Godzinsky and Frank, 1990; Maroudas et al., 1985; Mow et al., 1990). Collagen, principally of types II, VI, IX and XI, is present in a non-soluble (polymerized) fibrillar form, although it can also exist in an unpolymerized, albeit fibril-associated, state (FAZIT-collagen) (Bruckner and Vanderrest, 1994), particularly type IX (Muller Glauser et al., 1986), and typically in adult tissue. Other collagen types (e.g., I, III and V) occur mainly in immature articular cartilage and at low levels. The intervening space between the collagen fibrils and fibres is occupied mainly by dermatan- and keratan-sulphatecontaining proteoglycans (Bayliss et al., 1983; Hardingham et al., 1992) and by fibromodulin (Hedbom and Heinegard, 1993; Hedlund et al., 1994). But a number of proteins and glycoproteins have also been identified within the extracellular matrix of mature articular cartilage. These include cartilage matrix protein (Hauser and Paulsson, 1994), cartilage oligomeric matrix protein, fibronectin, link proteins and osteonectin (Aeschlimann et al., 1995; Mundlos et al., 1992). As with the fibrillar collagens, these non-collagenous proteins and glycoproteins are distributed in a highly characteristic manner (Lorenzo et al., 1998; Wiberg et al., 2003). The extracellular matrix is generally recognized to be subdivided into three major compartments ± the pericellular, the territorial and the interterritorial (Fig. 3.2) ± which were first described by Meachim and Roy (1967) and Meachim and Stockwell (1973), and later characterized in detail by several groups of investigators (Eggli et al., 1985; Poole, 1993; Poole et al., 1982; Szirmai, 1963,

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3.1 Light micrograph of vertically-sectioned adult human articular cartilage (femoral condyle), illustrating its subdivision into superficial (S), transitional (T), upper radial (U), lower radial (L) and calcified cartilage (M) zones; the latter abuts on the subchondral bone plate (B). 100 m-thick saw-cut, surface-stained with basic Fuchsine, McNeal's Tetrachrome and Toluidine Blue O. Bar = 100 m (reproduced with permission from Hunziker, E.B. (1992) Articular cartilage structure in humans and experimental animals. In: Articular Cartilage and Osteoarthritis. K.E. Kuettner, R. Schleyerbach, J.G. Peyron and V.C. Hascall (eds.) Raven Press, New York, pp. 183±199).

1969; Szirmai and Doyle, 1961; Weiss et al., 1968). In these studies, the pericellular matrix coat was generally described to be free of fibrillar collagen (Eggli et al., 1988; Meachim and Roy, 1967; Weiss et al., 1968), but rich in soluble components, mainly proteoglycans. When articular cartilage is conventionally fixed in the absence of a cationic dye, as was the case in these former studies, proteoglycans are extracted from the pericellular matrix, which, as a consequence of the ensuing cell shrinkage, appears as an optically `empty' lacuna (Davies et al., 1962; Freeman, 1973; Hunziker et al., 1982; Meachim and Roy, 1967). The pericellular space abuts on the territorial domain, which is characterized by a basket-like network of collagen fibrils that embraces not only individual chondrocytes, but also chondrocyte groups (chondrons) (Hunziker, 1992; Poole, 1993; Poole et al., 1987; Szirmai, 1963, 1969). The territorial

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3.2 Electron micrograph of a pair of chondrocytes (chondron) from the lower radial zone of adult human articular cartilage, processed by high-pressure freezing, freeze substitution and Epon embedding. At this relatively low level of magnification, the pericellular matrix compartment (PM) appears as a homogeneously stained mantle around each chondrocyte, in contrast to the territorial domain (TM), which forms a fibrillar coat around the chondron. Both of these compartments may vary considerably in width. The interterritorium (ITM), which is not clearly demarcated from the territorium, is generally distinguished from the latter by its higher proportion of parallel-oriented fibrils (see arrows): it occupies the bulk of the intercellular space. Arrowheads: fine cellular processes. Bar = 4 m (reproduced with permission from Hunziker et al., 1997).

matrix gives way to the interterritorial compartment, which constitutes the bulk of the extracellular space. It is characterized by a gradual increase in fibril diameter on moving away from any given chondrocyte (Bonucci et al., 1974; Davies et al., 1962; Dearden et al., 1974; Hedlund et al., 1994) and, on a more global basis, from the articular cartilage surface to the calcified tissue layer (spanning an eight- to ten-fold difference in the global case) (Davies et al., 1962; Ghadially, 1983; Hunziker, 1992). During the 1990s, improvements in cryotechnical tissue processing permitted a more precise and more detailed ultrastructural analysis of the extracellular matrix of articular cartilage (Hunziker et al., 1996, 1997; Studer et al., 1995, 1996). Using this methodology, it was possible to identify for the first time a network of cross-banded filaments, 10±15 nm in diameter, with a periodicity characteristic of collagen fibrils, throughout the truly vitrified substance (Fig. 3.3), even within the pericellular matrix compartment. This finding, which has been since neglected, should be of interest to tissue engineers, who are currently

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3.3 Electron micrograph of adult human articular cartilage, processed by highpressure freezing, freeze substitution and Epon embedding. Interterritorial matrix from the lower radial zone, illustrating part of a large longitudinallysectioned bundle of fibrils (LB), which traverse the picture diagonally from the lower left-hand edge to the upper right-hand one. The fibrillar units making up this bundle exhibit various diameters and are organized with a torsional twist around its longitudinal axis. The periodic banding of each fibril is aligned in register with that of its neighbour. A number of fine cross-banded filaments (F), with a diameter of 10±15 nm, can be seen dispersed throughout the entire extracellular space. Both filaments and collagen fibrils are mantled by an electron-lucent zone; this is most patent in cross-sectioned elements, where it is manifested as a halo, particularly when these units form bundles (demarcated by arrowheads). Bar = 0.5 m (reproduced with permission from Hunziker et al., 1997).

intent on demonstrating the usefulness of nanofibrillar meshworks in the engineering particularly of articular cartilage (Schindler et al., 2006), and they should bear in mind that nature has forestalled their `novel' idea. This fine crossbanded filamentous network has been identified in human as well as in bovine cartilage, and probably exists also in other mammalian species. Its existence adds to the complexity of the collagen architecture within articular cartilage (Fig. 3.4), and its presence has unknowingly influenced the conception of biomechanical models (Lai et al., 1993; Mow et al., 1980; Parsons and Black, 1977; Setton et al., 1993). The presence of this fine nanofibrillar meshwork may also help to account more satisfactorily for the diffusion characteristics of macromolecules within the extracellular space. Although the precise chemical composition of these filaments has yet to be determined, their cross-banded appearance and 67 nm periodicity afford strong indications that they are collagenous in nature (type-XI collagen is a very likely candidate). Other structural phenomena of collagen fibrils, which may be of relevance in a biomechanical context, include `kinking' and `brushing' (see Fig. 3.5). Also of relevance are variations in the internal structural make-up of individual fibres, which reflect differences in the types of collagen of which they are composed.

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3.4 Schematic representation of the matrix organization in adult articular cartilage. The pericellular compartment surrounds individual chondrocytes and is of variable width; it is free of fibrillar components but contains an abundance of isotropically arranged, cross-banded filaments which occur not only here but extend throughout the entire extracellular space. This domain abuts on the territorial compartment, which distinguishes the chondron as a distinct morphological entity. It contains a basket-like arrangement of collagen fibrils and, like the pericellular domain, is variable in width. The remaining, and bulk, portion of the extracellular space is referred to as the interterritorium; two subpopulations of fibrils and of fibril bundles are distinguishable here on the basis of their orientation: one exhibits the classic parallel arrangement, which distinguishes this compartment from the territorium and gives rise, on a broader scale, to the arcade-like architecture described by Benninghoff (1922, 1925); the other manifests a more random (isotropic) organization (reproduced with permission from Hunziker et al., 1997).

3.3

Dual function of immature articular cartilage during postnatal growth

During the postnatal development of a mammalian organism, the long bones undergo extensive growth and modelling in all regions, but particularly in the epiphysis. In the latter region, activities are governed by the joint cartilage, which thus acts not only as an articulating layer, but also as a surface growth plate for this portion of the bone during postnatal development (Fig. 3.6) (Carlsson et al., 1986). Hence, immature mammalian articular cartilage is sometimes referred to as an articular epiphyseal complex (Carlsson et al., 1985, 1986). The growth activities of the metaphysis and of the diaphysis are governed by the `true' growth plate, viz., by the physis (Hunziker et al., 1987). At the time of birth and during postnatal development, the layer of articular cartilage manifests a fairly isotropic structural organization (Hunziker et al.,

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3.5 Electron micrograph illustrating a portion of the interterritorial matrix compartment derived from the lower radial zone of adult human articular cartilage, processed by high-pressure freezing. Unstained ultra-thin cryosection. Cross-sectioned collagen fibrils (CF) exhibit variable size and shape. Their profile areas manifest a characteristic `staining' pattern, namely, a pale central region, a narrow, dark boundary and a perifibrillar electron-lucent halo, which features are more readily discerned in fibril bundles (demarcated by arrowheads). Longitudinally-sectioned fibrils, which exhibit a periodic banding of 67 nm, with a sub-banding of approximately 22.5 nm, sometimes exhibit brushing (Br), namely, a splitting up into finer elements; single fibrils are sometimes observed to undergo abrupt changes in their course, a phenomenon that is referred to as kinking (K). Note the occurrence of bubbling (B), which is induced by prolonged exposure of the section to the electron beam during microscopy. Magnification: 50 000. Insert: Electron-diffraction analysis of the section, revealing the central electron beam to be surrounded by diffuse concentric rings. This manifestation indicates that tissue water has been frozen in an amorphous (vitrified) state (reproduced with permission from Hunziker et al., 1997).

2007; Schenk et al., 1986), which bears little resemblance to that of the adult organism (Hunziker, 1992). In rabbits, the layer is already quite thick one month after birth (Hunziker et al., 2007), but the cells are distributed fairly randomly (Fig. 3.7a). Although the cells of the superficial zone tend to be oriented with their long axis running parallel to the articular cartilage surface, those of the underlying zones are arranged more isotropically. They exist singly or as small clusters, but with no preferential spatial orientation. During the ensuing (second) month, the structural organization of the articular cartilage layer does not change dramatically. However, the cells become more anisotropically arranged, and their numerical density increases (Fig. 3.7b). During the third postnatal month, which, in rabbits, marks the onset of puberty and the attainment of sexual maturity, a more dramatic change in the structural organization of the articular

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3.6 (a) Scheme representing postnatal growth of the epiphysis (E) of a long bone. The articular cartilage (A) acts not only as an articulating layer, but also as a surface growth plate for the longitudinal, radial and lateral growth of the epiphyseal bone (radially-oriented arrows). The true growth plate (G), which is located between the epiphysis (E) and the metaphysis (M), is responsible for the longitudinal growth of the metaphysis (M) and the diaphysis (D) (reproduced with permission from Hunziker et al., 2007). (b) Scheme illustrating the bidirectional replication of superficial-zone stem cells during the growth-activity phase of the articular cartilage layer. This slowly proliferating pool supplies daughter cells which can be displaced either horizontally (1) or vertically (2). Horizontally-displaced cells replenish the stem-cell pool and effect lateral growth of the articular cartilage layer. Vertically-displaced cells feed the rapidly proliferating pool of transitamplifying cells in the transitional and upper radial zones. These latter cells effect rapid clonal expansion in the vertical direction. Later, they hypertrophy and initiate matrix mineralization. Longitudinal bone growth is achieved both by rapid clonal expansion in the vertical direction and by cell hypertrophy (modified from Lavker and Sun, 2000) (reproduced with permission from Hunziker et al., 2007).

cartilage layer is apparent (Fig. 3.7c). The individual chondrocytes are highly oriented in space. Indeed, in the transitional zone and in the upper and lower radial zones, the anisotropy coefficient of the cells is comparable to that of chondrocytes in mature tissue (Fig. 3.8). A noteworthy change occurs also at the mineralization front. During the first and second postnatal months, only the longitudinal septa are mineralized (Fig. 3.9a). Hence, the region is still open. But at the end of the third month, by which time the animals have attained sexual maturity, the mineralization front is continuous, viz., the horizontal as well as the longitudinal septa are mineralized (Fig. 3.9b,c). This closure of the mineralization front coincides with the cessation of the growth activity of the articular cartilage layer. During the fourth to the eighth postnatal months, no noteworthy changes occur in the structural organization of the articular cartilage layer (Fig. 3.7d). At the ultrastructural level, the architecture of the collagen fibrils within the extracellular matrix changes in parallel with the temporal process of cellular

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3.7 Light micrographs of articular cartilage tissue derived from the medial femoral condyle of New Zealand white rabbits 1 month (a), 2 months (b), 3 months (c) and 8 months (d) after birth. The images illustrate the transition from an isotropic cellular organization 1 month after birth (a) to a highly anisotropic one by the third postnatal month (c). At this latter stage, the architecture resembles that in the adult animal (d). The change in structural organization is accompanied by a decrease in the overall height of the articular cartilage layer. One micrometre-thick sections stained with Toluidine Blue O. Scale bars: (a) ˆ 220 m; (b, c, d) ˆ 110 m (reproduced with permission from Hunziker et al., 2007).

reorganization. One month after birth, the fibrils are arranged randomly (isotropically) throughout the extracellular space (Fig. 3.10a) in all zones. By the end of the third month (Fig. 3.10b), and up until the eighth month (Fig. 3.10c), they are organized as in adult articular cartilage. Within the pericellular and the territorial matrix compartments, the fibrils are arranged in a basket-like fashion around the cells and cell groups. After short-term labelling of the articular cartilage layer at the different postnatal ages with tritiated thymidine, which tags solely the rapidly

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3.8 Graph depicting the anisotropy index (AQ) for articular cartilage chondrocytes (AQ ˆ cell diameter in the horizontal direction [D (hor)] divided by cell diameter in the vertical direction [D (vert)]) as a function of the tissue zone and the postnatal age of the New Zealand white rabbits. In the superficial zone, and especially in the transitional and upper radial zones, the anisotropy index decreases between the first and third postnatal months. The decrease in this quotient reflects the transition from an isotropic to an anisotropic organization of the cells. No further significant change in the anisotropy index occurs after the third postnatal month, which indicates that the mature structural organization of the chondrocytes is attained at this juncture (puberty). Mean values are represented together with the standard error of the mean (reproduced with permission from Hunziker et al., 2007).

proliferating cell pools, autoradiography reveals a positive reaction only in the transitional and the upper radial zones. Hence, it is within these zones that the cells undergo the rapid proliferation and clonal expansion that are required for the speedy growth and neoformation of articular cartilage tissue. The location of the slowly proliferating precursor cells, with an estimated cycling time of about 8 days, can be identified immunohistochemically after administering bromodeoxyuridine to the rabbits on a daily basis (via the drinking water) during the final 12 days prior to sacrifice. This analysis reveals a

3.9 Light micrographs of articular cartilage tissue derived from the medial femoral condyle of New Zealand white rabbits 1 month (a) and 3 months (b, c) after birth. Each image depicts the border between the hyaline articular cartilage layer and the mineralization front. One month after birth (a), this region is still open, since only the longitudinal septa are mineralized. The site is characterized by a high level of resorptive activity, as evidenced by the abundance of macrophages (M) and osteoclasts (O). By the third postnatal month (b), this resorption of the longitudinal (mineralized) septa has ceased. The mineralization front between hyaline and calcified cartilage (CC) is continuous (namely closed), since not only the longitudinal, but also the horizontal septa are mineralized. However, the calcified cartilage is subject to physiological remodelling by osteoclasts. The characteristically undulating course of the calcified cartilage layer (seen at higher magnification in (c)) is believed to improve its mechanical anchorage within the subchondral bone plate (Broom and Pole, 1982; Keinan-Adamsky et al., 2005). RC ˆ osteoclastic resorption channel; T ˆ bone tissue; arrowheads ˆ bone-cartilage interface. One micrometre-thick sections stained with Toluidine Blue O. Scale bars: (a, b) ˆ 30 m; (c) ˆ 15 m (reproduced with permission from Hunziker et al., 2007).

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3.10 Electron micrographs of chondrocytes within the radial zone of articular cartilage tissue derived from the medial femoral condyle of New Zealand white rabbits 1 month (a), 3 months (b) and 8 months (c) after birth. In 1-month-old rabbits (a), the collagen fibrils are distributed randomly (isotropically) and homogeneously throughout the entire extracellular space. No distinct compartmentalization of the matrix is apparent. In 3- (b) and 8-month-old (c) rabbits, the extracellular space is organized into distinct pericellular (P), territorial (T) and interterritorial (I) compartments. The narrow pericellular matrix compartment is highly electron-dense owing to the abundance of ruthenium-hexaamine-trichloride-precipitated proteoglycans. Within the territorial compartment, the collagen fibrils are arranged in a typically basketlike fashion around the cells. Within the interterritorium, which constitutes the bulk of the extracellular space, the collagen fibrils are oriented parallel to each other and in a predominantly longitudinal direction. Scale bars: (a) ˆ 10 m; (b, c) ˆ 5 m (reproduced with permission from Hunziker et al., 2007).

positive reaction within all cell nuclei of all zones, including the superficial one. Hence, the slowly proliferating pool of precursor cells (which resemble stem cells in this respect) is located within the superficial zone, in analogy to the resting zone (referred to in the older literature as the stem-cell zone) of a true growth plate (Hunziker et al., 1987; Kember, 1960).

3.4

Physiological mechanism underlying the evolution of a mature from an immature articular cartilage structure

Mature articular cartilage is characterized by a high degree of structural anisotropy, its cells being organized into well-defined vertical columns and horizontal septa, whereas immature articular cartilage is more isotropic in structure (Fig. 3.11). The mechanism underlying the evolution of the mature from the immature architecture has been only recently elucidated (Huniker et al., 2007). It was hypothesized that the articular cartilage layer of synovial joints underwent structural reorganization either by a process of internal tissue remodelling, or by one of controlled tissue resorption (at the vascular invasion front) that was synchronized with tissue renewal (on the basis of the proliferative activity of precursor cells within the superficial zone). The daily growth rate of the epiphyseal bone was determined in rabbits at monthly intervals from the first to the eighth postnatal months (Huniker et al., 2007) according to the tetracycline-labelling principle (Hulth and Olerud, 1962). The daily growth rate

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3.11 Schemes illustrating the structure of immature (left-hand picture) and mature (right-hand picture) articular cartilage tissue. During foetal and early postnatal life, the chondrocytes are distributed randomly (isotropically) in space. However, a vectoral gradient in cell size and shape is apparent: near the articular surface, the cells are small and horizontally flattened; with increasing depth, they become larger, rounder and ultimately (near the vascular invasion front) irregular in form. In adult articular cartilage, the chondrocytes are organized anisotropically into distinct vertical columns and horizontal zones (superficial/tangential, transitional, upper and lower radial, and calcified). Our data reveal this structural transformation to be achieved not by a process of internal remodelling, but by the resorption and neoformation of tissue. Arrows indicate the level of the mineralization front (namely, the tidemark) (reproduced with permission from Hunziker et al., 2007).

was highest 1 month after birth and decreased almost exponentially thereafter until the time of sexual maturity (between the third and fourth postnatal months). After the fourth month, growth activity ceased altogether (Fig. 3.12). The height of the articular cartilage layer (Fig. 3.13) decreased in parallel with the decrease in bone growth rate. The total bone length gain achieved during each of the first five postnatal months decreased with time (Fig. 3.14). However, during the growth phase (i.e., during the first three postnatal months), the monthly gain in bone length exceeded the height of the articular cartilage layer (from which that of the superficial zone had been subtracted). This finding indicates that immature articular cartilage does not become reorganized by a process of internal tissue remodelling. If this were the case, then the total bone length gain per month during the growth phase would be smaller than the corresponding height of the articular cartilage layer (after subtracting that of the superficial zone). Hence, immature articular cartilage must be completely resorbed and replaced by new tissue. The existing tissue is destroyed at the vascular invasion front. The superficial zone alone is spared, and serves as the source of the precursor cells which give rise to new tissue. One of the implications of these findings is that the postnatal structural reorganization of articular cartilage tissue occurs not at a fixed topographical location in space, but by a process of growth involving an elongation of the underlying bone. This circumstance is of importance in the engineering of articular cartilage within adult organisms. Adult articular cartilage lesions would be ideally repaired by tissue that manifests a high degree of structural anisotropy

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3.12 Graph depicting the daily growth rate of the epiphyseal bone as a function of the postnatal age of the New Zealand white rabbits. The growth rate declines precipitously up to the third postnatal month; by the fourth month, growth activity has ceased altogether. Mean values are represented together with the standard error of the mean (reproduced with permission from Hunziker et al., 2007).

3.13 Graph depicting the overall height of the articular cartilage layer as a function of the postnatal age of the New Zealand white rabbits. The height of the articular cartilage layer decreases precipitously and almost linearly up to the third postnatal month, at which juncture the mature structural organization of the tissue is achieved and the animals attain sexual maturity. Thereafter (between 4 and 8 months), the height of the articular cartilage layer does not change significantly. Mean values are represented together with the standard error of the mean (reproduced with permission from Hunziker et al., 2007).

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3.14 Bar graph comparing the net monthly length-gain in the epiphyseal bone (black columns) with the height of the articular cartilage layer from which that of the superficial zone has been substracted (grey columns) at each postnatal month. During the first 2 months, when the articular cartilage layer is undergoing structural reorganization, the net length-gain in the epiphyseal bone exceeds the height of the articular cartilage layer. This finding indicates that the articular cartilage layer is structurally reorganized not by a process of internal remodelling, but by the resorption of all zones except the superficial (stem-cell) one and their neoformation by appositional growth of the latter. If a process of internal remodelling were involved, the height of the articular cartilage layer (excluding the superficial zone) would exceed the net monthly length-gain in the epiphyseal bone. Mean values are represented together with the standard error of the mean (reproduced with permission from Hunziker et al., 2007).

from the very onset of the healing process, in order to ensure its longevity and mechanical competence (Wong et al., 1997). However, current cell-based approaches involve the implantation of immature cartilage, with a random distribution of cells (for review, see Hunziker, 2002). Since the structural reorganization of immature cartilage under physiological conditions does not take place at a fixed topographical position in space, it is highly unlikely that the current tissue-engineering approaches will lead to optimal repair results.

3.5

Inter-species differences in articular cartilage structure, and structure±function correlations in humans

A quantitative description of the three-dimensional structures (Cruz Orive and Hunziker, 1986) that make up the mature human articular cartilage layer is essential for a thorough understanding of its biochemical, biophysical and biomechanical properties, which determine its physiological functions. Such

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data are also necessary for the rational development of cartilage-engineering strategies. Furthermore, structural differences between mammalian species have to be borne in mind when selecting an animal model for the in-vivo testing of a potential repair strategy for human use. Currently, baseline structural data are available for only a few human joint types; most especially for the medial femoral condyle of young and middle-aged adults with no known pathological affections or history of joint disease (Hunziker et al., 2002; Shepherd and Seedhom, 1999). In this joint, the mean heights of the layers of hyaline and calcified cartilage, and of the subchondral bone plate, are 2±4 mm (coefficient of error: 22%), 0.13 mm (coefficient of error: 74%) and 0.19 mm (coefficient of error: 57%), respectively. In small and middle-sized experimental mammals, namely, in rabbits, sheep and goats, the hyaline cartilage layer in the corresponding joint is much thinner, viz., 0.25, 0.4 and 0.9 mm, respectively (Hulth and Olerud, 1962; Hunziker, 1999; Masoud et al., 1986). However, in larger mammals, such as bovine cows and horses, it is not thicker but of similar height. The calcified cartilage layer and the subchondral bone plate are, on the other hand, much thinner in humans than they are in rabbits, goats and sheep (Hulth and Olerud, 1962; Masoud et al., 1986). As may be seen from the data cited above, in the adult human medial femoral condyle, the heights of the calcified cartilage layer and the subchondral bone plate are characterized by very high coefficients of error. These great intra- and inter-individual variations around the mean heights reflect the undulating upperand lower-surface profiles of the calcified cartilage layer, which in turn reflect its interdigitation with the overlying hyaline cartilage layer and the underlying subchondral bone plate, and which facilitate anchorage. With respect to the junction between the hyaline and the calcified cartilage layers, this undulating profile could also enhance the diffusion of nutrients (from the former to the latter stratum). The volume density of chondrocytes within the articular cartilage layer of the adult human medial femoral condyle is overall very low (mean: 0.65%; coefficient of error: 9%), and decreases with increasing distance from the surface. Between the superficial zone and the lower portion of the lower radial zone, it drops by a factor of two (from 2.6% to 1.2%). Such low values have not been encountered in any other mammalian species. In rabbits and goats, for example, the overall value is about 12% (Hulth and Olerud, 1962; Hunziker, 1999). Among human bodily tissues, articular cartilage is unique in having such a low volume density of cells. In simple terms, the low volume density of chondrocytes indicates that the cells are very sparsely distributed, which in turn implies that the metabolic and synthetic activities of any given chondrocyte sustain a very large domain of the extracellular matrix. Given that articular cartilage is avascular, and that the chondrocytes thus depend upon the diffusion of oxygen and nutritients over a great distance (from blood capillaries within the synovium and the subchondral bone plate), it is not surprising that the metabolic activities of the cells are conducted chiefly along anaerobic pathways (Wong

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and Hunziker, 2001). The volume of the extracellular domain that is sustained by a single chondrocyte is 104 040 mm3 (coefficient of error: 4.9%) for the articular cartilage layer as a whole, which rises to 106 707 mm3 for the lower portion of the upper radial zone (Hunziker et al., 2002). These values are about ten-fold higher than those for the articular cartilage layer of the medial femoral condyle in rabbits (Eggli et al., 1988). And in the growth plate, such huge matrix domains have not been described for any mammalian species (Cruz Orive and Hunziker, 1986; Hunziker et al., 1987), including humans. Their great compass in human articular cartilage has an impact on the remodelling activity of the outskirts of the interterritorium, which is very low. The structure of the articular cartilage layer changes in response to the mode of biomechanical loading. A sustained increase in load accelerates tissue remodelling, which leads to a dramatic thinning of the articular cartilage layer (Vanwanseele et al., 2002). However, sustained decreases in load lead to only a minimal thickening of the layer (Eckstein et al., 2002; Shepherd and Seedhom, 1999). The adaptive potential of articular cartilage is probably limited by cellscale biophysical considerations. Since the activity of chondrocytes depends greatly on the diffusion of solutes through the avascular extracellular matrix, their metabolism is closely coupled with the local transport of oxygen and nutrients, which is partly governed by the organization of the matrix (Maroudas, 1975). This tenet is borne out by the finding that sustained loading of a weightbearing joint, which presumably induces local changes in the mechanical properties of the articular cartilage tissue, can give rise to highly circumscribed osteoarthritic lesions (Buckwalter and Mankin, 1998; Froimson et al., 1997; Shepherd and Seedhom, 1999). Furthermore, the metabolic activities of chondrocytes (Wong et al., 1996) and the biomechanical properties of the matrix (Chen et al., 2001) are known to vary between zones, and these differences represent the functional correlates of differences in structure. However, the thickness of human knee-joint cartilage varies between anatomical locations in a manner that is seemingly independent of the structural organization of the cells and the matrix (Quinn et al., 2005). Anatomical variations in knee-joint cartilage appear to be governed by factors such as the degree of congruency between the apposing bony elements (Simon et al., 1973) and topographical relationships with other tissues, for example, the meniscus (Shepherd and Seedhom, 1999; Vanwanseele et al., 2002). However, as aforeindicated, cartilage thickness is not trivially related to load bearing. A sustained increase in load accelerates tissue remodelling. This response can result in a decrease in joint surface area (Eckstein et al., 2002) and in site-specific differences in the mechanical properties of the articular cartilage (Froimson et al., 1997; Shepherd and Seedhom, 1999). The uniformity of cartilage structure in different anatomical regions of the knee joint, with different functional needs, indicates the existence of fundamental cell-scale constraints. For example, the metabolism of a given cell must be augmented if the matrix volume that it controls increases

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(assuming a constant rate of turnover); but it cannot do so indefinitely, owing to limitations in the transport of solutes both to and from the cell. A balance must be struck between decreased solute transport and increased mechanical stiffness, which is effected by an increase in matrix density. These cell-scale constraints may account for the observation that an increase in cartilage thickness is associated with a decrease in its mechanical stiffness (Froimson et al., 1997; Lyyra et al., 1999; Shepherd and Seedhom, 1999). The decrease in the cellularity of human articular cartilage that occurs with age (Mow et al., 1993) may contribute to its diminishing capacity for repair, since fewer cells govern an increasingly expanding volume of matrix. However, not only the numerical density of cells but also their organization influences the mechanical stiffness of articular cartilage: although foetal cartilage is characterized by a higher volume density of cells than is mature cartilage, its cells and matrix are organized isotropically, whereas in mature cartilage, they are arranged anisotropically; but the latter is mechanically stiffer than the former (Wong and Hunziker, 2001).

3.6

References

Aeschlimann, D., Kaupp, O., and Paulsson, M. Transglutaminase-catalyzed matrix crosslinking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate. J Cell Biol 129: 881±892; 1995. Bayliss, M. T., Venn, M., Maroudas, A., and Ali, S. Y. Structure of proteoglycans from different layers of human articular cartilage. Biochem J 209: 387±400; 1983. Benninghoff, A. Ueber den funktionellen Bau des Knorpels. Anat Anz Erg Heft 55: 250± 267; 1922. Benninghoff, A. Form und Bau der Gelenkknorpel in ihren Beziehungen zur Funktion. II. Der Aufbau des Gelenknorpels in seinen Beziehungen zur Funktion. Z Zellforsch Mikrosk Anat 2: 783±862; 1925. Bonucci, E., Cuicchio, M., and Dearden, L. C. Investigations of ageing in costal and tracheal cartilage of rats. Z Zellforsch Mikrosk Anat 147: 505±527; 1974. Broom, N. D., and Poole, C. A. A functional-morphological study of the tidemark region of articular cartilage maintained in a non-viable physiological condition. J Anat 135: 65±82; 1982. Bruckner, P., and Vanderrest, M. Structure and function of cartilage collagens. Microsc Res Technique 28: 378±384; 1994. Buckwalter, J. A., and Mankin, H. J. Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation. Instr Course Lect 47: 487±504; 1998. Carlson, C. S., Hilley, H. D., and Henrikson, C. K. Ultrastructure of normal epiphyseal cartilage of the articular±epiphyseal cartilage complex in growing swine. Am J Vet Res 46: 306±313; 1985. Carlson, C. S., Hilley, H. D., Henrikson, C. K., and Meuten, D. J. The ultrastructure of osteochondrosis of the articular±epiphyseal cartilage complex in growing swine. Calcif Tissue Int 38: 44±51; 1986. Chen, S. S., Falcovitz, Y. H., Schneiderman, R., Maroudas, A., and Sah, R. L. Depthdependent compressive properties of normal aged human femoral head articular cartilage: relationship to fixed charge density. Osteoarthritis Cartilage 9: 561±569; 2001.

102

Regenerative medicine and biomaterials in connective tissue repair

Cruz Orive, L. M., and Hunziker, E. B. Stereology for anisotropic cells: application to growth cartilage. J Microsc 143: 47±80; 1986. Davies, D. V., Barnett, C. H., Cochrane, W., and Palfrey, A. J. Electron microscopy of articular cartilage in the young adult rabbit. Ann Rheum Dis 21: 11±22; 1962. Dearden, L. C., Bonucci, E., and Cuicchio, M. An investigation of ageing in human costal cartilage. Cell Tissue Res 152: 305±337; 1974. Eckstein, F., Faber, S., Muhlbauer, R., Hohe, J., Englmeier, K. H., Reiser, M., and Putz, R. Functional adaptation of human joints to mechanical stimuli. Osteoarthritis Cartilage 10: 44±50; 2002. Eggli, P. S., Herrmann, W., Hunziker, E. B., and Schenk, R. K. Matrix compartments in the growth plate of the proximal tibia of rats. Anat Rec 211: 246±257; 1985. Eggli, P. S., Hunziker, E. B., and Schenk, R. K. Quantitation of structural features characterizing weight- and less-weight-bearing regions in articular cartilage: a stereological analysis of medial femoral condyles in young adult rabbits. Anat Rec 222: 217±227; 1988. Freeman, M. A. R. Adult Articular Cartilage. London: Pitman Medical; 1973. Froimson, M. I., Ratcliffe, A., Gardner, T. R., and Mow, V. C. Differences in patellofemoral joint cartilage material properties and their significance to the etiology of cartilage surface fibrillation. Osteoarthritis Cartilage 5: 377±386; 1997. Ghadially, F. N. Fine Structure of Synovial Joints. A Text and Atlas of the Ultrastructure of Normal and Pathological Articular Tissues. London: Butterworth & Co. (Publishers) Ltd; 1983. Grodzinsky, A. J., and Frank, E. H. Electromechanical and physicochemical regulation of cartilage strength and metabolism. In: D. W. L. Hukins (ed.), Connective Tissue Matrix Part 2, pp. 91±126. London: The Macmillan Press Ltd; 1990. Hardingham, T. E., Fosang, A. J., and Dudhia, J. Aggrecan: the chondroitin sulfate/ keratan sulfate proteoglycan from cartilage. In: K. E. Kuettner, R. Schleyerbach, J. G. Peyron, and V. C. Hascall (eds.), Articular Cartilage and Osteoarthritis, pp. 5± 20. New York: Raven Press; 1992. Hauser, N., and Paulsson, M. Native cartilage matrix protein (CMP) ± a compact trimer of subunits assembled via a coiled-coil alpha-helix. J Biol Chem 269: 25747±25753; 1994. Hedbom, E., and Heinegard, D. Binding of fibromodulin and decorin to separate sites on fibrillar collagens. J Biol Chem 268: 27307±27312; 1993. Hedlund, H., Mengarelliwidholm, S., Heinegard, D., Reinholt, F. P., and Svensson, O. Fibromodulin distribution and association with collagen. Matrix Biol 14: 227±232; 1994. Hulth, A., and Olerud, S. Tetracycline labelling of growing bone. Acta Soc Med Ups 67: 219±231; 1962. Hunziker, E. B. Articular cartilage structure in humans and experimental animals. In: K. E. Kuettner, R. Schleyerbach, J. G. Peyron, and V. C. Hascall (eds.), Articular Cartilage and Osteoarthritis, pp. 183±199. New York: Raven Press; 1992. Hunziker, E. B. Biologic repair of articular cartilage. Defect models in experimental animals and matrix requirements. Clin Orthop Relat Res, S135±S146; 1999. Hunziker, E. B. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage 10: 432±463; 2002. Hunziker, E. B., Herrmann, W., and Schenk, R. K. Improved cartilage fixation by ruthenium hexammine trichloride (RHT). A prerequisite for morphometry in growth cartilage. J Ultrastruct Res 81: 1±12; 1982. Hunziker, E. B., Schenk, R. K., and Cruz-Orive, L. M. Quantitation of chondrocyte

The structure of articular cartilage

103

performance in growth-plate cartilage during longitudinal bone growth. J Bone Joint Surg Am 69: 162±173; 1987. Hunziker, E. B., Wagner, J., and Studer, D. Vitrified articular cartilage reveals novel ultra-structural features respecting extracellular matrix architecture. Histochem Cell Biol 106: 375±382; 1996. Hunziker, E. B., Michel, M., and Studer, D. Ultrastructure of adult human articular cartilage matrix after cryotechnical processing. Microsc Res Tech 37: 271±284; 1997. Hunziker, E. B., Quinn, T. M., and Hauselmann, H. J. Quantitative structural organization of normal adult human articular cartilage. Osteoarthritis Cartilage 10: 564±572; 2002. Hunziker, E. B., Kapfinger, E., and Geiss, J. The structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development. Osteoarthritis Cartilage 15: 403±413; 2007. Keinan-Adamsky, K., Shinar, H., and Navon, G. The effect of detachment of the articular cartilage from its calcified zone on the cartilage microstructure, assessed by 2Hspectroscopic double quantum filtered MRI. J Orthop Res 23: 109±117; 2005. Kember, N. F. Cell division in endochondral ossification. A study of cell proliferation in rat bones by the method of tritiated thymidine autoradiography. J Bone Joint Surg (Br) 42: 824±839; 1960. Lai, W. M., Mow, V. C., and Zhu, W. B. Constitutive modeling of articular cartilage and biomacromolecular solutions. J Biomech Eng 115: 474±480; 1993. Lavker, R. M., and Sun, T. T. Epidermal stem cells: properties, markers, and location. Proc Natl Acad Sci USA 97: 13473±13475; 2000. Lorenzo, P., Bayliss, M. T., and Heinegard, D. A novel cartilage protein (CILP) present in the mid-zone of human articular cartilage increases with age. J Biol Chem 273: 23463±23468; 1998. Lyyra, T., Kiviranta, I., Vaatainen, U., Helminen, H. J., and Jurvelin, J. S. In vivo characterization of indentation stiffness of articular cartilage in the normal human knee. J Biomed Mater Res 48: 482±487; 1999. Maroudas, A. Biophysical chemistry of cartilaginous tissues with special reference to solute and fluid transport. Biorheology 12: 233±248; 1975. Maroudas, A., Ziv, I., Weisman, N., and Venn, M. Studies of hydration and swelling pressure in normal and osteoarthritic cartilage. Biorheology 22: 159±169; 1985. Masoud, I., Shapiro, F., Kent, R., and Moses, A. A longitudinal study of the growth of the New Zealand white rabbit: cumulative and biweekly incremental growth rates for body length, body weight, femoral length, and tibial length. J Orthop Res 4: 221± 231; 1986. Meachim, G., and Roy, S. Intracytoplasmic filaments in the cells of adult human articular cartilage. Ann Rheum Dis 26: 50±58; 1967. Meachim, G., and Stockwell, R. A. The matrix. In: M. A. R. Freeman (ed.), Adult Articular Cartilage, pp. 1±50. London: Pitman Medical; 1973. Mow, V. C., Kuei, S. C., Lai, W. M., and Armstrong, C. G. Biphasic creep and stress relaxation of articular cartilage in compression: theory and experiments. J Biomech Eng 102: 73±84; 1980. Mow, V. C., Ratcliffe, A., and Woo, S. L. Y. Biomechanics of Diarthrodial Joints. New York: Springer-Verlag; 1990. Mow, V. C., Ateshian, G. A., and Spilker, R. L. Biomechanics of diarthroidal joints ± a review of 20 years of progress. J Biomech Eng 115: 460±467; 1993.

104

Regenerative medicine and biomaterials in connective tissue repair

Muller Glauser, W., Humbel, B., Glatt, M., Strauli, P., Winterhalter, K. H., and Bruckner, P. On the role of type IX collagen in the extracellular matrix of cartilage: type IX collagen is localized to intersections of collagen fibrils. J Cell Biol 102: 1931±1939; 1986. Mundlos, S., Schwahn, B., Reichert, T., and Zabel, B. Distribution of osteonectin mRNA and protein during human embryonic and fetal development. J Histochem Cytochem 40: 283±291; 1992. Parsons, J. R., and Black, J. The viscoelastic shear behavior of normal rabbit articular cartilage. J Biomech 10: 21±29; 1977. Poole, A. R., Pidoux, I., Reiner, A., and Rosenberg, L. An immunoelectron microscope study of the organization of proteoglycan monomer, link protein, and collagen in the matrix of articular cartilage. J Cell Biol 93: 921±937; 1982. Poole, C. A. The structure and function of articular cartilage matrices. In: J. F. J. Woessner and D. S. Howell (eds.), Joint Cartilage Degradation. Basic and Clinical Aspects, pp. 1±35. New York: Marcel Dekker, Inc.; 1993. Poole, C. A., Flint, M. H., and Beaumont, B. W. Chondrons in cartilage: ultrastructural analysis of the pericellular microenvironment in adult human articular cartilages. J Orthop Res 5: 509±522; 1987. Quinn, T. M., Hunziker, E. B., and Hauselmann, H. J. Variation of cell and matrix morphologies in articular cartilage among locations in the adult human knee. Osteoarthritis Cartilage 13: 672±678; 2005. Schenk, R. K., Eggli, P. S., and Hunziker, E. B. Articular cartilage morphology. In: K. Kuettner, R. Schleyerbach, and V. C. Hascall (eds.), Articular Cartilage Biochemistry, pp. 3±22. New York: Raven Press; 1986. Schindler, M., Nur, E. K. A., Ahmed, I., Kamal, J., Liu, H. Y., Amor, N., Ponery, A. S., Crockett, D. P., Grafe, T. H., Chung, H. Y., Weik, T., Jones, E., and Meiners, S. Living in three dimensions: 3D nanostructured environments for cell culture and regenerative medicine. Cell Biochem Biophys 45: 215±227; 2006. Setton, L. A., Zhu, W. B., and Mow, V. C. The biphasic poroviscoelastic behavior of articular cartilage ± role of the surface zone in governing the compressive behavior. J Biomech 26: 581±592; 1993. Shepherd, D. E., and Seedhom, B. B. Thickness of human articular cartilage in joints of the lower limb. Ann Rheum Dis 58: 27±34; 1999. Simon, W. H., Friedenberg, S., and Richardson, S. Joint congruence. A correlation of joint congruence and thickness of articular cartilage in dogs. J Bone Joint Surg Am 55: 1614±1620; 1973. Studer, D., Michel, M., Wohlwend, M., Hunziker, E. B., and Buschmann, M. Vitrification of articular cartilage by high-pressure freezing. J Microsc 179: 321±332; 1995. Studer, D., Chiquet, M., and Hunziker, E. B. Evidence for a distinct water rich layer surrounding collagen II fibrils in articular cartilage extracellular matrix. J Structur Biol 117: 81±85; 1996. Szirmai, J. A. Quantitative approaches in the histochemistry of mucopolysaccharides. J Histochem Cytochem 11: 24±34; 1963. Szirmai, J. A. Structure of cartilage. In: A. Engel and T. Larsson (eds.), Thule International Symposium on Aging of Connective and Skeletal Tissue, pp. 163±184. Stockholm: Nordiska Boekhandelns; 1969. Szirmai, J. A., and Doyle, J. Quantitative histochemical and chemical studies on the composition of cartilage. J Histochem Cytochem 9: 611±618; 1961. Vanwanseele, B., Eckstein, F., Knecht, H., Stussi, E., and Spaepen, A. Knee cartilage of spinal cord-injured patients displays progressive thinning in the absence of normal

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joint loading and movement. Arthritis Rheum 46: 2073±2078; 2002. Weiss, C., Rosenberg, L., and Helfet, A. J. An ultrastructural study of normal young adult human articular cartilage. J Bone Joint Surg (Am) 50: 663±674; 1968. Wiberg, C., Klatt, A. R., Wagener, R., Paulsson, M., Bateman, J. F., Heinegard, D., and Morgelin, M. Complexes of matrilin-1 and biglycan or decorin connect collagen VI microfibrils to both collagen II and aggrecan. J Biol Chem 278: 37698±37704; 2003. Wong, M., and Hunziker, E. B. Articular cartilage biology and biomechanics. In: C. Erggelet and M. Steinwachs (eds.), Gelenkknorpeldefekte, pp. 15±28. Darmstadt: Steinkopff Verlag; 2001. Wong, M., Wuethrich, P., Eggli, P., and Hunziker, E. B. Zone-specific cell biosynthetic activity in mature bovine articular cartilage: a new method using confocal microscopic stereology and quantitative autoradiography. J Orthop Res 14: 424± 432; 1996. Wong, M., Wuthrich, P., Buschmann, M. D., Eggli, P., and Hunziker, E. B. Chondrocyte biosynthesis correlates with local tissue strain in statically compressed adult articular cartilage. J Orthop Res 15: 189±196; 1997.

4

Measuring the biomechanical properties of cartilage cells D . L . B A D E R and M . M . K N I G H T , Queen Mary University of London, UK

Abstract: Current techniques are described to deform chondrocytes and derive both quasi-static and viscoelastic parameters. The influence of intracellular structures, such as cytoskeletal elements and the nucleus, on these parameters is highlighted. The chapter also describes the metabolic response of chondrocytes to biomechanical conditioning. Output parameters, including cell proliferation and matrix synthesis, are estimated with respect to cell source, scaffolds and culture conditions. Key words: chondrocytes, loading techniques, cell mechanics, biomechanical conditioning, metabolic response.

4.1

Introduction

Mechanical loading is essential for the development, health and homeostasis of articular cartilage. This occurs primarily through a process of cellular mechanotransduction whereby the chondrocytes sense and respond to their mechanical environment by modulating the synthesis and catabolism of the extracellular matrix. Normal physiological loading of articular cartilage produces depthdependent compression of the tissue with associated deformation of the chondrocytes. Whilst the precise process of cellular mechanotransduction is as yet unclear, cell deformation is believed to be one of the primary stimuli. Hence it is of major importance to elucidate the deformation behaviour of cartilage cells in health and disease, which is governed by the inherent cellular and subcellular biomechanics. Measurements of cellular biomechanical parameters generally involve deformation of the cell surface, at least in part, by a known force or stress and simultaneous visualisation and measurement of the resulting cell deformation. Alternatively, a prescribed cellular deformation may be applied and the resulting force measured. Both approaches enable the force±displacement (F±x) relationship to be plotted for a single living cell, from which the apparent stiffness (k) may be calculated from the resulting gradient (k ˆ F=x). Frequently experimental data is combined with some sort of theoretical or computational model in order to derive a fundamental cell modulus. However, it is also

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necessary to take into account the fact that living cells exhibit characteristic viscoelastic behaviour. Hence any calculated cellular modulus is highly dependent on the temporal conditions of measurement. Finally, the process of mechanotransduction means that chondrocytes themselves actively respond to mechanical loading by remodelling intracellular structures resulting in changes in both cellular and intracellular biomechanics. Indeed it has been suggested that cells show behaviour similar to Wolf's Law in bone, namely that the biomechanical remodelling occurs in response to altered mechanical loading. There are numerous techniques to impart some form of deformation on living cells whilst simultaneously enabling direct microscopic visualisation and/or measurement of cell deformation (for review see Bader and Knight, 2008). This chapter reviews those techniques primarily used for analysing the biomechanics of chondrocytes. In addition, there have been many studies describing specialist loading rigs and bioreactor systems designed to apply in vitro mechanical stimuli to a variety of cell types in order to study their metabolic or injury responses (for review see Brown, 2000). This is further explored with reference to chondrocytes in the second part of this chapter, with particular emphasis on the influence of loading regimens, cell source and scaffold materials.

4.2

Measurement of chondrocyte biomechanics

4.2.1

Chondrocyte biomechanics in situ within cartilage explants

Various studies have investigated the chondrocyte deformation in cartilage explants subjected to mechanical loading in the form of compression or indentation. Consequently, a variety of loading rigs have been developed. These systems, such as the one shown schematically in Fig. 4.1, typically mount upon the stage of an inverted microscope enabling simultaneous visualisation of the resulting deformation to the tissue and cells. By visualising the tissue in both the unstrained and strained state and using the cells as displacement markers it is possible to calculate the local strain fields in terms of compressive, tensile and shear strains (Guilak et al., 1995; Schinagl et al., 1996). It is clear that the depthdependent mechanical properties of articular cartilage results in heterogeneous levels of local deformation, such that the local tissue strains will inevitably differ from the gross strain. Furthermore the local strain also differs from the cellular strain due to the mechanical properties of the pericellular matrix (PCM) associated with the chondrocyte, a functional unit termed the chondron (Poole et al., 1987). Cartilage deformation is transferred to the chondrocytes in two ways: (i) directly, via the deformation of the extracellular matrix (ECM), and (ii) indirectly, due to the compression-induced increase in extracellular osmolarity which produces a reduction in cell volume (Chao et al., 2005; Erickson et al.,

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2003; Hing et al., 2002). It is also possible that the compression-induced loss of cell volume in situ may be the result of mechanical deformation squeezing fluid from the cell, although this has not yet been established. In addition fluid flow during cartilage loading may also generate shear forces, which will impart a more subtle deformation at the cell surface. However, in order to determine cellular biomechanical properties based on cell deformation behaviour in cartilage explants, computational models have to be developed in which the physical properties of the cell can be adjusted to match the experimental data.

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4.2.2

109

Micropipette aspiration

The most published approach for measuring chondrocyte biomechanics is that of micropipette aspiration, which is used to deform the membrane of the cell with a known suction pressure (for review see Hochmuth, 2000; Sato et al., 1987). The micropipettes, with inner diameters typically between 5 and 15 m, are coated with a silicone solution (Sigmacote, Sigma, MO) to prevent cell adhesion. A cell chamber containing approximately 1 mL of cell suspension permits the entry of the micropipette through a side wall. The micropipette is fixed to the stage of an inverted microscope and connected to a reservoir as shown in Fig. 4.1(a). Both the micropipette and the pressure control system need to be filled with a physiological saline solution, such as phosphate buffered saline (PBS). With the control of a hydraulic micro-manipulator (e.g. MO-203, Narishige, Tokyo, Japan), the micropipette is moved into contact with the cell surface and a tare pressure (typically 0.01 kPa) is applied to draw the chondrocyte against the mouth of the micropipette and to define the reference position. Aspiration pressure is then applied in a series of step increments, typically up to 5 cm H2O (0.49 kPa). At each pressure increment the cell is allowed to equilibrate for about 60±120 seconds, before a brightfield microscopy image reveals the extent of the cell deformation into the micropipette. This deformation is quantified by the aspirated length, L. A typical series of brightfield images of an isolated cartilage cell subjected to incremental levels of aspiration pressure is shown in Fig. 4.1(b)

4.1 (opposite) Micropipette aspiration. (a) Schematic diagram illustrating the micropipette aspiration system for quantifying cellular biomechanics. Suction pressure is applied to an isolated chondrocyte via a micropipette, by lowering a fluid-filled reservoir. A manometer or pressure sensor is used to provide a reading of the applied aspiration pressure. The syringes are used to fill the silicone tubing and to provide high positive pressure for releasing cells from the pipette. Cells are visualised during the aspiration process using an inverted microscope which may be connected to a confocal system (PBS ˆ phosphate buffered solution). (b) Representative brightfield and corresponding confocal microscopy images of a single isolated chondrocyte visualised during micropipette aspiration at pressures of 0, 1, 2, 3 and 4 cm of water. Scale bar indicates 5 m. Arrows indicate the aspiration of the cell into the micropipette. The cell was transfected with enhanced green fluorescent protein (eGFP) actin to examine mechanically induced changes in actin remodelling. (c) Corresponding plot of aspiration length, L, normalised to pipette radius, a, and plotted against aspiration pressure. A linear model has been fitted to the data with a gradient of 0.053 from which the cellular Young's modulus may be estimated at a value of 0.2 kPa. (d) Transient viscoelastic behaviour of a single chondrocyte immediately following a step increase in aspiration pressure to approximately 3.5 cm of water (data from Trickey et al., 2000). A theoretic model has been fitted to the data based on equation 4.2, thereby enabling the calculation of the instantaneous modulus, Ei, and the relaxation modulus, Er, at 0.41 and 0.24 kPa respectively (see text for details).

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alongside the corresponding confocal fluorescence images showing deformation of the actin cytoskeleton labelled using eGFP (enhanced green fluorescent protein) transfection. Prior to aspiration, the initial diameter of the chondrocye is measured, as well as the pipette inner diameter using calibrated brightfield microscopy. However, it is necessary to compensate for the distortion caused by the refractive index mismatch between the micropipette glass and the surrounding buffer solution. This is best achieved by multiplying the measured micropipette diameter by a factor of 0.92 (EngstroÈm et al., 1992). On the basis of the experimental data, the apparent Young's modulus can be determined using a theoretical elastic model previously developed to analyse the material properties of endothelial cells (Theret et al., 1988). In this model, the cell is assumed to be a homogeneous, elastic half-space material and the Young's modulus, E, is therefore given as:   3…†
P Eˆ …4:1† 2 L=a where a and b are the inner and outer radii of the micropipette and …† is defined as the wall function with  ˆ …b ÿ a†=a. This so-called `rigid punch model' employs a boundary condition of no axial displacement of the cell at the micropipette mouth, corresponding to …† ˆ 2:1 for the practical range used in a typical study. The Young's modulus can be determined from the slope of the linear regression of the normalised length L=a versus the negative pressure P as shown in Fig. 4.1(c). Further models and experimental approaches have been developed to calculate the viscoelastic properties of chondrocytes based on an analytical solution of micropipette aspiration (Sato et al., 1987). For this technique, the chondrocyte is aspirated into the micropipette with a single step aspiration pressure ranging from approximately 1 to 10 cm H2O. The aspiration length is then recorded over a time period of up to 300 seconds (Fig. 4.1d). The associated model assumes that the chondrocyte behaves as a homogeneous linear viscoelastic three-parameter solid half-space. Using this model the aspiration length, L, and the relaxation constant,  can be predicted at time, t, based on the following equation:   …†a
P k2 …4:2† 1ÿ et= L…t† ˆ k1 ‡ k2 k1 ˆ

…k1 ‡ k2 † k1 k2

…4:3†

The viscoelastic parameters k1 , k2 and  can be calculated by fitting experimental aspiration length data to equation 4.2 using a non-linear regression. The parameter k1 is termed the equilibrium or relaxation modulus (Er or E1 ), k1 ‡ k2 is the instantaneous modulus (Ei) and  is the apparent viscosity. Using

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this viscoelastic model it is also possible to determine the apparent Young's model given by: Eˆ

3 2k1

…4:4†

It should be recognised that although both the elastic and viscoelastic models benefit from being simple, they neglect geometrical factors, such as finite cell dimensions, evolution of cell-micropipette contact region and curvature of the micropipette edges. Thus other models incorporating these geometric factors into a computational form have been developed, which will also account for the inhomogeneities in the cellular properties (Haider and Guilak, 2000). Extensive studies by Guilak's group have used pipette aspiration to investigate the biomechanics of chondrocyte isolated from both healthy and diseased human articular cartilage (Guilak et al., 1999; Jones et al., 1999; Trickey et al., 2004). These studies have quantified both the `pseudo' elastic properties and the viscoelastic time-dependent behaviour. The results of these and other micropipette aspiration studies suggest that chondrocytes have a Young's modulus of approximately 0.5 kPa, and instantaneous and relaxation moduli of 0.4 and 0.2 kPa respectively, as summarised in Table 4.1. Some studies suggest that cells from osteoarthritic (OA) cartilage are stiffer than those from normal tissue, particularly with respect to the equilibrium moduli (Trickey et al., 2000, 2004). In addition, this group has used the same experimental approach to investigate the biomechanics of the chondrocyte nucleus (Guilak et al., 2000; Vaziri and Mofrad, 2007). Indeed micropipette aspiration provides a valuable tool for examining intracellular biomechanical behaviour including nucleus deformation, cytoplasmic biomechanics (Bomzon et al., 2006; Ohashi et al., 2006) and cytoskeletal deformation and remodelling (Fig. 4.1b). Finally, pipette aspiration has also been used to quantify the biomechanical properties of the pericellular matrix associated with isolated chondrons. These studies estimate the Young's moduli of enzymatically isolated chondrons at approximately 25 kPa (Alexopoulos et al., 2005; Guilak et al., 2005). Such studies provide essential data for developing hierarchical models of articular cartilage biomechanics. However, it has to be appreciated that the isolation process may well damage the inherent biomechanics of the chondron, which may be substantially stiffer than the estimated values from these studies (Knight et al., 2001).

4.2.3

Atomic force microscopy (AFM)

Atomic force microscopy (AFM) systems, which were developed from a simple cell poking approach, are now available in both laboratory-based and commercial systems (e.g. Veeco Instruments). Their use in cell biomechanics involves the indentation of the cell surface with a small probe, whose movement

Table 4.1 Summary of compressive stiffness values for chondrocytes, as estimated from a range of different measurement techniques Reference

Modulus

Technique/model

Cell type

Micropipette aspiration Jones et al. (1999)

E ˆ 0:65 kPa

Elastic half space

Trickey et al. (2000, 2004), Guilak et al. (2002) Bader et al. (2002) Ohashi et al. (2006)

El ˆ 0:41 kPa Er ˆ 0:24 kPa E ˆ 0:81 kPa E ˆ 0:97 kPa

Viscoelastic half space

Human chondrocytes, knee, hip, ankle, elbow, normal and OA Human chondrocytes, knee, hip, normal and OA Adult bovine chondrocytes, MCPJ Adult bovine chondrocytes, MCPJ

K ˆ 0:02 N/m El ˆ 0:29±0.55 kPa Er ˆ 0:17±0.31 kPa

No model used Viscoelastic, isotropic surface

Adult bovine chondrocytes, MCPJ Porcine femoral condyle, superficial and deep zone cells

E ˆ 1:10 kPa El ˆ 8:00 kPa Er ˆ 1:09 kPa

Elastic half space Viscoelastic half space

Adult bovine chondrocytes, distal surface of first metatarsal

E ˆ 2:55 kPa El ˆ 2:47 kPa Er ˆ 1:48 kPa Ea ˆ 1:48 kPa El ˆ 1:06 kPa Er ˆ 0:78 kPa

Linear elastic model Viscoelastic model

Adult bovine chondrocytes, distal surface of first metatarsal

Compression in 3D scaffolds Freeman et al. (1994)

E ˆ 4 kPa

Bader et al. (2002) Knight et al. (2002)

Er ˆ 2:7 kPa E ˆ 3:2 kPa

Compression in agarose and elastic FEM Relaxation in 1% agarose Relaxation in 2% alginate

Atomic force microscopy Bader et al. (2002) Darling et al. (2006) Cytoindentation Koay et al. (2003) Cytocompression Leipzig and Athanasiou (2005)

Shieh and Athanasiou (2006)

Elastic half space Elastic half space

Linear biphasic model Viscoelastic half space

Adult bovine chondrocytes, distal surface of first metatarsal Swarm rat chondrosarcoma cells Adult bovine chondrocytes, MCPJ Adult bovine chondrocytes, MCPJ

E, Young's modulus; Er, relaxation (equilibrium)modulus; El, instantaneous modulus; Ea, aggregate modulus; FEM, finite element model; MCPJ, metacarpal phalangeal joint (proximal surface); OA, osteoarthritic chondrocytes.

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is controlled at a constant velocity (Radmacher et al., 1996; Sato et al., 2000). The tip is typically pyramidal or hemispherical in shape and is carefully moved towards the surface of an individual chondrocyte, imaged through a conventional light microscope. The tip can probe various locations on the cell surface with the force indirectly recorded under indentation control. Hence structural properties, in the form of the force±deformation relationship, can be obtained. Previous studies employing AFM to quantify chondrocyte biomechanics have demonstrated a typical force±displacement relationship with a characteristic toe-in followed by a more linear region with increasing indentation (Bader et al., 2002; Darling et al., 2006). The resulting stiffness of articular chondrocytes has been estimated from the gradient of the linear region with values ranging from 0.02 to 0.10 N/m (Bader et al., 2002), with cells from OA cartilage being significantly softer than those from normal tissue (Hsieh et al., 2008). However, interpretation of the results from AFM deformation is complicated by the tapered shape of its probe tip and its small size relative to the depth of indentation. Therefore to determine biomechanical material properties, such as the cell modulus from this experimental approach, finite element models have been developed (Costa and Yin, 1999). Using a theoretical solution for stress relaxation of a viscoelastic, incompressible, isotropic surface indented with a hard spherical indenter, previous AFM studies have estimated the chondrocyte modulus, as summarised in Table 4.1. In particular, the instantaneous modulus of isolated superficial zone cells has been estimated at a 0.55 kPa whilst the value for middle/deep zone cells is significantly lower at 0.23 kPa (Darling et al., 2006). It should be noted that these values are in broad agreement with those obtained using pipette aspiration.

4.2.4

Cytoindentation and cytocompression

A few studies, notably those from Athanasiou and co-workers, have developed specialist single cell cytoindentation (Koay et al., 2003; Shin and Athanasiou,1999) or cytocompression rigs (Leipzig and Athanasiou, 2005; Shieh and Athanasiou, 2002, 2006). Both approaches have been used to determine viscoelastic creep properties of individual isolated chondrocytes. However, as with micropipette aspiration and AFM, these techniques require assumptions to be made so that theoretical models may be used to derive the cellular biomechanical properties. The cytoindentation tests yield values for instantaneous and relaxation moduli of articular chondrocytes at 8.00 and 1.09 kPa respectively (Table 4.1) (Koay et al., 2003). These values are significantly greater than those obtained using micropipette aspiration or AFM. Similar elevated values for instantaneous and relaxation moduli are reported from cytocompression tests with superficial cells appearing stiffer than those isolated from the middle and deep zones (Shieh and Athanasiou, 2006). The results were broadly similar when derived using three different continuum models (Leipzig and Athanasiou, 2005) (Table 4.1).

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Compression of isolated chondrocytes in 3D scaffolds

One disadvantage with the techniques described above is that the nature of the applied localised cell deformation is distinctly different from that experienced in vivo during physiological loading of articular cartilage. However, to derive cellular biomechanical parameters from experiments with cartilage explants is also problematic, as discussed above. Therefore an alternative approach for examining cellular biomechanics involves compression of living chondrocytes in homogeneous 3D scaffolds. This can yield biomechanical data at both cellular and subcellular levels, associated with a more physiological gross chondrocyte deformation. Numerous studies have used the well-characterised chondrocyte-agarose model to investigate the role of cell deformation. This in vitro model system consists of isolated chondrocytes seeded within agarose gel, typically prepared at a concentration of 2±4% (w/v) (Lee and Bader, 1995, 1997). The cells adopt a spherical morphology with a cortical arrangement of the actin cytoskeletal similar to that observed in situ. Thus the isolated cells maintain their chondrocytic phenotype as shown by the synthesis of type II collagen and the proteoglycan, aggrecan. Compressive strain can be applied to cells seeded within agarose or other low modulus scaffolds, such as alginate, using relatively simple microscope-mounted loading rigs such as that shown in Fig. 4.2(a). These devices enable simultaneous visualisation of cells at different levels of applied gross compression. Studies using confocal microscopy to measure the deformation of viable chondrocytes in agarose demonstrate that gross compression results in deformation to an oblate ellipsoid morphology with significant lateral expansion and conservation of cell volume (Fig. 4.2b) (Lee et al., 2000b). Although this mode of loading is more physiological than that associated with micropipette aspiration, AFM or cytoindentation, the deformation behaviour differs from that in situ within cartilage explants where compression occurs with a reduction in cell volume (Section 4.2.1) (Guilak, 1994; Guilak et al., 1995). The viscoelastic properties of the scaffold relative to the cell can lead to temporal changes in cell deformation during either static or cyclic compression (Knight et al., 1998a). This phenomenon has been exploited to investigate cellular biomechanics by monitoring the reduction in cell deformation over a 60 minute period of static compression in 2% (w/v) alginate gel (GMB low viscosity, Kelco, UK) (Knight et al., 2002). Cell strain measurements were plotted against the corresponding viscoelastic stress relaxation in the gel, measured using a 2.5 N load cell. It was therefore possible to estimate the cell compressive modulus (E ˆ stress/strain) at a value of approximately 3.2 kPa (Table 4.1). A similar approach has also been used for deriving cell moduli from measurements of cell deformation in compressed agarose gels (Table 4.1) (Bader et al., 2002; Freeman et al., 1994). However, these approaches do not take into account the long-term viscoelastic behaviour of the cell, as opposed to

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4.2 Compression of chondrocytes in 3D scaffolds. (a) Schematic cross-section of loading rig for compression of a cartilage explants or a chondrocyte seeded scaffold. The device mounts upon the stage of an inverted microscope and allows simultaneous visualisation of cells during compression thereby enabling analysis of cellular and intracellular biomechanics (Knight et al., 1998b). The specimen is placed on a coverslip and hydrated in medium. Compression is applied via one or two sliding platens connected to stepping linear actuators controlled via a PC. (b) Representative confocal images of a single isolated chondrocyte visualised in an agarose construct at 0, 5, 10, 15 and 20% gross compression strain. The top row shows a cell labelled with calcein AM whilst the second row shows a separate cell labelled with mitotracker green and syto16 to label the mitochondria and nucleus respectively. Images demonstrate the deformation of the cell and intracellular structures during gross compression. Scale bar indicates 5 m. (c) The associated intracellular local strains distribution parallel to the axis of compression was calculated from the mitochondria images using digital image correlation. The magnitude and direction of the local strains are shown on a pseudocolour scale relative to the uncompressed images (Knight et al., 2006).

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that of the scaffold, as demonstrated in previous cell biomechanics studies (Darling et al., 2006; Trickey et al., 2004). Intracellular biomechanics may also be examined based on analysis of cell deformation in 3D scaffolds. Indeed, previous studies have investigated the biomechanics of the nucleus and mitochondria based on compression of chondrocytes in agarose gel (Bomzon et al., 2006; Knight et al., 2006; Lee et al., 2000b). However, with increasing time in culture, the matrix synthesised by the isolated chondrocytes forms a pericellular shell which is stiffer than the surrounding agarose and thus prevents cell deformation during gross compression, thereby providing indirect analysis of pericellular matrix biomechanics (Knight et al., 1998b, 2001).

4.3

Intracellular biomechanics

Whilst the measurement of cell deformation is essential for analysing gross chondrocyte biomechanics, an understanding of intracellular biomechanics provides additional, important information. In particular, it can generate a clearer understanding of both the structures which provide cells with their viscoelastic time-dependent biomechanical properties and the mechanotransduction signalling pathways through which mechanical loading is translated into an alteration in cell activity. Studies have reported that mechanical loading of cartilage explants induces cell deformation with associated distortion of cellular organelles including the rough endoplasmic reticulum (Szafranski et al., 2004), mitochondria (Knight et al., 2006), nucleus (Guilak, 1995) and potentially the primary cilium (Jensen et al., 2004), all of which may have a role in mechanotransduction. However, of all the intracellular structures, mechanical deformation of the nucleus has been most commonly examined since it may be involved in mechanotransduction through changes in gene expression and nuclear transport (Buschmann et al., 1996). Studies using pipette aspiration have estimated the viscoelastic biomechanical properties of isolated chondrocyte nuclei, with moduli values approximately 5±10 times stiffer than the surrounding cytoplasm (Guilak et al., 2000; Vaziri and Mofrad, 2007). Thus, the levels of nucleus deformation are typically less than that of the cell (Fig. 4.2b) (Guilak, 1995; Knight et al., 2002; Lee et al., 2000b). However, changes in the biomechanical properties of the nucleus may occur during differentiation or in the diseased state, with associated changes in mechanotransduction. In addition, the relative stiffness of the nucleus means that where cell deformation is sufficient to induce nuclear distortion, the nucleus is likely to provide a significant contribution to the gross biomechanical stiffness of the chondrocyte. Nucleus morphology and deformation are typically heterogeneous and nonuniform in nature (Knight et al., 2002). This needs to be considered when quantifying nucleus deformation ideally performed using live cell imaging. Cytoskeletal integrity is important for strain transfer to the nucleus (Djabali,

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1999; Janmey, 1998) and thus changes in cytoskeletal organisation will influence nucleus deformation and other aspects of intracellular biomechanics (Knight et al., 2002; Lee et al., 2000b). For the study of intracellular deformations, the optical sectioning capability of confocal laser scanning microscopy enables clear, blur-free imaging of intracellular structures. The scope of this technology has been greatly enhanced by the ever-increasing range of fluorescent compounds for labelling intracellular structures and organelles in living cells (see Molecular Probes: http:// probes.invitrogen.com/). Thus viable fluorescent markers are now available for organelles including the nucleus, mitochondria and endoplasmic reticulum. Whilst most of these compounds are cell permeable, labelling of other structures, such as the cytoskeletal protein networks, is possible using microinjection of fluorescent analogues (for review see Goldman and Spector, 2004). Alternatively the development of transfection techniques involving fluorescent tags, such as GFP, provides a powerful tool for visualising intracellular structural dynamics and biomechanics within living chondrocytes (Fig. 4.1b). The complexity and heterogeneity of intracellular structures, such as the cytoskeleton, frequently require advanced computational techniques, such as digital image correlation (DIC), to quantify the local biomechanics. DIC can analyse, with sub-pixel resolution, the movement and distortion within pairs of images and has been used for measuring deformation in a wide range of structures including articular cartilage (Chahine et al., 2004; Wang et al., 2003). More recently, the technique has been optimised for measuring the deformation and biomechanics of fluorescently labelled intracellular structures, visualised in living chondrocytes using confocal microscopy (Delhaas et al., 2002; Helmke et al., 2000, 2001, 2003; Hu et al., 2003). Intracellular displacements and strains can be automatically computed and graphically displayed in the form of pseudocolour maps (Fig. 4.2c). The technique is able to determine the local compressive and tensile strains as well as shear strains, area strains, von-Mises strains and the magnitude and direction of the principal strains. It should be noted that this approach does not distinguish between percentage changes in dimensions resulting from true biomechanical deformation, or strain, and inherent temporal movement. However, with appropriate experimental controls it is possible to use this information to generate computational models to describe biomechanical properties of intracellular elements (Bomzon et al., 2006).

4.4

Biomechanical conditioning of chondrocytes

4.4.1

Introduction

In addition to the many studies focused on chondrocyte biomechanics, others have proposed the use of in vitro biomechanical conditioning strategies for chondrocyte-seeded scaffolds as an essential feature for the long-term

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functionality of tissue engineered implants for cartilage repair (e.g. Guilak et al., 2001). This requires the development of suitable bioreactors, incorporating mechanical loading modules for use in a controlled biological environment. These can provide appropriate mechanical conditioning regimes to stimulate the formation of a functional neo-cartilage, thereby improving the efficacy and efficiency of production of tissue engineered implants for cartilage defects. Two fundamental approaches have been adopted to examine the response of chondrocytes to biomechanical stimuli in vitro, similar to those described earlier in the chapter. One involves the use of cartilage explants in which the chondrocytes are associated with ECM mimicking the in situ case (Gray et al., 1989; Jin et al., 2001; Sah et al., 1989). The alternative involves model systems incorporating isolated chondrocytes, which may be maintained in various culture systems, including pellet cultures, suspension cultures or monolayer cultures of either high or low cell densities, or the cells may be seeded within a 3D construct, typically comprising a hydrogel or a porous scaffold (Buschmann et al., 1995; Freeman et al., 1994; Kisiday et al., 2002; Lee and Bader, 1997). Both model systems have their proponents. It is evident that cartilage explants are appropriate for studies investigating fundamental mechanotransduction events associated with normal turnover and pathology. However, they are less suitable to examine individual extracellular components of mechanotransduction, such as cell deformation, due to the inherent coupling of mechanical and physicochemical processes in the charged ECM. Alternatively model systems, although non-physiological in nature, are generally considered appropriate for tissue engineered therapeutic strategies. Accordingly, a wide variety of model systems have been proposed in the literature (Table 4.2). One of the most popular systems involves chondrocytes seeded in 3D agarose constructs, as discussed earlier in the chapter. The system maintains a rounded chondrocyte phenotype over extended culture periods (Aydelotte et al., 1990; Benya and Shaffer, 1982; Hauselmann et al., 1994), by preventing the formation of actin stress fibres, which are evident when chondrocytes are cultured in monolayer. Thus agarose or similar model systems such as alginate, which can be characterized in mechanical terms (Knight et al., 1998a,b; Lee and Bader 1995), provide an ideal construct for the application of gross compression. Although compression of cell±agarose constructs will initiate transient changes in hydrostatic pressure and fluid flow, which have both been implicated in mechanotransduction, it is widely believed that cell deformation is the primary mediator. The effects of loading on cell morphology and deformation during the application of physiological levels of compressive strain has been discussed (Fig. 4.2b). Various factors have been shown to influence the level of cell deformation, including the presence and mechanical properties of the elaborated PCM, the modulus of the scaffold relative to that of the cells and matrix and the loading regime and viscoelastic properties of the scaffold (Bader et al., 2002; Knight et al., 1998a, 2002).

Table 4.2 Summary of experimental protocols used to assess in vitro biomechanical conditioning of chondrocytes in model systems Source

Cell source

Sample/scaffold material

Loading conditions/Culture conditions

Lee and Bader (1997), Lee et al. (1998a,b) Chowdhury et al. (2006)

Bovine (adult)

Agarose

15%, 0.3, 1 and 3 Hz, 48 h

Human

Agarose

Bonassar et al. (2001) Kisiday et al. (2002)

Bovine (calf) Bovine (calf)

Cartilage discs Agarose and self-assembling peptide gel

Buschmann et al. (1995) Buschmann et al. (1999)

Bovine (calf) Bovine (calf)

Agarose Explants

Sah et al. (1989), Li et al. (2001) Mauck et al. (2000)

Bovine (calf)

Explants

Bovine (calf)

Agarose

Millward-Sadler et al. (1999) Guilak et al. (1994), Fermor et al. (2001) Altman et al. (2002)

Human normal or OA ± passaged Bovine (calf)/porcine

Monolayer

15%, 1 Hz, 48 h 10 ng mlÿ1 TGF 2%, 0.1 Hz, 48 h 2.5±3%, 1 Hz, 1 h on/1 h off, days 9±12 or 1 h on/7 h off, days 27±34 0.001 Hz to 1 Hz, 10 h 0.001 Hz, 23 h, days 4 and 6 0.01 Hz, 23 h, days 3 and 5 2.4%, 0.01 Hz (200 kPa) 2 h on/2 h off, 23 h, 0.88±1 mm compression 10%, 1 Hz, 3  1 h intermittent/day, 5 days/week for 4 weeks 0.33 Hz, 20 min

Explants

0.1, 0.5 MPa, 0.5 Hz, 24 h

Human bone marrow stromal cells ± passaged Bovine (calf)

Silk fibre matrices

0.0167 Hz, 2 mm compression

Martin et al. (2000) Hunter et al. (2002) Hunter and Levenston (2002a) Waldman et al. (2003, 2007)

Bovine (calf) Bovine (calf)

Xie et al. (2007)

Young rabbit

Bovine

Polyglycolic acid

Compressive deformation for samples 1.3±3 mm thickness Collagen I Oscillatory 25  4%, 1 Hz, 24 h Either core explant with Dynamic compression at 10  4% at 0.3 or 1.0 Hz, agarose core or agarose alone after 3 or 15 days of pre-culture Porous calcium Static culture for 4 weeks ± various combinations polyphosphate of compression and/or shear strain at 1 Hz for 6 min, followed by 48 h recovery Microporous PLL±PCL Dynamic compression at 10% (continuous and elastomeric scaffolds intermittent) at 0.01±0.5 Hz

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Biomechanical loading systems

Conventional in vitro mechanical tests require the soft tissues to be kept in a moist environment and are performed on either a Universal materials test facility or a specially designed test system. However, if tissue explants or chondrocyteseeded 3D constructs are to be examined, viability must be maintained in an environment similar to a conventional CO2 incubator, whilst the system is subjected to either static or dynamic loading. With regards to commercial systems, that produced by Flexercell has gained widespread use for subjecting specimens to a range of tensile loading regimens. Although there is a compressive system equivalent, as reported by Graff and colleagues (2000), most researchers have used custom-made bioreactor systems to apply compression to chondrocyte cultures. Others describe bioreactors which incorporate shear, fluidinduced shear, hydrostatic pressure or a combination of loading modalities (Wernike et al., 2008). One of the few compression-type bioreactors available commercially (Zwick Testing Machines Ltd, Leominster, UK) has been used to apply static and dynamic loading to biomaterial constructs seeded with chondrocytes. The system, detailed over a decade ago (Lee and Bader, 1997), consists of a conventional loading frame with an hydraulic actuator-controlled vertical assembly, which enters a tissue culture incubator (Heraeus Instruments, Brentwood, UK). The assembly is connected to a central rod which is attached to a mounting plate located within a Perspex box, as shown in Fig. 4.3. The box, in turn, is placed on a circular platten fixed to the base of the loading frame. The mounting plate holds 24 loading pins, half of which are unconstrained to move vertically in harmony with the loading assembly. Each loading pin incorporates an 11 mm circular Perspex indenter, which applies compressive strain to samples located within separate wells of a 24-well tissue culture plate. An important aspect of any prolonged culturing period is the maintenance of cell viability. During a 48-hour culture period in the compressive cell strain system (Fig. 4.3), the chondrocyte viability has been shown to remain above 95% within both unstrained and strained constructs (Lee and Bader, 1997). Thus differences in metabolism could not be attributed to alterations in chondrocyte viability. Accordingly, this loading system has been used by the authors to examine key variables associated with the application of mechanical conditioning to chondrocyte-seeded 3D constructs. Specific studies have aimed at elucidating the influence of mechanical loading regimes, cell sub-populations and scaffold materials on the efficacy of mechanical conditioning strategies.

4.4.3

Mechanical loading regimens

An initial series of experiments on full-depth adult bovine chondrocytes seeded in agarose investigated the influence of both static and dynamic continuous

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4.3 Photograph (a) and schematic representation (b) of the compressive cell strain system.

compression at a strain amplitude of 15%. The normalised data for tritiated ([3H]) thymidine incorporation, a measure of cell proliferation, are presented in Fig. 4.4(b). It is evident that constructs subjected to dynamic strain at all three frequencies exhibited significant stimulation in comparison with unstrained control values, although there were no statistically significant differences between the dynamic frequencies. The corresponding data for glycosaminoglycan (GAG) synthesis values, normalised to unstrained control levels, are presented in Fig. 4.4(a). It can be seen that at the low frequency (0.3 Hz) dynamic strain inhibited GAG synthesis, while a frequency of 1 Hz induced a significant stimulation of GAG synthesis. At the higher frequency of 3 Hz GAG synthesis returned to unloaded control levels. These findings suggested that, using the chondrocyte±agarose system at the strain level of 15%, there is a frequency range which can induce GAG synthesis, with a lower cut-off frequency between 0.3 and 1 Hz and an upper cut-off between 1 and 3 Hz. This frequency will be dependent on the strain level, the mechanical properties of the matrix and the size and shape of the individual specimens. Indeed it has been established that increasing frequency will increase the rate of fluid flow in the periphery of the specimens, but will reduce the effective width of the peripheral ring in which it occurs (Sah et al., 1989). It is conceivable, therefore, that the influence of the central core may mask stimulatory effects within the peripheral ring at the higher frequency of 3 Hz. Although all loading regimens yielded an inhibition in protein synthesis, the analysis of data revealed an association between the frequency rate and the level of inhibition (data not presented, Lee and Bader, 1997). The findings also implied that each metabolic parameter may be influenced by dynamic strain regimens in a distinct manner, implying that the associated signalling mechanisms are uncoupled (Lee and Bader, 1997). A subsequent study examined the metabolic response of adult bovine cells from different zones of cartilage under dynamic compression (Lee et al., 1998a). Thus slices of cartilage from the uppermost 15±20% of the total uncalcified tissue

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4.4 GAG synthesis and [3H]-thymidine incorporation by full-depth bovine chondrocytes (a and b, respectively, and surface and deep bovine chondrocytes (c and d) embedded in agarose constructs and subjected to 15% dynamic compressive strain amplitude at various frequencies for 48 h. The values are presented as % change from unstrained control levels. Each value represents the mean and standard error of at least 12 replicates from at least two separate experiments. Unpaired Student's t-test results indicate differences from control values as follows: * ˆ p  0:05.

depth were removed from the proximal joint surface, and the `superficial cells' isolated and seeded into agarose constructs. `Deep cells' from the residual tissue were seeded into separate constructs. Both groups of constructs were cultured under a continuous compressive amplitude of 15% for 48 hours. Normalised GAG synthesis data, as presented in Fig. 4.4(c), revelaled that all three frequencies produced an inhibition in GAG synthesis by superficial cells, the differences being statistically significant at 0.3 and 3 Hz. With reference to the GAG synthesis by deep cells, 0.3 Hz produced a significant reduction, and 1 Hz induced a highly significant stimulation of 50% (Fig. 4.4c). By contrast, all three dynamic frequencies induced a significant increase in [3H]-thymidine incorporation by superficial cells (Fig. 4.4d). There was no statistically significant difference in the level of stimulation between the three dynamic strain regimens. However, [3H]thymidine incorporation by deep cells was not greatly influenced by the application of compressive strain. The only increase corresponded with the 0.3 Hz dynamic regime when compared with unstrained controls (Fig. 4.4d).

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Data from this study demonstrated that the control of GAG synthesis and proliferation in response to dynamic compression are not merely uncoupled, but occur in different sub-populations of chondrocytes within the full-depth cell isolate (Lee et al., 1998a). This conclusion raises the possibility of the involvement of distinct intracellular mechanotransduction mediators. One possible mediator is nitric oxide, which is known to influence both GAG synthesis and proliferation in chondrocytes, and may be modulated by physical stimuli. This has been examined by the authors in a series of papers (Chowdhury et al., 2001, 2003a, 2006; Lee et al., 1998b, 2000a), which have all indicated a downregulation of both nitric oxide and associated PGE2 by dynamic compression. Additionally in the presence of a pro-inflammatory cytokine, it has been shown that dynamic compression counteracts the IL1- -induced release of nitric oxide and PGE2 by superficial zone chondrocytes cultured in agarose constructs (Chowdhury et al., 2003a). This has important implications in the potential of exercise regimens, involving controlled biomechanical loading in vivo, for those subjects with inflammatory joint disease. It has been previously shown with different cell types that dynamic loading for short periods can stimulate cellular activity (Fermor et al., 2001; Robling et al., 2002; Rubin and Lanyon, 1992). This prompted an examination of the temporal response of full-depth bovine chondrocytes seeded in agarose constructs to both intermittent and continuous compression regimens (Chowdhury et al., 2003b). Intermittent compression was applied for 1.5 (denoted by I 1.5), 3 (I 3), 6 (I 6) and 12 (I 12) hours at 15% strain at 1 Hz with equivalent unloaded periods for a total of 48 hours. Each of the intermittent loading regimens, involving 86 400 duty cycles, resulted in significant increases in both sulphate and thymidine incorporation (Fig. 4.5). However there were clear differences in the optimal profiles over the 48-hour culture period. For example, there was a monotonic increase in stimulation of sulphate incorporation with increasing strain durations, such that differences were found to be statistically significant between the values at 1.5 h compared to those at 6 and 12 h (p < 0:01). By contrast, thymidine was maximal after 1.5 h of intermittent loading with values of 197% compared with unstrained controls. Longer bursts of cyclic compression were associated with a decrease in the absolute proliferative response (Fig. 4.5b). Similar findings were observed with extended periods of cyclic compression (Chowdhury et al., 2003b). It was postulated that dynamic compression acts as a competence and/or progression factor for DNA synthesis in chondrocytes. Once the cell has been stimulated to enter the cell cycle it will take several days to progress through the complete cycle. Thus stimulation of cell proliferation within the timescale of this study is a unique event, as opposed to repeated stimulation, which is required to up-regulate proteoglycan synthesis. It is proposed that the frequency of dynamic strain does not affect the response, as it appears from the continuous compression data that a finite number of cycles of dynamic strain are sufficient for stimulation. This suggests that the cells are temporally processing the mechanical stimulus.

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4.5 The percentage change from unstrained control values for 35 SO 4 incorporation (a) and [3H]-thymidine incorporation (b), by chondrocytes seeded in 3% agarose and subjected to different periods of intermittent compression of 15% dynamic strain at a frequency of 1 Hz. Error bars represent the mean and SEM of 24 replicates from two separate experiments (based on Chowdhury et al., 2003b).

A similar approach was adopted in a recent study, which examined the effects of both intermittent and continuous compression on young rabbit chondrocytes seeded in microporous elastomeric scaffolds made from a poly--caprolactone± poly-L-lysine (PLL±PCL) co-polymer cultured for up to 6 days (Xie et al., 2007). Both ECM and appropriate genes were monitored. Data indicated a mechanical-induced up-regulation in both GAG and collagen secretion up to a maximum at day 3. The mRNA expression of collagen type II was noted to be up-regulated with intermittent stimulation in the short term although, over an extended time period, there was a redundancy of stimulation leading to a downregulation of cell biosynthesis. The benefits of intermittent compression were also demonstrated for bovine chondrocytes grown on porous ceramic substrates (Waldman et al., 2003). After a free swelling culture period of 4 weeks, the cultures were subjected to short bursts (400 duty cycles at 1 Hz) of either compression or shear loading. Biochemical analysis indicated an up-regulation in both proteoglycan and collagen content, which was more significant under dynamic shear stimulation at 2%.

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This work was extended to examine the intermittent effects of combinations of compression and shear stimulation (Waldman et al., 2007). The findings again revealed an increase in up-regulation of ECM components with stimulation, which correlated with an increase in the mechanical integrity of the cultured constructs. A recent study (Wernike et al., 2008) examined the influence of dynamic mechanical stimulation at a different oxygen tensions. They used a custom-built joint stimulator, which permitted the application of both axial compression and simulated surface torsion. Calf chondrocytes were seeded in porous polyurethane scaffolds and subjected to 1 hour stimulation per day for up to 34 days in either a normoxic or anoxic (5% O2) environment. There were clear differences in results of both gene and protein analyses in the two conditions. For example, under reduced oxygen tension, there was an increase in mRNA levels of type II collagen and aggrecan, with an associated increase in GAG/ DNA content. Mechanical compression yielded an enhanced level of Type II collagen gene. Interestingly a combination of biomechanical stimulation and hypoxia produced a significant down-regulation of type I collagen gene expression, suggesting that their combination could prove an effective tool for maintaining chondrocyte phenotype in culture.

4.4.4

Cell source

Autologous chondrocyte implantation (ACI or ACT) represents a wellestablished approach to repair full or partial thickness cartilage defects (Brittberg et al., 1994). The technique is detailed in Chapter 9. To review briefly, chondrocytes are isolated from a small biopsy, removed from a low load-bearing site at the periphery of the joint surface. The chondrocytes are then expanded, typically ten-fold, in monolayer culture for approximately 4 weeks to ensure the practicality and financial viability of the technique (Brittberg et al., 1994). While these methods are extremely effective at inducing cell proliferation, chondrocytes are known to dedifferentiate and adopt a fibroblastic phenotype in monolayer culture and this process is only slowly reversible (Benya and Shaffer, 1982; deHaart et al., 1999; Mayne et al., 1976). Additionally, the induction of cell proliferation during expansion will increase the telomeric age of the cells, which, in turn, may influence their response to biomechanical stimulation. Accordingly, a number of cell-related factors need to be addressed, particularly given the diverse nature of the experimental protocols employed in previous studies (Table 4.2), which ultimately can provide only phenomenological information. Nonetheless, a number of studies by the authors and others have attempted to address these issues in a systematic fashion. One study examined the effects of continuous dynamic compression to agarose constructs containing full-depth bovine chondrocytes isolated from either the femoral condyle and the patella groove of the equine knee joint

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(Wiseman et al., 2003). These tissue locations were selected to represent areas experiencing different loading conditions, as identified during normal articulation of the joint (Athanasiou et al., 1991) and thus correspond to the sites of lesion and donor tissue, respectively, in the ACI protocol (Brittberg et al., 1994). Results indicated differences between the response from the two selected tissue locations, in terms of both the absolute levels of proteoglycan synthesis and cell proliferation and also changes induced by dynamic compression. Additionally, nitric oxide was inhibited by the application of loading for cells isolated from both anatomical regions for all equine samples tested. However the heterogeneous response may have been compounded by the use of tissue of different ages and unknown exercise history, a factor which is highly relevant for the translation of tissue engineered cartilage repair systems from the laboratory to the clinic. The influence of passage in monolayer on the response of full-depth bovine chondrocytes to the application of dynamic compression was examined (Wiseman et al., 2004). The chondrocytes were either seeded directly into 3D agarose constructs or were passaged up to four times at weekly intervals prior to seeding into the constructs. It is well established that cells at passage 2 and beyond express a fibroblastic phenotype even when cultured in alginate beads, as evidenced by the presence of type I collagen and the absence of type II collagen staining (Wiseman et al., 2004). On application of dynamic compression (15% strain at 1 Hz), chondrocytes at early passages (P1 and P2) exhibited a stimulation of both proteoglycan synthesis and cell proliferation, as illustrated in Fig. 4.6(a) and (b), respectively. However, beyond passage 2 (P3± P4) the reverse was evident with dynamic compression acting to inhibit these factors, which are essential for the re-population of defect sites and the formation of a cartilaginous neo-tissue. Thus the study demonstrated a clear relationship between passage number and mechanical sensitivity, with mechanicalinduced stimulation only evident during early passage. Many studies have demonstrated that the provision of specified differentiation factors, such as fibroblast growth factor (FGF) and transforming growth factor beta (TGF- ), either during expansion or subsequent culture in 3-D, can influence the re-expression of the chondrogenic phenotype (deHaart et al. 1999; Jakob et al., 2001; Lemare et al., 1998; Yaeger et al., 1997). This was further investigated in a study by Chowdhury et al. (2004) utilising monolayerexpanded human chondrocytes surplus to requirement for clinical ACI repair procedure (expanded and supplied by Verigen AG, Leverkusen, Germany). The cells were seeded into agarose constructs and subjected to dynamic compression during incubation in either standard medium comprising Dulbecco's modified eagle's medium (DMEM) + 20% fetal calf serum (FCS), or a defined chondrogenic medium comprising DMEM + insulin plus transferring and selenous acid (ITS) + 10 ng mLÿ1 TGF- . Absolute levels of both proteoglycan synthesis and cell proliferation were elevated during incubation in the presence of the con-

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4.6 Sulphate incorporation (a) and [3H]-thymidine incorporation (b) by freshly isolated bovine chondrocytes (designated passage 0) and chondrocyte passaged in monolayer between one and four times. The cells were subsequently embedded in agarose constructs and subjected to 15% dynamic compressive strain amplitude at 1 Hz for 24 hours. The values are presented as % change from unstrained control levels. Each value represents the mean and standard error of at least 12 replicates. Unpaired Student's t-test results indicate differences from control values as follows: * ˆ p < 0:05 (based on Wiseman et al., 2004).

ditioned medium. In addition, a further up-regulation was achieved by application of dynamic compression (Fig. 4.7). Importantly, during incubation in DMEM + 20% FCS alone, dynamic compression failed to stimulate either metabolic parameter, similar to the response demonstrated for monolayerexpanded bovine chondrocytes reported above P2 (Fig. 4.6). These data provide further evidence for a link between the expression of a chondrocytic phenotype

4.7 Sulphate incorporation (a) and [3H]-thymidine incorporation (b) by human monolayer-expanded chondrocytes embedded in agarose constructs and subjected to 15% dynamic compressive strain amplitude at 1 Hz for 48 hours (black) or remained unstrained (white). The constructs were maintained in DMEM + 20% FCS or a defined medium comprising DMEM + ITS + 10 ng mLÿ1 TGF- . Each value represents the mean and standard error of at least 12 replicates. Unpaired Student's t-test results indicate differences from control values as follows: * ˆ p < 0:05 (based on Chowdhury et al., 2004).

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and a beneficial response to dynamic compression. In addition this study demonstrates the importance of interactions between biophysical and biochemical stimuli, an understanding of which is essential for the successful utilisation of biomechanical conditioning strategies.

4.4.5

Scaffold materials

As indicated in Table 4.2, there are a wide range of scaffold materials which have been employed to examine the metabolic effects of chondrocytes when subjected to dynamic loading. It might be supposed that the cell±scaffold interactions in agarose gels are substantially different from those in protein scaffolds (Hunter and Levenston, 2002b). In the former the cells preferentially bind to the synthesised PCM as it is deposited, while in protein scaffolds, cell adhesion molecules such as integrins, will enable direct interactions between the cell and the scaffold. Cell receptor binding is well known to alter both mechanical behaviour and the manner in which cells respond to mechanical conditioning. Indeed in the study involving chondrocytes seeded in fibrin glue, results suggested that early sustained oscillatory compression, inhibited both cell proliferation and matrix accumulation (Hunter et al., 2002). These results are in marked contrast to those found in other systems (Lee and Bader, 1997; Sah et al., 1989). An alternative series of scaffolds proposed for cartilage tissue engineering is based on poly(ethylene glycol) (PEG) hydrogels (Bryant and Anseth, 2001), which exhibit a range of mechanical properties, for example compressive stiffness ranging from 60 to 670 kPa, and controlled degradation profiles. Previous work has reported that such photo-crosslinkable matrix maintains chondrocyte viability and promotes deposition of both proteoglycans and type II collagen (Elisseeff et al., 2000). The effects of changes in the hydrogel crosslinking density on the metabolic response of chondrocytes to continuous dynamic compression of 15% at a 1 Hz frequency for 48 hours were investigated (Bryant et al., 2004). Adult bovine chondrocytes were seeded into two PEG dimethacrylate (PEGDM) gels crosslinked with final concentrations of 10% and 20% (w/w). An increase in crosslinking density resulted in an inhibition in cell proliferation and proteoglycan synthesis. The normalised data for both sulphate and thymidene incorporation and nitrite production are presented in Fig. 4.8. Dynamic compression only marginally influenced GAG synthesis in the 10% gel, although for the 20% gel, there was a marked decrease in PG production. Cell proliferation was inhibited in both crosslinked gels but particularly in the highly crosslinked gel. By contrast, nitrite release was slightly increased as a result of dynamic stimulation. These trends in the metabolic response are in marked contrast to those found for 3% agarose constructs seeded with bovine chondrocytes (Fig. 4.8). It is clear that the interactions between cells and scaffold materials must be well characterised before successful strategies for biomechanical conditioning can be adopted.

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4.8 Sulphate incorporation (a) and [3H]-thymidine incorporation (b) by full depth bovine chondrocytes embedded in 3% agarose constructs (grey) or constructs comprising 10% (white) or 20% (black) PEGDM. All constructs were subjected to 15% dynamic compressive strain amplitude at 1 Hz for 48 hours. The values are presented as % change from unstrained control levels. Each value represents the mean and standard error of at least 12 replicates. Unpaired Student's t-test results indicate differences from control values as follows: * ˆ p < 0:05.

4.5

Future trends

Chondrocyte biomechanics has developed considerably in recent years in terms of both experimental and computational modelling techniques. With regards to the latter, future work is likely to involve the development of more sophisticated triphasic cell biomechanical models at a variety of hierarchical levels associated with tissues, cells, intracellular structures, such as the nucleus or cytoskeletal networks, and individual proteins, such as stretch activated ion channels. This approach involving different length scales will provide a more realistic model of chondrocyte biomechanics and response to loading, including the associated activation of putative mechanoreceptors. In addition future biomechanical models must also take into account the active biological response to mechanical loading, such as mechanically induced remodelling of the actin cytoskeletal, with resulting changes in chondrocyte biomechanics. Thus it should be possible to develop the equivalent of Wolff's Law for single chondrocytes and to incorporate previous loading history into any model of cellular biomechanics.

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In addition new experimental techniques such as magnetic twist cytometry and scanning acoustic microscopy, may be adapted for cellular and intracellular biomechanics. However, these and existing measurement techniques are designed to examine isolated chondrocytes only. Therefore a major challenge involves the development of minimally invasive experimental techniques that are able to determine the viscoelastic properties of chondrocytes within the native cartilage PCM microenvironment and for cells subject to more appropriate physiological loading. To extend this further the ultimate goal would be to quantify cellular biomechanics in vivo during physiological joint loading, to aid the development of new techniques for diagnosis and treatment of cartilage disease and injury. The biomechanical conditioning studies highlight the complex interplay between stimuli and metabolic parameters, which appear to be distinctive and uncoupled. Nonetheless provided suitable monitoring systems are available, there remains a possibility of fine-tuning the mechanical stimulation to elicit a specific cellular response during the in vitro conditioning period. Future developments will include the use of mini-bioreactor systems that permit the application of defined mechanical loading regimes to constructs maintained in an uninterrupted and defined oxygen environment. These constructs might contain either chondrocytes and/or chondrons and be stimulated for extended time periods. This permits the provision of a more physiological mechano/ metabolic environmental conditions that will be a powerful tool for tissue engineering and for studying pathophysiological processes by providing more relevant 3D model systems that could be used for drug discovery applications. Future studies will interrogate key signalling pathways that are known to be both mechano- and oxygen-sensitive. These include the pathophysiological release of nitric oxide and prostaglandin E2 by chondrocytes (Chowdhury et al., 2006, 2008) and the potential of mesenchymal stem cells to differentiate into the chondrogenic lineage (Campbell et al., 2006; Terraciano et al., 2007). The adoption of a systematic approach can ultimately result in the definition of underlying mechanistic parameters leading to the derivation of predictive strategies, incorporated within computational models, to control the optimisation of neo-cartilage formation.

4.6

Acknowledgements

The authors acknowledge the invaluable assistance of Professor David Lee, Dr Tina Chowdhury, Dr Toshiro Ohashi and other colleagues, who have contributed to much of the experimental work described within this chapter.

4.7

References

Alexopoulos LG, Williams GM, Upton ML, Setton LA, Guilak F (2005) Osteoarthritic changes in the biphasic mechanical properties of the chondrocyte pericellular matrix in articular cartilage. J. Biomech. 38: 509±517.

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Altman GH, Lu HH, Horan RL, Calabro T, Ryder D, Kaplan DL, Stark P, Martin I, Richmond JC, Vunjak-Novakovic G (2002) Advanced bioreactor with controlled application of multi-dimensional strain for tissue engineering. J. Biomech Eng. 124: 742±749. Athanasiou KA, Rosenwasser MP, Buckwalter JA, Malinin TI, Mow VC (1991) Interspecies comparisons of in situ intrinsic mechanical properties of distal femoral cartilage. J. Orthop. Res. 9: 330±340. Aydelotte MB, Schumacher BL, Kuettner KE (1990) In Methods in Cartilage Research. Maroudas A and Kuettner KE (eds), pp. 90±92, Academic Press, London. Bader DL, Knight MM (2008) Biomechanical analysis of structural deformation in living cells. Med. Biol. Eng. Comput. 46: 951±963. Bader DL, Ohashi T, Knight MM, Lee DA, Sato M (2002) Deformation properties of articular chondrocytes: a critique of three separate techniques. Biorheology 39: 69± 78. Benya PD, Shaffer JD (1982) Dedifferentiated chondrocytes reexpress the differentiated collagen phentotype when cultured in agarose gels. Cell 30: 215±224. Bomzon Z, Knight MM, Bader DL, Kimmel E (2006) Mitochondrial dynamics in chondrocytes and their connection to the mechanical properties of the cytoplasm. J. Biomech. Eng. 128: 674±679. Bonassar LJ, Grodzinsky AJ, Frank EH, Davila SG, Bhaktav NR, Trippel SB (2001) The effect of dynamic compression on the response of articular cartilage to IGF-1. J. Orthop. Res. 19: 11±17. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L (1994) Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. New Eng. J. Med. 331: 889±895. Brown TD (2000) Techniques for mechanical stimulation of cells in vitro: a review. J. Biomech. 33: 3±14. Bryant SJ, Anseth KS (2001) Hydrogel properties influence ECM production by chondrocyte photoencapsulated in poly(ethylene glycol) hydrogels. J. Biomed. Mater. Res. 59: 63±72. Bryant SJ, Chowdhury TT, Lee DA, Bader DL, Anseth KS (2004) Crosslinking density influences chondrocyte metabolism in dynamically loaded photocrosslinked poly(ethylene glycol) hydrogels. Annals Biomed. Eng. 32: 407±417. Buschmann MD, Gluzband YA, Grodzinsky AJ, Hunziker EB (1995) Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture. J. Cell Sci. 108: 1497±1508. Buschmann MD, Hunziker EB, Kim YJ, Grodzinsky AJ (1996) Altered aggrecan synthesis correlates with cell and nucleus structure in statically compressed cartilage. J. Cell Sci. 109: 499±508. Buschmann MD, Kim YJ, Wong M, Frank E, Hunziker EB, Grodzinsky AJ (1999) Stimulation of aggrecan synthesis in cartilage explants by cyclic loading is localized to regions of high interstitial fluid flow. Arch. Biochem. Biophys. 366: 1± 7. Campbell JJ, Lee DA, Bader DL (2006) Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells. Biorheology 43: 455±470. Chahine NO, Wang CC, Hung CT, Ateshian GA (2004) Anisotropic strain-dependent material properties of bovine articular cartilage in the transitional range from tension to compression. J. Biomech. 37: 1251±1261. Chao PG, Tang Z, Angelini E, West AC, Costa KD, Hung CT (2005) Dynamic osmotic

132

Regenerative medicine and biomaterials in connective tissue repair

loading of chondrocytes using a novel microfluidic device. J. Biomech. 38: 1273± 1281. Chowdhury TT, Bader DL, Lee DA (2001) Dynamic compression inhibits the synthesis of nitric oxide and PGE2 by IL-1 stimulated chondrocytes cultured in agarose constructs. Biochem. Biophys. Res. Commun. 285: 1168±1174. Chowdhury TT, Bader DL, Lee DA (2003a) Dynamic compression counteracts IL1- induced release of nitric oxide and PGE2 by superficial zone chondrocytes cultured in agarose constructs. Osteoarthritis Cartilage 11: 688±696. Chowdhury TT, Bader DL, Shelton JC, Lee DA (2003b) Temporal regulation of chondrocyte metabolism in agarose constructs subjected to dynamic compression. Arch. Biochem. Biophys. 417: 105±111. Chowdhury TT, Salter DM, Bader DL, Lee DA (2004) 5 1 Integrin-mediated mechanotransduction processes in TGF -stimulated monolayer-expanded chondrocytes. Biochem. Biophys. Res. Commun. 318: 873±881. Chowdhury TT, Bader DL, Lee DA (2006) Dynamic compression counteracts IL-1 induced iNOS and COX-2 activity by human chondrocytes cultured in agarose constructs. Biorheology 43: 413±429. Chowdhury TT, Arghandawi S, Brand J, Akanji OO, Bader DL, Salter DM, Lee DA (2008) Dynamic compression counteracts IL-1 inducible nitric synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs. Arthritis Rheum. Therapy 10: R35 available on line. Costa KD, Yin FC (1999) Analysis of indentation: implications for measuring mechanical properties with atomic force microscopy. J. Biomech. Eng. 121: 462±471. Darling EM, Zauscher S, Guilak F (2006) Viscoelastic properties of zonal articular chondrocytes measured by atomic force microscopy. Osteoarthritis Cartilage 14: 571±579. Delhaas T, Van Engeland S, Broers J, Bouten C, Kuijpers N, Ramaekers F, Snoeckx LH (2002) Quantification of cytoskeletal deformation in living cells based on hierarchical feature vector matching. Am. J. Physiol. Cell Physiol. 283: C639± C645. Djabali K (1999) Cytoskeletal proteins connecting intermediate filaments to cytoplasmic and nuclear periphery. Histol. Histopathol. 14: 501±509. Elisseeff J, McIntosh W, Anseth KS, Riley S, Ragan P, Langer R. (2000) Photoencapsulation of chondrocytes in poly(ethylene oxide)-based semiinterpenetrating networks. J. Biomed. Mater. Res. 51: 164±171. Erickson GR, Northrup DL, Guilak F (2003) Hypo-osmotic stress induces calciumdependent actin reorganization in articular chondrocytes. Osteoarthritis Cartilage 11: 187±197. EngstroÈm KG, MoÈller B, Meiselman HJ (1992) Optical evaluation of red blood cell geometry using micropipette aspiration. Blood Cells 18: 241±257. Fermor B, Weinberg JB Pisetsky DS Misukonis MA Banes AJ, Guilak F (2001) The effects of static and intermittent compression on nitric oxide production in articular cartilage explants. J. Orthop. Res. 19: 729±737. Freeman PM, Natarajan RN, Kimura JH, Andriacchi TP (1994) Chondrocytes respond mechanically to compressive loads J. Orthop. Res. 12: 311±320. Goldman RD, Spector DL (2004) Live Cell Imaging ± A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Graff RD, Lazarowski ER, Banes AJ, Lee GM (2000) ATP release by mechanically loaded porcine chondrons in pellet culture. Arthritis Rheum. 43(7): 1571±1579. Gray ML, Pizzanelli AM, Lee RC, Grodzinsky AJ, Swann DA (1989) Kinetics of the

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chondrocyte biosynthetic response to compressive load and release. Biochim. Biophys. Acta 991: 415±425. Guilak F (1994) Volume and surface area measurement of viable chondrocytes in situ using geometric modelling of serial confocal sections. J. Microsc. 173: 245±256. Guilak F (1995) Compression-induced changes in the shape and volume of the chondrocyte nucleus. J. Biomech. 28: 1529±1541. Guilak F, Meyer BC, Ratcliffe A and Mow VC (1994) The effects of matrix compression on proteoglycan metabolism in articular cartilage explants. Osteoarthritis Cartilage 2: 91±101. Guilak F, Ratcliffe A, Mow VC (1995) Chondrocyte deformation and local tissue strain in articular cartilage: a confocal microscopy study. J. Orthop. Res. 13: 410±421. Guilak F, Jones WR, Ting-Beall P, Lee GM (1999) The deformation behavior and mechanical properties of chondrocytes in articular cartilage. Osteoarthritis Cartilage 7: 59±70. Guilak F, Tedrow JR, Burgkart R (2000) Viscoelastic properties of the cell nucleus. Biochem. Biophys. Res. Commun. 269: 781±786. Guilak F, Butler DL, Goldstein SA (2001) Functional tissue engineering: the role of biomechanics in articular cartilage repair. Clin. Orthop. 391 (Suppl): S295±305. Guilak F, Erickson GR, Ting-Beall HP (2002) The effects of osmotic stress on the viscoelastic and physical properties of articular chondrocytes. Biophys. J. 82: 720±727. Guilak F, Alexopoulos LG, Haider MA, Ting-Beall HP, Setton LA (2005) Zonal uniformity in mechanical properties of the chondrocyte pericellular matrix: micropipette aspiration of canine chondrons isolated by cartilage homogenization. Ann. Biomed. Eng. 33: 1312±1318. deHaart M, Marijnissen WJCM, van Osch GJVN, Verhaar JAN (1999) Optimization of chondrocyte expansion in culture: effects of TGF- , bFGF and L-ascorbic acid on bovine articular chondrocytes. Acta Orthop. Scand. 70: 55±61. Haider MA, Guilak F (2000) An axisymmetric boundary integral model for incompressible linear viscoelasticity: application to the micropipette aspiration contact problem. J. Biomech. Eng. 122: 236±244. Hauselmann HJ et al. (1994) Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads. J. Cell Sci. 107: 17±27. Helmke BP, Goldman RD, Davies PF (2000) Rapid displacement of vimentin intermediate filaments in living endothelial cells exposed to flow. Circ. Res. 86: 745±752. Helmke BP, Thakker DB, Goldman RD, Davies PF (2001) Spatiotemporal analysis of flow-induced intermediate filament displacement in living endothelial cells. Biophys. J. 80: 184±194. Helmke BP, Rosen AB, Davies PF (2003) Mapping mechanical strain of an endogenous cytoskeletal network in living endothelial cells. Biophys. J. 84: 2691±2699. Hing WA, Sherwin AF, Poole CA (2002) The influence of the pericellular microenvironment on the chondrocyte response to osmotic challenge. Osteoarthritis Cartilage 10: 297±307. Hochmuth RM (2000) Micropipette aspiration of living cells. J. Biomech. 33: 15±22. Hsieh CH, Lin YH, Lin S, Tsai-Wu JJ, Herbert Wu CH, Jiang CC (2008) Surface ultrastructure and mechanical property of human chondrocyte revealed by atomic force microscopy. Osteoarthritis Cartilage 16: 480±488. Hu S, Chen J, Fabry B, Numaguchi Y, Gouldstone A, Ingber DE, Fredberg JJ, Butler JP, Wang N (2003) Intracellular stress tomography reveals stress focusing and structural anisotropy in cytoskeleton of living cells. Am. J. Physiol. Cell Physiol.

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285: C1082±C1090. Hunter CJ, Levenston ME (2002a) The influence of repair tissue maturation on the response to oscillatory compression in a cartilage defect repair model. Biorheology 39(1±2): 79±88. Hunter CJ, Levenston ME (2002b) Native/engineered cartilage adhesion varies with scaffold material and does not correlate to gross biochemical content. Trans. Orthop. Res. Soc. 27: 479. Hunter CJ, Imler SM, Malaviya P, Nerem RM, Levenston ME (2002) Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels. Biomaterials 23: 1249±59. Jakob M, Demarteau O, Schafer D, Hintermann B, Dick W, Heberer M, Martin I (2001) Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro. J. Cell Biochem. 81: 368±77. Janmey PA (1998) The cytoskeleton and cell signalling: component localization and mechanical coupling. Physiol. Rev. 78: 763±781. Jensen CG, Poole CA, McGlashan SR, Marko M, Issa ZI, Vujcich KV, Bowser SS (2004) Ultrastructural, tomographic and confocal imaging of the chondrocyte primary cilium in situ. Cell Biol. Int. 28: 101±110. Jin M, Frank EH, Quinn TM, Hunziker EB, Grodzinsky AJ (2001) Tissue shear deformation stimulates proteoglycan and protein biosynthesis in bovine cartilage explants. Arch. Biochem. Biophys. 395: 41±48. Jones WR, Ting-Beall HP, Lee GM, Kelley SS, Hochmuth RM, Guilak F (1999) Alterations in the Young's modulus and volumetric properties of chondrocytes isolated from normal and osteoarthric human cartilage. J. Biomech. 32: 119±127. Kisiday J, Jin M, Grodzinsky AJ (2002) Effects of dynamic compressive loading duty cycle on in vitro conditioning of chondrocyte seeded peptide and agarose scaffolds. Trans. Orthop. Res. Soc. 27: 216. Knight MM, Ghori SA, Lee DA, Bader DL (1998a) Measurement of the deformation of isolated chondrocytes in agarose subjected to cyclic compression. J. Med. Eng. Phys. 20: 684±688. Knight MM, Lee DA, Bader DL (1998b) The influence of elaborated pericellular matrix on the deformation of isolated articular chondrocytes cultured in agarose. Biochim. Biophys. Acta 1405: 67±77. Knight MM, Ross JM, Sherwin AF, Lee DA, Bader DL, Poole CA (2001) Chondrocyte deformation within mechanically and enzymatically extracted chondrons compressed in agarose. Biochim. Biophys. Acta 1526: 141±146. Knight MM et al. (2002) Cell and nucleus deformation in compressed chondrocyte± alginate constructs: temporal changes and calculation of cell modulus. Biochim. Biophys. Acta 1570: 1±8. Knight MM, Bomzon Z, Kimmel E, Sharma AM, Lee DA, Bader DL (2006) Chondrocyte deformation induces mitochondrial distortion and heterogeneous intracellular strain fields. Biomech. Model Mechanobiol. 5: 180±191. Koay EJ, Shieh AC, Athanasiou KA (2003) Creep indentation of single cells. J. Biomech. Eng. 125: 334±341. Lee DA, Bader DL (1995) The development and characterization of an in vitro system to study strain-induced cell deformation in isolated chondrocytes. In Vitro Cell. Dev.Am. 31: 828±835. Lee DA, Bader DL (1997) Compressive strains at physiological frequencies influence the metabolism of chondrocytes seeded in agarose. J. Orthop. Res. 15: 181±188.

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Lee DA et al. (1998a) Response of chondrocyte sub-populations cultured within unloaded and loaded agarose. J. Orthop. Res. 16: 726±733. Lee DA, Frean S, Lees P, Bader DL (1998b) Dynamic mechanical compression influences nitric oxide production by articular chondrocytes seeded in agarose. Biochem. Biophys. Res. Commun. 251: 580±585. Lee DA, Noguchi T, Frean SP, Lees P, Bader DL (2000a) The influence of mechanical loading on isolated chondrocytes seeded in agarose constructs. Biorheology 37: 149±161. Lee DA et al. (2000b) Chondrocyte deformation within compressed agarose constructs at the cellular and sub-cellular levels. J. Biomech. 33: 81±95. Leipzig ND, Athanasiou KA (2005) Unconfined creep compression of chondrocytes. J. Biomech. 38: 77±85. Lemare F, Steinberg N, Le Griel C, Demignot S, Adolphe M (1998) Dedifferentiated chondrocytes cultured in alginate beads: restoration of the differentiated phenotype and of the metabolic response to interleukin-1 beta. J. Cell Physiol. 176: 303±313. Li K.W, Williamson AK, Wang AS, Sah RL (2001) Growth responses of cartilage to static and dynamic compression. Clin. Orthop. 391: S34±48. Martin I, Obradovic B, Treppo S, Grodzinsky AJ, Langer R, Freed LE, VunjakNovakovic G (2000) Modulation of the mechanical properties of tissue engineered cartilage. Biorheology 37: 141±147. Mauck RL, Soltz MA, Wang CC, Wong DD, Chao PH, Valhmu WB, Hung CT, Ateshian GA (2000) Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J. Biomech. Eng. 122: 252±260. Mayne R, Vail MS, Mayne PM, Miller EJ (1976) Changes in the type of collagen synthesised as clones of chick chondrocytes grow and eventually lose division capacity. Proc. Nat. Acad. Sci. USA 73: 1674±1678. Millward-Sadler SJ, Wright MO, Lee H, Nishida K, Caldwell H, Nuki G, Salter DM (1999) Integrin-regulated secretion of interleukin 4: a novel pathway of mechanotransduction in human articular chondrocytes. J. Cell Biol. 145: 183±189. Ohashi T, Hagiwara M, Bader DL, Knight MM (2006) Intracellular mechanics and mechanotransduction associated with chondrocyte deformation during pipette aspiration. Biorheology 43: 201±214. Poole CA, Flint MH, Beaumont BW (1987) Chondrons in cartilage: ultrastructural analysis of the pericellular microenvironment in adult human articular cartilages. J. Orthop. Res. 5: 509±522. Radmacher M, Fritz M, Kacher CM, Cleveland JP, Hansma PK (1996) Measuring the viscoelastic properties of human platelets with the atomic force microscope. Biophys. J. 70: 556±567. Robling AG, Hinant FM, Burr DB, Turner CH (2002) Improved bone structure and strength after long-term mechanical loading is greatest if loading is separated into short bouts. J. Bone Miner. Res. 17: 1545±1554. Rubin CT, Lanyon LE (1992) Regulation of bone formation by applied dynamic loads. J. Bone Joint Surg. Am. 66: 397±402. Sah RL, Kim YJ, Doong JY, Grodzinsky AJ, Plaas AH, Sandy JD (1989) Biosynthetic response of cartilage explants to dynamic compression. J. Orthop. Res. 7: 619±636. Sato M, Levesque MJ, Nerem RM (1987) An application of the micropipette technique to the measurement of the mechanical properties of cultured bovine aortic endothelial cells. J. Biomech. Eng. 109: 27±34. Sato M, Nagayama K, Kataoka N, Sasaki M, Hane K (2000) Local mechanical properties measured by atomic force microscopy for cultured bovine endothelial cells exposed

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to shear stress. J. Biomech. 33: 127±135. Schinagl RM, Ting MK, Price JH, Sah RL (1996) Video microscopy to quantitate the inhomogenous strain within articular cartilage during confined compression. Ann. Biomed. Eng. 24: 500±512. Shieh AC, Athanasiou KA (2002) Biomechanics of single chondrocytes and osteoarthritis. Crit. Rev. Biomed. Eng. 30: 307±343. Shieh AC, Athanasiou KA (2006) Biomechanics of single zonal chondrocytes. J. Biomech. 39: 1595±1602. Shin D, Athanasiou K (1999) Cytoindentation for obtaining cell biomechanical properties. J. Orthop. Res. 17: 880±90. Szafranski JD, Grodzinsky AJ, Burger E, Gaschen V, Hung HH, Hunziker EB (2004) Chondrocyte mechanotransduction: effects of compression on deformation of intracellular organelles and relevance to cellular biosynthesis. Osteoarthritis Cartilage 12: 937±946. Terraciano V, et al. (2007) Differential response of adult and embryonic progenitor cells to mechanical compression in hydrogels. Stem Cells 25: 2730±2738. Theret DP, Levesque MJ, Sato M, Nerem RM, Wheeler LT (1988) The application of a homogeneous half-space model in the analysis of endothelial cell micropipette measurements. J. Biomech. Eng. 110: 190±199. Trickey WR, Lee GM, Guilak F (2000) Viscoelastic properties of chondrocytes from normal and osteoarthritic human cartilage. J. Orthop. Res. 18: 891±898. Trickey WR, Vail TP, Guilak F (2004) The role of the cytoskeleton in the viscoelastic properties of human articular chondrocytes. J. Orthop. Res. 22: 131±139. Vaziri A, Mofrad MR (2007) Mechanics and deformation of the nucleus in micropipette aspiration experiment. J. Biomech. 40: 2053±2062. Waldman SD, Spiteri CG, Grynpas MD, Pilliar RM, Hong J, Kandel RA (2003) Effect of biomechanical conditioning on cartilaginous tissue formation in vitro. J. Bone Joint Surg. Am. 85: 101±105. Waldman SD, Couto DC, Grynpas MD, Pilliar RM, Kandel RA (2007) Multi-axial mechanical stimulation of tissue engineered cartilage: review. Eur. Cell Mater. 12± 13: 66±73. Wang CC, Chahine NO, Hung CT, Ateshian GA (2003) Optical determination of anisotropic material properties of bovine articular cartilage in compression. J. Biomech. 36: 339±353. Wernike E, Li Z, Alini M, Grad S (2008) Effect of reduced oxygen tension and long-term mechanical stimulation on chondrocyte-polymer constructs. Cell Tissue Res. 331(2): 473±483. Wiseman M, Henson F, Lee DA, Bader DL (2003) Dynamic compressive strain inhibits nitric oxide synthesis by equine chondrocytes isolated from different areas of the cartilage surface. Equine Vet. J. 35: 451±56. Wiseman M, Bader DL, Reisler T, Lee DA (2004) Passage in monolayer influences the response of chondrocytes to dynamic compression. Biorheology 41: 283±298. Xie J, Han Z, Kim SH, Kim YH, Matsuda T (2007) Mechanical loading-dependence of mRNA expressions of extracellular matrices of chondrocyte inoculated into elastomeric microporous poly( L)-lactide-co--caprolactone scaffold. Tissue Engineering 13: 29±40. Yaeger PC, Masi TL, deOrtiz JL, Binette F, Tubo R, McPherson JM (1997) Synergistic action of transforming growth factor-beta and insulin-like growth factor-I induces expression of type II collagen and aggrecan genes in adult human articular chondrocytes. Exp. Cell Res. 237: 318±325.

5

Understanding tissue response to cartilage injury

F . D E L L ' A C C I O , Barts and The London, Queen Mary's School of Medicine and Dentistry, UK and T . L . V I N C E N T , Kennedy Institute of Rheumatology, UK

Abstract: It is widely accepted that cartilage injury leads to osteoarthritis (OA), although the mechanisms by which this occurs are still poorly understood. Research in this area has been somewhat neglected in recent years, but it was a highly fashionable academic pursuit in the 18th, 19th and early 20th centuries and many seminal observations were made during this time. These included the findings that acute cartilage injury induces an active chondrocytic response, involving both degradative as well as synthetic processes that resemble OA. There was also evidence of a repair response, determined to be both from the substance of the tissue and from the underlying bone marrow. In patients, injury to cartilage is defined as either direct, e.g. following intra-articular fracture, or indirect by repetitive wear on the tissue, with age or following joint destabilisation. Although both are associated with the risk of developing OA, this risk is variable and there are emerging data to suggest that some focal cartilage lesions not only do not progress, but may actually heal spontaneously. Recent in vitro studies have begun to unravel the molecular basis for these responses, and these are identifying potentially important pathways which may be involved in driving OA, as well as those that stimulate cartilage repair. Key words: joint surface injury, osteoarthritis, explantation, intrinsic repair, impact load.

5.1

Introduction

Very little is known about how tissues respond to injury, apart from the wellestablished activation of the clotting cascade upon vascular damage: this is a highly orchestrated process that is initiated by platelets binding to the damaged vascular endothelium. Activated platelets regulate the catalytic activities that initiate thrombin generation, which is then sustained at the site of injury by the recruitment of circulation monocytes and neutrophils. Monocytes are capable of driving thrombin production through expression of tissue factor, a protein also expressed by subendothelial cells and activated endothelium which, when in contact with factor VIIa activates the clotting cascade zymogens. The combination of damaged endothelium, the activated surface of the platelet and activated leukocytes provide the optimal environment for locally contained control of haemostasis.1

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In skin, wounding evokes a coordinated cellular response which is critical upon resident cells as well as invading immune cells, which are essential for resolution of the healing response.2,3 Clearly, skin wounding also causes endothelial damage, as it is a vascular tissue, but whether there are other triggers that initiate this response, is not known. What triggers injury responses in tissues that are avascular is very poorly understood. Articular cartilage injury is considered to be one of the most important risk factors for osteoarthritis (OA); disease is increased in aged joints (chronic wear and tear-associated injury), and occurs prematurely in individuals who have sustained injury to the cartilage surface or to other associated structures within the joint. Damage to the menisci and cruciate ligaments is thought to lead to increased wear across the articular surfaces through induction of joint instability. The response of articular cartilage to injury was a highly fashionable pursuit in the 18th and 19th centuries as well as the early 20th century, but it is surprising how `out of vogue' this area of research has become. This is perhaps surprising in view of the increasing prevalence of OA in our society associated with increased longevity and the obesity epidemic. As funding bodies focus their attention to neglected prevalent diseases such as OA, so it is likely that research into basic mechanisms of cartilage injury will increase. This chapter will address what is known about the natural history of clinical cartilage injury in vivo, and will discuss the work that has been undertaken to understand tissue responses in animal models of in vivo injury, and in vitro injury systems.

5.2

Clinical in vivo cartilage injury

In vivo injury of cartilage is often referred to as joint surface injury (JSI) and results in joint surface defects (JSD). It includes a broad range of conditions involving damage to the articular cartilage, with or without involvement of the subchondral bone. Such lesions are very common, being reported in over 60% of all arthroscopic procedures.4,5 Chronic JSDs represent a clinical problem because (1) they can be symptomatic and disabling, with pain and/or locking of the joint, and (2) they predispose to further cartilage loss and development of OA.6 Chondral lesions vary greatly in their morphology and topography, and this variation likely influences their outcome and clinical manifestations. Broadly speaking, lesions can be divided into being localised, such as those due to trauma, or diffuse, such as those that characterise OA. A second important division is that between superficial, partial thickness cartilage defects, which do not involve the subchondral bone, and full-thickness lesions which cross the ostoechondral junction (Outerbridge grade 3 or 4). Superficial cartilage defects, particularly if linear, without tissue loss, have a poor repair capacity. Although much emphasis has been given to this factor in the field of cartilage biology, in clinical practice such defects are rarely considered an indication for chondral

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surgery, since they are often asymptomatic and the evidence available today does not indicate that they predispose to cartilage loss and OA progression.7±10 Indeed, chondral surgery, such as autologous chondrocyte implantation or microfracture, is usually reserved for chronic, symptomatic, full thickness chondral or osteochondral defects, especially where injury is the most likely cause of the symptoms.10±13

5.2.1

The aetiology of JSI

The most important risk factors in the development of cartilage loss in OA development are age, trauma, malalignment, meniscal or cruciate ligament injury, and a family history.14±17 Such factors point towards mechanical factors contributing to JSD development. Although we generally attribute meniscal injury as causing OA through joint destabilisation, the clinical data for this are missing. For instance joint instability in benign hypermobility syndrome does not predispose to OA, nor is it the case that individuals with repair to their menisci have a reduce risk of developing OA compared with those who have not had a repair (Lohmander, personal communication). Trauma has traditionally been regarded as the most important aetiological factor in the development of focal chondral or osteochondral defects.18 However, in a large study of 1000 consecutive arthroscopies, 39% of patients with a focal defect in their knee cartilage failed to remember a previous traumatic episode to their joint.5 In addition, it has been shown that up to 43% of healthy subjects without a family history of OA have knee chondral lesions as evaluated by magnetic resonance imaging (MRI).19 These data point to the fact that chondral or osteochondral defects are more common than previously thought and that a traumatic episode may not always be apparent from the history.

5.2.2

The natural history of JSI

Over 250 years ago Hunter stated: `If we consult the standard Chirurgical Writers from Hippocrates down to the present Age, we shall find, that an ulcerated Cartilage is universally allowed to be a very troublesome disease and when destroyed, it is never recovered.'20 Of course, this statement most likely referred to severely symptomatic lesions that had acquired a chronic and disabling course, leading patients to the attention of a surgeon. Through the years, however, such a paradigm has extended to all chondral injuries. This is mainly because of the lack of diagnostic tools capable of identifying, and prospectively following up smaller and, in particular, acute lesions. It is also the case that published studies are capable of fuelling intuitive preconceptions; reporting, for instance, that the risk of developing OA by the age of 65 is 13% in individuals with a history of trauma compared with 6% in those without a history of trauma.21 As a consequence the cartilage biology/repair literature

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often concludes that `cartilage lesions never heal spontaneously', and that they necessarily `predispose to osteoarthritis'. The recent development of cell-based therapies for the repair of chondral lesions such as microfracture, mosaicplasty and autologous chondrocyte implantation (ACI) has generated interest in the natural history of these lesion to define indications for such invasive procedures.12 Improved cartilage imaging using MRI has contributed significantly to the generation of longitudinal data in all groups of patients. In 1996, Messner and Maletius8 reported that 22 out of 28 young athletes who had an isolated chondral injury in a weight-bearing part of their knees diagnosed by arthroscopy had good or excellent knee function at 14 years follow-up as evaluated clinically and radiographically. No specific treatment had been preformed except Pridie drilling (similar to microfracture) in three cases and occasional debridement. Twenty-one patients were able to return to pre-injury level sports activities. Although at the end of follow-up 12 patients had some radiographic joint space reduction. No control group (without JSD) was included and therefore we do not know whether joint space reduction would have occurred in the absence of a JSD. In this study, all patients had an isolated Outerbridge grade 2 (most cases) or 3 chondral defect (diameter > 1 cm), without any damage to other joint structures including menisci, ligaments and the remaining cartilage. No patient had instability, or a previous history of knee surgery. What we learn from this study is that isolated chondral or osteochondral lesions, in young active patients, in otherwise healthy knees, have a favourable natural history leading to long-term functional restoration. We do not know whether (good) structural repair is required for functional outcome or whether these lesions become asymptomatic or repair with scar tissue. In either case, such a good outcome after 14 years follow-up in 78% of the lesions, suggest that an aggressive approach for all such lesions is not justified. The study discussed above focused on isolated (osteo) chondral defects in otherwise normal knees. However, isolated chondral defects are present in only 36.6% of symptomatic knees requiring arthroscopy. In the majority of cases, other lesions, including those to menisci4 or ligaments,9,22 co-exist. In a longitudinal study, Shelbourne et al.9 asked the question whether the presence of a chondral injury detected in young athletes undergoing ACL reconstruction modifies the clinical outcome. In this study they selected two groups of such patients, one that had a single chondral or osteochondral injury at the time of arthroscopy, and an age and sex matched group who had no chondral injury. The cartilage injury was left untreated, and the clinical outcome was monitored for 8.7 years, clinically and radiographically. Throughout follow-up, the patients with a chondral injury had more subjective symptoms that those without. This difference was statistically significant, but small in size and more than 79% of patients returned to pre-injury levels of sports activities involving jumping, twisting and pivoting. The radiological score was not different in the two groups. There was no correlation between the size of the defect and the

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outcome, although the size of some defects might have been underestimated since lesions were not debrided. Finally, they reported that, in each individual patient, the severity of symptoms fluctuated significantly during the follow-up. Again, there was no information as to whether structural repair of the chondral injury was a prerequisite for good clinical outcome. The recent improvement in imaging of the articular cartilage with 3D fast spin-echo, or fat suppressed spoiled gradient-echo MRI have allowed detection of chondral lesions diagnosed at arthroscopy with a sensitivity approaching 95%, and specificity sometimes reaching 100%.23±27 MRI has therefore allowed monitoring of chondral defects to obtain prospective clinical and structural outcomes in symptomatic and asymptomatic groups. These studies have yielded surprising results. Ding et al.28 reported a longitudinal study in which the presence and the natural history of chondral defects had been studied by MRI in asymptomatic subjects with a family history of joint replacement, and in an age and sex matched population without family history of OA. This study revealed that 43% of the subjects without a family history of OA and 57% of subjects with a family history of OA19 had chondral defects. At 2.3 years follow-up, 33% of all subjects had a worsening of the defects as graded by MRI, 37% an improvement and the rest remained stable. A worse outcome was associated with female sex, age and body mass index at baseline. Although factors associated with the reproducibility of the MRI grading may have contributed to the defect variation, in general, measurement error was considered to be very low. Importantly, only 18% of the subjects with a cartilage defect had a history of knee trauma. These data show three very important points. Firstly, that chondral defects, including full thickness ones, are often asymptomatic; secondly, that the majority of these lesions may not be related to traumatic injury as previously thought. Thirdly, and most importantly, a number of these lesions may improve (and possibly heal) spontaneously. The presence of chondral defects in these patients predicted a rate of cartilage loss of 2±3% instead of 1±2% of subjects without chondral defects.7 Since the rate of cartilage loss is an independent predictor of joint replacement in patients with OA,29 it is arguable that at least a number of such asymptomatic defects may predispose to OA. In a separate paper, Davies-Tuck et al. reported on the natural history of similar chondral lesions in a cohort of patients with OA.30 In this cohort, chondral injuries worsened in 81% of the cases and improved in only 4% over 2 years. In a similar prospective study, Wluka et al. showed that the presence of cartilage defects in patients with OA was associated with disease severity, correlated with the rate of cartilage loss within 2 years and was a predictor of joint replacement within 4 years.31 We could summarise these data by saying that cartilage defects are often but not always due to acute mechanical injury, and can complicate and accelerate the course of OA, but may be present in otherwise normal knees, where, at least

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in some cases, they may accelerate the physiological rate of cartilage loss that takes place after the age of 40 years. Importantly, particularly in the absence of OA, some of these defects may undergo at least a partial healing. Although age, female sex and body mass index are generally associated with progression, it is not presently possible to predict the outcome of one individual defect. These considerations have important clinical consequences in deciding when to treat a chondral defect. Although general common sense would suggest that chronic, symptomatic, isolated defects are a good indication for interventions such as microfracture or autologous chondrocyte transplantation, in most cases we do not have good evidence that defects are associated with progression to OA. Nor do we know whether asymptomatic isolated defects go on to become chronic and predispose to cartilage loss and OA. There is therefore a clinical need for criteria or biomarkers that could predict the outcome of JSD accurately. Such tools would not only be precious for the clinician, but would also improve the sensitivity of clinical trials and therefore avoid the floor effect due to a number of patients who might improve spontaneously in the absence of treatment.

5.2.3

Chronic joint injury

Chronic injuries include repetitive trauma, excessive loading, as well as direct and indirect altered biomechanics. All these factors have been associated with cartilage loss, pain and disability.14 Subjects exposed to heavy work such as farming and repetitive lifting of heavy weight, as well as athletes practising sports involving high levels of twisting and torsional loading are at higher risk of developing OA.32±36 It is worth stressing, however, that both heavy work and sport activities also predispose to meniscal and ligament injuries, which are independent risks for cartilage loss.37 The way in which damage to menisci and ligaments predisposes to OA is thought to be due to loss of cartilage protection, and creation of joint instability, leading to increased wear of the articulating surfaces.14,38,39 Certainly there is evidence that patients with such lesions have an increased risk of developing OA.37 Indeed, even simple extrusion of the medial meniscus is a risk factor in for progression of knee OA, and is predictive of joint replacement.38,39 Malalignment is another important risk factor for cartilage loss and OA.37,40 Cartilage loss occurs in dysplastic joints in which weight-bearing is not evenly distributed.14,40 Interestingly, even in the case of chronic cartilage ulcers in adult individuals with varus knees, correcting malalignment by tibial osteotomy was able to partially restore the articular surface,40±44 though mostly by fibrocartilage.40 How acute or chronic mechanical injury results in progressive breakdown of the cartilage tissue is not completely understood. Ultimately, the degradation of the cartilage matrix is believed to be mediated by enzymes such as

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metalloproteinases, aggrecanases and other proteolytic enzymes, which may be produced by inflamed synovium, by infiltrating haematopoietic cells,45 and also by injured chondrocytes.45±47

5.3

Animal models of cartilage injury

The development of animal models has contributed enormously to our understanding of the pathophysiology of cartilage injury. Injury in such models can be described as direct or indirect, and are discussed below.

5.3.1

Direct injury to the articular surface

Despite the earlier observations of Hunter in the 18th century, Kolliker and Paget were the first to recognise the ability of cartilage lesions to mend. At the time they attributed repair to cells outside the articular cartilage (reviewed in Campbell48). These early observations heralded an explosion in experimental in vivo cartilage injury models, which became a fashionable scientific pursuit for the next 150 years. Many of the seminal papers in this field are derived from these early years. Peter Redfern in 1851 (reprinted in 196949) was one of the first to describe the tissue response following incisions to the articular cartilage of dogs. Redfern's observations apparently contradicted the concensus view, by demonstrating that cuts in articular cartilage were capable of `uniting with a highly cellular, disorganised, granular mass'. He described a reactive chondrocyte phenotype; superficial cells were enlarged and those close to the lesion showed evidence of proliferation. He concluded that there was `no longer . . . the slightest doubt that wounds in articular cartilages are capable of perfect union by formation of fibrous tissue out of the texture of the cut surfaces'. Whilst these findings have held true over the years, his interpretation of the repair response was probably incorrect. Shands in 1931 observed that there was a significant difference in repair responses between full thickness defects, where the cut extended across the osteochondral junction, and superficial lesions where the cut did not breech the junction.50 In the 1970s, Meachim proposed that there were two responses to articular cartilage injury; an intrinsic and extrinsic repair response. Extrinsic repair was stimulated by bone marrow cells when the cut had breeched the osteochondral junction, and it was these cells that contributed to what we now regard as a fibrocartilage scar.51 Intrinsic repair was by cells of the cartilage and was more effective at generating a scar-free hyaline cartilage between cut surfaces, even though Bennett and co-workers recognised that `the powers of such regeneration are feeble and not always demonstrable'.52,53 Calandruccio went further to suggest that both sorts of lesions were capable of stimulating intrinsic repair, but that intrinsic repair was suppressed in the presence of a dominant extrinsic response from the bone marrow.54 What these studies as well as those of others clearly indicated was that there was

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considerable variability in repair responses in individual animals which could not, and has not, been explained. Nonetheless, these studies collectively demonstrated that cartilage tissue was able to stimulate a repair response. In 1963, Meachim performed a comprehensive study following the effects of superficial scarification of articular cartilage in adult rabbits. On histological examination of the tissue and by sulphate incorporation, he found an early increase in metachromasia around superficial chondrocytes near the cuts, and subsequent loss of inter-territorial proteoglycan over the next few weeks. Cell clusters were apparent at 16 weeks especially in the deep layers.51 Cell clustering was shown to be due to chondrocyte division by Mankin55 who concluded that this represented intrinsic repair, albeit late, and unlikely to be contributing to healing in the presence of a more rapid extrinsic reaction. More recent studies have confirmed these original observations but few have added much more to how cartilage repair is controlled and coordinated. Shapiro et al.56 generated small osteochondral defects in young rabbit cartilage, which resulted in invasion of bone marrow cells into the lesion within 2 weeks of injury. Interestingly, the process that led to restoration of the articular surface within 24 weeks involved a coordinated and polarised morphogenetic process highly reminiscent of endochondral bone formation during development. This experiment suggested that mechanisms similar to those governing embryonic skeletogenesis were reactivated during postnatal repair processes. This finding is supported by the observation that mutations or allelic variants of several morphogens and growth factors known to play a role in skeletal development are associated with an increased risk of OA.57±62 Wei et al.63 studied repair responses in rabbits of different ages, from adolescent to fully mature, and reported results similar to that of Shapiro et al. in young rabbits. In adult rabbits the outcome of repair was considerably worse, thereby paralleling clinical data in humans. The role of inflammatory cells in cartilage injury was suggested by Hembry et al. when they demonstrated macrophage recruitment to the damaged surface.45 They proposed that these cells were responsible for release of MMP9, which probably activated other metalloproteinases of joint origin, causing activation of chondrocytic MMP-3 and MMP-13, as well as aggrecanases, at the site of injury. Matrix metalloproteinase (MMP) inhibitors such as tissue inhibiting metalloproteinase 1 (TIMP-1) were also induced by injury suggesting a tight regulation of proteolysis. The subchondral bone in adult animals has also been shown to react promptly to cartilage injury with activation of intense remodelling. In a goat model, Vasara et al. reported that, even partial thickness lesions caused extensive bone remodelling with distortion of the trabeculae and reduced bone volume.64 Towards the end of the 20th century a number of groups tested the responses induced by injurious loading of the joint. This might be regarded as more physiological in vivo damage. Donohue et al. impact loaded closed canine joints

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and studied the histological response 2, 4 and 6 weeks later.65 They found that there was clonal expansion of cells in the cartilage, with increased vascular invasion and increased water content. They also calculated a 40% reduction in proteoglycan associated with collagen. A similar closed joint transarticular load to the patella was used by Thompson et al. who extended the period of observation to 24 weeks post-injury. They described the presence of fissures in the cartilage, and late loss of proteoglycan in the tissue associated with deep clefts.66 Haut and co-workers added that there was significant softening of the articular cartilage early post-impact,67 and in a follow-up study.68 They determined that there was increased tissue thickness in the first 5 months following injury, but by 36 months a significant (45%) reduction in tissue thickness was measured. They also documented a significant increase in the thickness of the subchrondral bone. Whilst the histological chronology of articular cartilage lesions has been well characterised over the past 200 years, the molecular and cellular mechanisms that control such events are far from clear. We may be able to attribute this in part to the recent decline in the use of such in vivo models at a time when molecular tools were becoming increasingly sensitive. As experimental JSI was becoming unfashionable, many were turning their attention to indirect models of cartilage damage in vivo.

5.3.2

Indirect cartilage injury

Modern approaches to look at cartilage injury in vivo have tended to involve indirect injury, through, for instance, joint destabilisation. Such models have the advantage of more closely mimicking clinical osteoarthritis. Joint instability can be induced through surgery (a list of some such models is given in Table 5.1), or through enzymatic treatment of the joint with collagenase,69 which is thought to weaken the supporting ligaments. It is worth stressing that all of these models, to a greater or lesser extent, induce joint inflammation, and so cartilage injury is due to a combination of inflammation and mechanical factors. Table 5.1 Surgical models of osteoarthritis Animal

Method of induction

Rabbit

Medial meniscectomy70 Meniscectomy and cruciate transaction71

Dog

Cruciate transection (Pond-Nuki)72

Guinea pig

Gluteal resection (myectomy and tendotomy)73

Mouse

Partial meniscectomy and transaction of med collateral ligament74 Meniscotibial ligament transaction (DMM)75

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One recently described model deserves detailed attention. This murine model, which is induced by destabilisation of the medial meniscus (DMM), was described by Glasson et al. in 2005.75 A robust and predictable degeneration of the articular cartilage is seen in male mice within 4±8 weeks of transection of the medial menisco-tibial ligament. Synovitis is rarely present, and bone changes are a late feature. As meniscal injury is a strong risk factor for the development of OA in humans, this model is highly clinically relevant.37,39 There are a number of advantages in using this model over other historical ones: · · · · · · ·

100% penetrance (all male mice develop disease); cartilage loss from 4 weeks post-surgery; mice are cheap and easy to house; mouse experimentation is less emotive than that using higher species; can combine model in mice which have been genetically modified; less inflammatory than other historical models of cartilage degeneration; could be used for drug screening.

Of paramount importance for understanding pathogenesis of OA and early responses to cartilage injury is the fact that this model allows one to combine OA progression in genetically modified mice. This was done to good effect by Glasson et al., who demonstrated that mice deficient in ADAMTS5, but not ADAMTS4 (aggrecan degrading enzymes in cartilage) were partially protected from osteoarthritis.75 This not only told us that cartilage degeneration was dependent upon aggrecanase activity, but also that ADAMTS5 was the principal cartilage aggrecanase in mice. The model has provided functional data on a large number of molecules in addition to ADAMTS5.76 Partial protection was seen in mice deficient in IL-1 beta, although unexpectedly MMP9 null mice developed accelerated cartilage degradation after DMM surgery, suggesting that it was protective in vivo. Quoting Sonya Glasson, `although the results in the mouse will not always transpose to the human condition, the track record of mouse knockouts corresponding to the human phenotype have been excellent'.76

5.4

In vitro cartilage injury

Much recent interest has focused on the responses of articular cartilage to injury in vitro. For the sake of simplicity we will focus on direct mechanical injury rather than chemical or inflammatory. There are a number of ways of inducing cartilage injury including explantation, re-cutting, impact loading and high magnitude (low velocity) loading. Each of these will be addressed in more detail below.

5.4.1

Explantation

Explantation is perhaps the `purest' injury in cartilage, because it is closest to how injury might be perceived in vivo. The articular surface of cartilage is

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normally bathed in synovial fluid which contains plasma proteins as well as some of the cellular constituents of blood. It is hardly surprising that washing plasma proteins from cartilage explants (which occurs when explants are cultured in medium) will change the response of the tissue to different stimuli. Although direct comparisons of explantation versus re-cutting cartilage explants in vitro have not been performed, some observations have been made. For instance, there is a strong activation of the extracellularly regulated kinsae (ERK), p38 and c-jun-terminal kinase (JNK), members of the mitogen activated protein (MAP) kinases upon explantation.77 The activation of the ERK pathway is due to release of FGF2, which also occurs when explants are rested then recut.78 Activation of JNK and p38 does not occur on re-cutting,78 and does not appear to be due to the release of a soluble factor (J. Saklatvala, personal communication). Activation of inflammatory MAP kinases upon explantation is sufficient to activate inflammatory response genes such as IL-1 and at the mRNA and protein level.77

5.4.2

Re-cutting

Re-cutting cartilage is usually performed when the cartilage has been explanted into medium (either serum containing or serum free) and rested for a variable amount of time ± anywhere from 12 hours to 7 days. The rationale for this experimental system is to allow any cellular response which occurred upon explantation to return to baseline before the cartilage is re-cut. The timing of the re-cutting is probably critical as a recent gene array study has demonstrated that it takes 7 days for chondrocyte gene expression to return to in vivo (resting) levels.79 In this study the authors rested human articular cartilage in medium for 7 days prior to re-cutting. They identified 690 genes that were significantly regulated at least two-fold following re-cutting. These included genes previously reported to be differentially expressed in OA versus normal cartilage, or having allelic variants genetically linked to OA. A systematic analysis of the Wnt signalling pathway revealed up-regulation of Wnt-16, down-regulation of FRZB, and up-regulation of Wnt target genes. They were able to demonstrate increased Wnt pathway activity in wounded as well as osteoarthritic cartilage, by demonstrating accumulation of cellular beta catenin.

5.4.3

Impact loading

Conservative mechanobiologists make a clear distinction between injurious mechanical loading and impact loading.80 The latter is characteristically a high velocity load with consequent cellular and matrix damage in the absence of significant tissue strain (the amount the tissue deforms). Such injury to cartilage is a feature of acute mechanical trauma to cartilage, say following a road traffic accident, or a fall from a height. Load is often delivered via a drop tower and is

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usually a single impact. Such injury causes cell loss associated with microscopic splitting in the superficial layer of the cartilage and early surface delamination.81 This same group also studied the effect of impact load on sulphate incorporation and loss of proteoglycan from the matrix. They found that in the 2 weeks following impact, 25±40% of sulphated proteoglycan was lost from the tissue. Although recovery was associated with an increase in proteoglycan synthesis, synthetic responses were suppressed early following injury.82

5.4.4

Low velocity injurious load

Low velocity injuries tend to be characterised by high strain (say 50%) with relatively low velocities, e.g. 100%/s. Such cartilage injury also causes significant proteoglycan loss in the first few days following load.83 Loss of proteoglycan was determined to be independent of MMP activity in these early stages, although later losses could be suppressed by the presence of MMP inhibitors.84 Microarray analysis of injured cartilage showed strong upregulation of MMP3 and ADAMTS5, as well as the negative regulator of metalloproteinase activity, suggesting that these enzymes may be responsible for active proteolytic degradation of tissue proteoglycan.47 Another response to cartilage injury that has been extensively investigated in vitro is cell death. From the early observations of Redfern, Bennett and others, cell death along the cut surface has been a robust feature. Tew et al. determined that cell death in explants following trephine injury was due to a combination of apoptosis and necrosis. They also were able to show increased cell proliferation in the regions adjacent to the cut surface.85 The same group went on to show that cartilage wounding responses were essentially similar between immature and mature cartilage,86 but that increased cell death was a feature of blunt wounds rather more so than those generated by a scalpel blade.87 Others have shown similar results in cartilage explants subjected to injurious load.88,89 Quinn et al. observed a mixture of cell death and enlarged viable cells adjacent to the dead chondrocytes, which, by autoradiography, showed increased proteoglycan synthesis. There is little doubt that such a loading regime induces significant cellular and tissue damage. Stevens et al. studied the pattern of secreted proteins 5 days following injurious load of bovine explants. They identified a number of proteins by mass spectrometry of sodium dodecyl sulphate±polyacrylamide gel electrophoresis (SDS-PAGE) separated proteins. Their main findings were the release of a large number of intracellular proteins indicating significant cell injury, as well as release of fragments of matrix proteins such as type VI collagen, dermatan sulphate proteoglycan 3 and fibronectin implying matrix damage.90

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5.5

149

Conclusions

Chondral injury in patients is still poorly understood, although recent emerging data suggest that small chondral defects may have the capacity to heal spontaneously. It remains the case that joint surface injury is a risk factor for development of OA probably through both the mechanical disruption of the tissue, cell death, as well as through activation of proteolytic pathways. The early in vivo injury observations of Redfern, Calandruccio, Meachim, Mankin and others have made an unrivalled contribution to our understanding of how cartilage responds to injury. In view of the advances in modern science over recent years it is perhaps surprising that our understanding of the cellular and molecular events of cartilage injury is not greater. A small number of novel molecular pathways of cartilage injury have been described. Such molecules are likely to be important in the development of molecular tools to support repair and, at the same time, may help identify those patients who would benefit from chondral surgery/tissue engineering. An exciting challenge in such studies will be finding strategies that not only stimulate production of hyaline cartilage, but that will allow integration of neocartilage into the existing matrix.

5.6

References

1 Bouchard, B.A., and Tracy, P.B. 2001. Platelets, leukocytes, and coagulation. Curr Opin Hematol 8: 263±269. 2 Martin, P., and Leibovich, S.J. 2005. Inflammatory cells during wound repair: the good, the bad and the ugly. Trends Cell Biol 15: 599±607. 3 Stramer, B.M., Mori, R., and Martin, P. 2007. The inflammation-fibrosis link? A Jekyll and Hyde role for blood cells during wound repair. J Invest Dermatol 127: 1009±1017. 4 Curl, W.W., Krome, J., Gordon, E.S., Rushing, J., Smith, B.P., and Poehling, G.G. 1997. Cartilage injuries: a review of 31,516 knee arthroscopies. Arthroscopy 13: 456±460. 5 Hjelle, K., Solheim, E., Strand, T., Muri, R., and Brittberg, M. 2002. Articular cartilage defects in 1,000 knee arthroscopies. Arthroscopy 18: 730±734. 6 Ding, C., Cicuttini, F., and Jones, G. 2008. How important is MRI for detecting early osteoarthritis? Nat Clin Pract Rheumatol 4: 4±5. 7 Ding, C., Cicuttini, F., Scott, F., Boon, C., and Jones, G. 2005. Association of prevalent and incident knee cartilage defects with loss of tibial and patellar cartilage: a longitudinal study. Arthritis Rheum 52: 3918±3927. 8 Messner, K., and Maletius, W. 1996. The long-term prognosis for severe damage to weight-bearing cartilage in the knee: a 14-year clinical and radiographic follow-up in 28 young athletes. Acta Orthop Scand 67: 165±168. 9 Shelbourne, K.D., Jari, S., and Gray, T. 2003. Outcome of untreated traumatic articular cartilage defects of the knee: a natural history study. J Bone Joint Surg Am 85-A Suppl 2: 8±16. 10 Smith, G.D., Knutsen, G., and Richardson, J.B. 2005. A clinical review of cartilage repair techniques. J Bone Joint Surg Br 87: 445±449.

150 11 12 13 14 15 16 17 18 19 20 21 22 23

24

25

26

27

Regenerative medicine and biomaterials in connective tissue repair Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O., and Peterson, L. 1994. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 331: 889±895. Brittberg, M., Peterson, L., Sjogren-Jansson, E., Tallheden, T., and Lindahl, A. 2003. Articular cartilage engineering with autologous chondrocyte transplantation. A review of recent developments. J Bone Joint Surg Am 85-A Suppl 3: 109±115. Peterson, L., Minas, T., Brittberg, M., Nilsson, A., Sjogren-Jansson, E., and Lindahl, A. 2000. Two- to 9-year outcome after autologous chondrocyte transplantation of the knee. Clin Orthop Relat Res: 212±234. Buckwalter, J.A., Saltzman, C., and Brown, T. 2004. The impact of osteoarthritis: implications for research. Clin Orthop Relat Res 427: S6±15. Loeser, R.F. 2006. Molecular mechanisms of cartilage destruction: mechanics, inflammatory mediators, and aging collide. Arthritis Rheum 54: 1357±1360. Sweeney, S.E., and Firestein, G.S. 2004. Rheumatoid arthritis: regulation of synovial inflammation. Int J Biochem Cell Biol 36: 372±378. van den Berg, W.B., van Lent, P.L., Joosten, L.A., Abdollahi-Roodsaz, S., and Koenders, M.I. 2007. Amplifying elements of arthritis and joint destruction. Ann Rheum Dis 66 Suppl 3: iii45±48. Morscher, E. 1979. [Traumatic cartilage lesions in the knee joint]. Der Chirurg 50: 599±604. Ding, C., Cicuttini, F., Scott, F., Stankovich, J., Cooley, H., and Jones, G. 2005. The genetic contribution and relevance of knee cartilage defects: case-control and sibpair studies. J Rheumatol 32: 1937±1942. Hunter, W. 1743. On the structure and diseases of the articular cartilages. Philos Trans Roy Soc 42B: 514±521. Gelber, A.C., Hochberg, M.C., Mead, L.A., Wang, N.Y., Wigley, F.M., and Klag, M.J. 2000. Joint injury in young adults and risk for subsequent knee and hip osteoarthritis. Ann Intern Med 133: 321±328. Indelicato, P.A., and Bittar, E.S. 1985. A perspective of lesions associated with ACL insufficiency of the knee. A review of 100 cases. Clin Orthop Relat Res 198: 77±80. Bredella, M.A., Tirman, P.F., Peterfy, C.G., Zarlingo, M., Feller, J.F., Bost, F.W., Belzer, J.P., Wischer, T.K., and Genant, H.K. 1999. Accuracy of T2-weighted fast spin-echo MR imaging with fat saturation in detecting cartilage defects in the knee: comparison with arthroscopy in 130 patients. AJR Am J Roentgenol 172: 1073±1080. Broderick, L.S., Turner, D.A., Renfrew, D.L., Schnitzer, T.J., Huff, J.P., and Harris, C. 1994. Severity of articular cartilage abnormality in patients with osteoarthritis: evaluation with fast spin-echo MR vs arthroscopy. AJR Am J Roentgenol 162: 99± 103. Disler, D.G., McCauley, T.R., Wirth, C.R., and Fuchs, M.D. 1995. Detection of knee hyaline cartilage defects using fat-suppressed three-dimensional spoiled gradientecho MR imaging: comparison with standard MR imaging and correlation with arthroscopy. AJR Am J Roentgenol 165: 377±382. Kawahara, Y., Uetani, M., Nakahara, N., Doiguchi, Y., Nishiguchi, M., Futagawa, S., Kinoshita, Y., and Hayashi, K. 1998. Fast spin-echo MR of the articular cartilage in the osteoarthritic knee. Correlation of MR and arthroscopic findings. Acta Radiol 39: 120±125. Recht, M.P., Piraino, D.W., Paletta, G.A., Schils, J.P., and Belhobek, G.H. 1996. Accuracy of fat-suppressed three-dimensional spoiled gradient-echo FLASH MR imaging in the detection of patellofemoral articular cartilage abnormalities. Radiology 198: 209±212.

Understanding tissue response to cartilage injury

151

28 Ding, C., Cicuttini, F., Scott, F., Cooley, H., Boon, C., and Jones, G. 2006. Natural history of knee cartilage defects and factors affecting change. Arch Intern Med 166: 651±658. 29 Cicuttini, F.M., Jones, G., Forbes, A., and Wluka, A.E. 2004. Rate of cartilage loss at two years predicts subsequent total knee arthroplasty: a prospective study. Ann Rheum Dis 63: 1124±1127. 30 Davies-Tuck, M.L., Wluka, A.E., Wang, Y., Teichtahl, A.J., Jones, G., Ding, C., and Cicuttini, F.M. 2008. The natural history of cartilage defects in people with knee osteoarthritis. Osteoarthritis Cartilage 16: 337±342. 31 Wluka, A.E., Ding, C., Jones, G., and Cicuttini, F.M. 2005. The clinical correlates of articular cartilage defects in symptomatic knee osteoarthritis: a prospective study. Rheumatology (Oxford) 44: 1311±1316. 32 Anderson, J.J., and Felson, D.T. 1988. Factors associated with osteoarthritis of the knee in the first national Health and Nutrition Examination Survey (HANES I). Evidence for an association with overweight, race, and physical demands of work. Am J Epidemiol 128: 179±189. 33 Buckwalter, J.A., and Lane, N.E. 1997. Athletics and osteoarthritis. Am J Sports Med 25: 873±881. 34 Croft, P., Coggon, D., Cruddas, M., and Cooper, C. 1992. Osteoarthritis of the hip: an occupational disease in farmers. BMJ 304: 1269±1272. 35 Felson, D.T., Hannan, M.T., Naimark, A., Berkeley, J., Gordon, G., Wilson, P.W., and Anderson, J. 1991. Occupational physical demands, knee bending, and knee osteoarthritis: results from the Framingham Study. J Rheumatol 18: 1587±1592. 36 Yoshimura, N., Sasaki, S., Iwasaki, K., Danjoh, S., Kinoshita, H., Yasuda, T., Tamaki, T., Hashimoto, T., Kellingray, S., Croft, P., et al. 2000. Occupational lifting is associated with hip osteoarthritis: a Japanese case-control study. J Rheumatol 27: 434±440. 37 Lohmander, L.S., Englund, P.M., Dahl, L.L., and Roos, E.M. 2007. The long-term consequence of anterior cruciate ligament and meniscus injuries: osteoarthritis. Am J Sports Med 35: 1756±1769. 38 Ding, C., Martel-Pelletier, J., Pelletier, J.P., Abram, F., Raynauld, J.P., Cicuttini, F., and Jones, G. 2007. Meniscal tear as an osteoarthritis risk factor in a largely nonosteoarthritic cohort: a cross-sectional study. J Rheumatol 34: 776±784. 39 Ding, C., Martel-Pelletier, J., Pelletier, J.P., Abram, F., Raynauld, J.P., Cicuttini, F., and Jones, G. 2007. Knee meniscal extrusion in a largely non-osteoarthritic cohort: association with greater loss of cartilage volume. Arthritis Res Ther 9: R21. 40 Koshino, T., Wada, S., Ara, Y., and Saito, T. 2003. Regeneration of degenerated articular cartilage after high tibial valgus osteotomy for medial compartmental osteoarthritis of the knee. Knee 10: 229±236. 41 Fujisawa, Y., Masuhara, K., and Shiomi, S. 1979. The effect of high tibial osteotomy on osteoarthritis of the knee. An arthroscopic study of 54 knee joints. Orthop Clin North Am 10: 585±608. 42 Matsui, N., Moriya, H., and Kitahara, H. 1979. The use of arthroscopy for follow-up in knee joint surgery. Orthop Clin North Am 10: 697±708. 43 Odenbring, S., Egund, N., Lindstrand, A., Lohmander, L.S., and Willen, H. 1992. Cartilage regeneration after proximal tibial osteotomy for medial gonarthrosis. An arthroscopic, roentgenographic, and histologic study. Clin Orthop Relat Res 277: 210±216. 44 Schultz, W., and Gobel, D. 1999. Articular cartilage regeneration of the knee joint after proximal tibial valgus osteotomy: a prospective study of different intra- and

152 45

46

47 48 49 50 51 52 53 54 55 56 57

58

59 60

61

Regenerative medicine and biomaterials in connective tissue repair extra-articular operative techniques. Knee Surg Sports Traumatol Arthrosc 7: 29±36. Hembry, R.M., Dyce, J., Driesang, I., Hunziker, E.B., Fosang, A.J., Tyler, J.A., and Murphy, G. 2001. Immunolocalization of matrix metalloproteinases in partialthickness defects in pig articular cartilage. A preliminary report. J Bone Joint Surg Am 83-A: 826±838. Flannelly, J., Chambers, M.G., Dudhia, J., Hembry, R.M., Murphy, G., Mason, R.M., and Bayliss, M.T. 2002. Metalloproteinase and tissue inhibitor of metalloproteinase expression in the murine STR/ort model of osteoarthritis. Osteoarthritis Cartilage 10: 722±733. Lee, J.H., Fitzgerald, J.B., Dimicco, M.A., and Grodzinsky, A.J. 2005. Mechanical injury of cartilage explants causes specific time-dependent changes in chondrocyte gene expression. Arthritis Rheum 52: 2386±2395. Campbell, C.J. 1969. The healing of cartilage defects. Clin Orthop Related Res 64: 45± 63. Redfern, P. 1851 reprinted in 1969. On the healing of wounds in articular cartilage. Clin Orthop 64: 4±6. Shands, A.R. 1931. Regeneration of hyaline cartilage in joints. Arch Surg 22: 137. Meachim, G. 1963. The effect of scarification on articular cartalige in the rabbit. J Bone Joint Surgery 45B: 150±161. Bennett, G.A., and Bauer, W. 1935. Further studies concerning the repair of articular cartilage in dog joints. Bone Joint Surg 17: 141. Bennett, G.A., Bauer, W., and Maddock, S.J. 1932. A study of the repair of articular cartilage and reactions of normal joints of adult dogs to surgically created defects of articular cartilage, `joint mice', and patellar displacement. Am J Pathol 8: 499. Calandruccio, R.A., and Gilmer, W.S. 1962. Proliferation, regeneration, and repair of articular cartilage of immature animals. J Bone Joint Surg 44A: 431. Mankin, H.J. 1962. Localisation of tritiated thymidine in articular cartilage of rabbits. II: Repair in immature cartilage. J Bone Joint Surg 44A: 688. Shapiro, F., Koide, S., and Glimcher, M.J. 1993. Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. J Bone Joint Surg Am 75: 532±553. Chapman, K., Takahashi, A., Meulenbelt, I., Watson, C., Rodriguez-Lopez, J., Egli, R., Tsezou, A., Malizos, K.N., Kloppenburg, M., Shi, D., et al. 2008. A metaanalysis of European and Asian cohorts reveals a global role of a functional SNP in the 50 UTR of GDF5 with osteoarthritis susceptibility. Hum Mol Genet May 15; 17, 10: 1497±1504. Loughlin, J., Dowling, B., Chapman, K., Marcelline, L., Mustafa, Z., Southam, L., Ferreira, A., Ciesielski, C., Carson, D.A., and Corr, M. 2004. Functional variants within the secreted frizzled-related protein 3 gene are associated with hip osteoarthritis in females. Proc Natl Acad Sci USA 101: 9757±9762. Loughlin, J. 2006. Osteoarthritis linkage scan: more loci for the geneticists to investigate. Ann Rheum Dis 65: 1265±1266. Miyamoto, Y., Mabuchi, A., Shi, D., Kubo, T., Takatori, Y., Saito, S., Fujioka, M., Sudo, A., Uchida, A., Yamamoto, S., et al. 2007. A functional polymorphism in the 50 UTR of GDF5 is associated with susceptibility to osteoarthritis. Nat Genet 39: 529±533. Southam, L., Rodriguez-Lopez, J., Wilkins, J.M., Pombo-Suarez, M., Snelling, S., Gomez-Reino, J.J., Chapman, K., Gonzalez, A., and Loughlin, J. 2007. An SNP in the 50 -UTR of GDF5 is associated with osteoarthritis susceptibility in Europeans and with in vivo differences in allelic expression in articular cartilage. Hum Mol Genet 16: 2226±2232.

Understanding tissue response to cartilage injury

153

62 Valdes, A.M., Van Oene, M., Hart, D.J., Surdulescu, G.L., Loughlin, J., Doherty, M., and Spector, T.D. 2006. Reproducible genetic associations between candidate genes and clinical knee osteoarthritis in men and women. Arthritis Rheum 54: 533±539. 63 Wei, X., Gao, J., and Messner, K. 1997. Maturation-dependent repair of untreated osteochondral defects in the rabbit knee joint. J Biomed Mater Res 34: 63±72. 64 Vasara, A.I., Hyttinen, M.M., Lammi, M.J., Lammi, P.E., Langsjo, T.K., Lindahl, A., Peterson, L., Kellomaki, M., Konttinen, Y.T., Helminen, H.J., et al. 2004. Subchondral bone reaction associated with chondral defect and attempted cartilage repair in goats. Calcif Tissue Int 74: 107±114. 65 Donohue, J.M., Buss, D., Oegema, T.R., Jr, and Thompson, R.C., Jr 1983. The effects of indirect blunt trauma on adult canine articular cartilage. J Bone Joint Surg Am 65: 948±957. 66 Thompson, R.C., Jr., Oegema, T.R., Jr., Lewis, J.L., and Wallace, L. 1991. Osteoarthritic changes after acute transarticular load. An animal model. J Bone Joint Surg Am 73: 990±1001. 67 Haut, R.C., Ide, T.M., and De Camp, C.E. 1995. Mechanical responses of the rabbit patello-femoral joint to blunt impact. J Biomech Eng 117: 402±408. 68 Ewers, B.J., Weaver, B.T., Sevensma, E.T., and Haut, R.C. 2002. Chronic changes in rabbit retro-patellar cartilage and subchondral bone after blunt impact loading of the patellofemoral joint. J Orthop Res 20: 545±550. 69 Rudolphi, K., Gerwin, N., Verzijl, N., van der Kraan, P., and van den Berg, W. 2003. Pralnacasan, an inhibitor of interleukin-1beta converting enzyme, reduces joint damage in two murine models of osteoarthritis. Osteoarthritis Cartilage 11: 738± 746. 70 Moskowitz, R.W., Davis, W., Sammarco, J., Martens, M., Baker, J., Mayor, M., Burstein, A.H., and Frankel, V.H. 1973. Experimentally induced degenerative joint lesions following partial meniscectomy in the rabbit. Arthritis Rheum 16: 397±405. 71 Telhag, H., and Lindberg, L. 1972. A method for inducing osteoarthritic changes in rabbits' knees. Clin Orthop Relat Res 86: 214±223. 72 Pond, M.J., and Nuki, G. 1973. Experimentally-induced osteoarthritis in the dog. Ann Rheum Dis 32: 387±388. 73 Layton, M.W., Goldstein, S.A., Goulet, R.W., Feldkamp, L.A., Kubinski, D.J., and Bole, G.G. 1988. Examination of subchondral bone architecture in experimental osteoarthritis by microscopic computed axial tomography. Arthritis Rheum 31: 1400±1405. 74 Clements, K.M., Price, J.S., Chambers, M.G., Visco, D.M., Poole, A.R., and Mason, R.M. 2003. Gene deletion of either interleukin-1beta, interleukin-1beta-converting enzyme, inducible nitric oxide synthase, or stromelysin 1 accelerates the development of knee osteoarthritis in mice after surgical transection of the medial collateral ligament and partial medial meniscectomy. Arthritis Rheum. 48: 3452± 3463. 75 Glasson, S.S., Askew, R., Sheppard, B., Carito, B., Blanchet, T., Ma, H.L., Flannery, C.R., Peluso, D., Kanki, K., Yang, Z., et al. 2005. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature. 434: 644± 648. 76 Glasson, S.S. 2007. In vivo osteoarthritis target validation utilising geneticallymodified mice. Curr Drug Targets 8: 367±376. 77 Gruber, J., Vincent, T.L., Hermansson, M., Bolton, M., Wait, R., and Saklatvala, J. 2004. Induction of interleukin-1 in articular cartilage by explantation and cutting. Arthritis Rheum. 50: 2539±2546.

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Vincent, T., Hermansson, M., Bolton, M., Wait, R., and Saklatvala, J. 2002. Basic FGF mediates an immediate response of articular cartilage to mechanical injury. Proc Natl Acad Sci USA 99: 8259±8264. 79 Dell'accio, F., De Bari, C., Eltawil, N.M., Vanhummelen, P., and Pitzalis, C. 2008. Identification of the molecular response of articular cartilage to injury, by microarray screening: Wnt-16 expression and signaling after injury and in osteoarthritis. Arthritis Rheum 58: 1410±1421. 80 Aspden, R.M., Jeffrey, J.E., and Burgin, L.V. 2002. Impact loading of articular cartilage. Osteoarthritis Cartilage 10: 588±589; author reply 590. 81 Jeffrey, J.E., Gregory, D.W., and Aspden, R.M. 1995. Matrix damage and chondrocyte viability following a single impact load on articular cartilage. Arch Biochem Biophys 322: 87±96. 82 Jeffrey, J.E., Thomson, L.A., and Aspden, R.M. 1997. Matrix loss and synthesis following a single impact load on articular cartilage in vitro. Biochim Biophys Acta 1334: 223±232. 83 Patwari, P., Cheng, D.M., Cole, A.A., Kuettner, K.E., and Grodzinsky, A.J. 2007. Analysis of the relationship between peak stress and proteoglycan loss following injurious compression of human post-mortem knee and ankle cartilage. Biomech Model Mechanobiol 6: 83±89. 84 DiMicco, M.A., Patwari, P., Siparsky, P.N., Kumar, S., Pratta, M.A., Lark, M.W., Kim, Y.J., and Grodzinsky, A.J. 2004. Mechanisms and kinetics of glycosaminoglycan release following in vitro cartilage injury. Arthritis Rheum 50: 840±848. 85 Tew, S.R., Kwan, A.P., Hann, A., Thomson, B.M., and Archer, C.W. 2000. The reactions of articular cartilage to experimental wounding: role of apoptosis. Arthritis Rheum 43: 215±225. 86 Tew, S., Redman, S., Kwan, A., Walker, E., Khan, I., Dowthwaite, G., Thomson, B., and Archer, C.W. 2001. Differences in repair responses between immature and mature cartilage. Clin Orthop Relat Res 391: 142±152. 87 Redman, S.N., Dowthwaite, G.P., Thomson, B.M., and Archer, C.W. 2004. The cellular responses of articular cartilage to sharp and blunt trauma. Osteoarthritis Cartilage 12: 106±116. 88 Loening, A.M., James, I.E., Levenston, M.E., Badger, A.M., Frank, E.H., Kurz, B., Nuttall, M.E., Hung, H.H., Blake, S.M., Grodzinsky, A.J., et al. 2000. Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis. Arch Biochem Biophys 381: 205±212. 89 Quinn, T.M., Schmid, P., Hunziker, E.B., and Grodzinsky, A.J. 2002. Proteoglycan deposition around chondrocytes in agarose culture: construction of a physical and biological interface for mechanotransduction in cartilage. Biorheology 39: 27±37. 90 Stevens, A.L., Wishnok, J.S., Chai, D.H., Grodzinsky, A.J., and Tannenbaum, S.R. 2008. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis-liquid chromatography tandem mass spectrometry analysis of bovine cartilage tissue response to mechanical compression injury and the inflammatory cytokines tumor necrosis factor alpha and interleukin-1beta. Arthritis Rheum 58: 489±500.

6

Understanding osteoarthritis and other cartilage diseases T . A I G N E R , Medical Center Coburg, Germany, N . S C H M I T Z , University of Leipzig, Germany È D E R , University of Erlangen-Nurnberg, Germany and S . S O

Abstract: The most relevant pathology of the articular cartilage is osteoarthritis (OA), i.e. degenerative joint cartilage destruction. Other important conditions are osteochondrosis dissecans, a focal subchondral bone destructive process leading to focal cartilage defects, as well as crystallopathies, which affect the joints in particular. Inflammatory conditions originating mostly from the synovial membrane as well as developmental malformations (chondrodysplasias) are not discussed. Besides the considerations of pathogenetic events and the description of pathological changes taking place during the disease process, a major emphasis will be put on the outline and discussion of grading systems of cartilage degeneration and repair. Key words: cartilage, osteoarthritis, osteochondrosis dissecans, grading, staging, typing, crystallopathies.

6.1

Introduction

The major relevant pathology of articular cartilage is degenerative joint disease, `osteoarthritis' (OA), which deals with all various forms of joint cartilage destruction. OA is in most cases primary, i.e. there are no clear reasons for its development and progression. Less frequently, OA is secondary due to inflammatory and endocrine conditions. However, (inflammatory) rheumatoid diseases are not within the scope of this chapter: they are primarily involving the synovial membrane (synovitis) and potentially bone and only secondarily involve the articular cartilage, largely similar to osteoarthritic cartilage degeneration. One important initiating event ± which might also be considered to lead to secondary OA ± is repetitive or focal trauma. Inflammatory events directly related to articular cartilage do not exist. Thus, for example, purulent arthritis, a rather rare condition, which can, however, have dramatic destructive consequences to the joints, is thought to result from septic granulocytic inflammation within the joint space. An important condition ± in particular as a potential target for repair ± is osteochondrosis dissecans, a focal subchondral bone destructive process leading to focal cartilage defects. Crystallopathies represent interesting conditions

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special to joints and the articular cartilage within the body. In contrast to most other tissues, there are no known neoplasias originating from articular cartilage. Besides considerations of pathogenetic events and the description of pathological changes taking place during the disease process, a major emphasis will be put on the outline and discussion of grading systems of cartilage degeneration and repair.

6.2

The normal joint

Joints are highly specialized organs (Fig. 6.1) that allow repetitive pain-free and largely frictionless movements. These properties are provided by the articular cartilage and its extracellular matrix, which under physiological conditions is capable of sustaining high cyclic loading. Articular cartilage covers the joint surfaces and is mainly responsible for the unique biomechanical properties of the joints. Joints are, however, complex composites of different types of connective tissue including (subchondral) bone, cartilage surfaces, ligaments and the joint capsule. Thus, the capsule, together with the ligaments, is extremely important for the mechanical stability of the joint as a whole. If malaligned, the cartilage is loaded abnormally and degenerates rather dramatically, as seen in misalignment syndromes of the joints (e.g. genu valgum et varum). All the different joint tissues together provide their own functional capacities in order to allow the correct functioning of the joint. The articular cartilage is a highly specialized and uniquely designed biomaterial that forms the smooth, gliding surface of the diarthrodial joints. It

6.1 Schematic representation of the main structures of a healthy (left side) and degenerated (right side) joint in osteoarthritis: in particular the articular cartilage is lost or severely thinned, the (subchondral) bone is sclerotic, the joint capsule thickened and the synovial membrane activated.

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is largely an avascular, aneural and alymphatic matrix, which is synthesized by sparsely distributed resident cells ± the chondrocytes. The major constituents of the articular cartilage matrix are collagens, proteoglycans as well as the very heterogeneous group of non-collagenous non-proteoglycaneous proteins.1 In fact, most of the physiological (wet) weight of articular cartilage comes from water bound to the proteoglycans (namely aggrecan). The synovial capsule and in particular the synovial membrane (i.e. the synovial lining cell layer) represent important portions of the joint as an organ. As already mentioned, it is the capsule together with the ligaments which provide the mechanical stability of the joint and determine the flexibility of the possible movement. Overflexibility (e.g. after traumatic ligament rupture) clearly increases the risk for joint degeneration with time.2 The synovial membrane with its metabolically highly active surface cells (synoviocytes) plays a crucial role in nourishing the chondrocytes as well as removing metabolites and (matrix) degradation products form the synovial space. The synoviocytes maintain the basic metabolic homeostasis of the joints. Furthermore, the synoviocytes produce large amounts of hyaluronic acid, which provides the joint surfaces with its gliding capacity.

6.3

Major cartilage pathology and pathobiology

Most prominent conditions of articular joint cartilage are OA, whether primary or secondary to trauma, inflammation, etc., osteochondrosis dissecans and crystal deposition disease. Exceptionally, chondrodysplasia in particular of the hips occurs (for review, see Daneshpouy et al., 20023), which at least in part leads to early OA development.4 Many of them are, however, lethal and/or lead to severe malformation of the body.

6.3.1

The pathology and pathobiology of OA

Osteoarthritis, the degeneration of the joints, is the most common disabling condition in the Western world. Clinically, degeneration affects mostly the large weight-bearing joints of the legs (i.e. hips and knees), but can in principle affect any joint of the body including, notably, the finger joints. Osteoarthritis is not a single disease entity, but represents a disease group with rather different underlying pathophysiological mechanisms. In this respect, primary osteoarthritis has to be distinguished from secondary forms of the disease, which are due to traumatic events, endocrine or metabolic disorders, etc. Clinically, pain and loss of joint functioning are the major issues leading to a significantly reduced quality of life for patients suffering from the disease. Primary OA of the large weight-bearing joints is generally the result of an imbalance between applied mechanical stress and the physicochemical ability of the articular cartilage to resist this stress. In the end, osteoarthritis results from

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the destruction and failure of the extracellular matrix, the functional element of articular cartilage. However, osteoarthritis is a disease of the joint as an organ system (Fig. 6.1), not only of the articular cartilage. This includes all connective tissues within and around the joints as well as the respective musculature, the nervous system and even portions of the body more remote from the joint site, such as the central nervous system. The latter is particularly important for the symptomatic aspects of the disease and in fact the innervation and the processing are important to pain, the major symptom of the disease process. In terms of joint tissues, clearly the synovial capsule including the synovial membrane plays a very important role in the scenario of joint functioning and tissue maintenance. Overall, recognition that the joint does not only consist of articular cartilage, but also a number of adjacent tissues is not only important for the understanding of joint physiology, but also joint pathology. All these tissues are more or less affected by degenerative changes or their consequences (Fig. 6.1). In conventional radiology, in particular changes to the bone and (indirectly via the loss of joint space) to articular cartilage are visible (Fig. 6.2a). This, however, changed dramatically with the introduction of magnetic resonance imaging (MRI) into joint imaging.5,6

6.2 (a) Radiographic appearance of hip osteoarthritis displaying distorted joint architecture, loss of joint space as well as osteophyte formation in the joint margins. (b) Arthroscopic picture of a cartilage defect of the femoral condyle within the knee joint (courtesy Dr Eger, Rummelsberg). (c±d) Macroscopic appearance of femoral condyles of the knee: normal (c) and severely damaged (d).

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The cartilage matrix Macroscopically, hyaline articular cartilage is a rather unruffled white to yellowish overlay coating the joint surface (Fig. 6.2c). The synovial fluid makes it appear sloppy and provides its gliding properties. Microscopically, hyaline cartilage consists of evenly stained (`hyaline') proteoglycan-rich ground substance with the cartilage cells (`chondrocytes') lying sparsely in between. The cells represent less than 5% of the total volume of articular cartilage, but are of obvious importance for the maintenance of the tissue. Chondrocytes are surrounded in most parts by a specialized pericellular matrix forming a biomechanical and biochemical interface in between the rigid interterritorial matrix and the cells. The mechanical properties of articular cartilage largely depend on the biochemical composition of the extensive interterritorial (extracellular) cartilage matrix. Macroscopically, osteoarthritic cartilage is often yellowish or brownish and is typically soft. The surface shows roughening in the early stages and overt fibrillation and matrix loss in the later stages until the eburnated subchondral bone plate is visible (Fig. 6.2b,d). These changes are visualized in more detail on the histological level (Fig. 6.3b,d,e; a is normal articular cartilage for comparison). Besides the total destruction of matrix areas, the degradation of matrix molecules also plays an important role preceding and driving the final loss of the respective matrix areas (Fig. 6.3d,e: loss of toluidine blue staining reflecting the loss of proteoglycans in damaged cartilage areas). Apart from the degradation of molecular components, destabilization of supramolecular structures also takes place. For example, destabilization of the collagen network results in microscopically and finally macroscopically visible matrix destruction. Both mechanical wear and enzymatic degradation appear to play a pivotal role during the disease process. Together, these result in the destruction of cartilage matrix on the molecular (e.g. proteoglycan depletion) and the macromolecular (e.g. network loosening), the microscopic (e.g. fissuring) and the macroscopic (e.g. cartilage tear) levels. The destruction of articular cartilage and the loss of its biomechanical function are largely due to the destruction and loss of the interterritorial cartilage matrix, which result from an imbalance between degradation and de novo synthesis of matrix components on the molecular and supramolecular level in spite of the compensatory attempts of the chondrocytes. The cartilage cells (chondrocytes) Despite the importance of the extracellular matrix for the functioning of articular cartilage, the cells are not `functionless' in connective tissues, as they are the only viable players within the tissue. Thus, they are centrally responsible for the balanced turnover of the extracellular matrix, which is necessary for maintenance of the integrity of the extracellular cartilage. During the

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6.3 Conventional histology shows fibrillation and matrix loss in osteoarthritic cartilage (b) compared to the normal (a). In severely damaged areas nearly all articular cartilage is destructed (e). Also a moderate (d) to severe (e) loss of proteoglycans is found as visualized by toluidine blue staining (d,e). Besides changes in articular cartilage, changes in the subchondral bone are also prominent, namely thickening of the bone trabeculae (f, osteoarthritic; c, normal).

osteoarthritic disease process, the cellular reaction patterns are altered. At first sight these are rather pleomorphic, but can be basically summarized in three categories. First, the chondrocytes can undergo cell death or they can proliferate to compensate for cell loss or to increase their synthetic activity. Secondly, chondrocytes activate or deactivate their synthetic-anabolic activity by increasing or decreasing anabolic gene expression. Lastly, chondrocytes undergo phenotypic modulation implicating an overall severely altered gene expression profile of the cells in the diseased tissue. In addition, osteoarthritic chondrocytes are heterogeneous and nearly all observed cellular changes are region- and zonespecific, and also dependent on the degradation stage. One straightforward explanation for osteoarthritic cartilage degeneration would be a mere loss of viable cells at the beginning and during the disease process (for review, see Aigner and co-workers7,8). However, this has only limited impact on the pathology of early osteoarthritis or ageing of human articular

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cartilage.9 The only zone in which a large number of empty lacunae were found was the calcified cartilage layer. The continuous progression of cartilage calcification in osteoarthritic cartilage might explain, at least partly, the increasing number of empty lacunae reported, particularly in high grade lesions.10,11 One striking phenomenon in many OA cartilages is the focal proliferation of chondrocytes, forming so-called chondrocyte `clusters': this clearly reflects the increased proliferative activity within the diseased tissue and might represent an attempt of cells to compensate for an increased need of cellular activity or even cell death (similar to other tissues). Overall, however this increased proliferative activity might not be of much help to maintain tissue homeostasis as these cell clusters are often anabolically inactive12 and in fact, might represent rather further empty holes destabilizing the cartilage matrix. Also, increased proliferation might lead to (focal) shortening of the telomeres of the cells which might itself be problematic for cell integrity.13 The synovial membrane, the joint capsule and inflammation OA research traditionally concentrates on the understanding of events within the degenerated articular cartilage as the tissue in which the initiating events presumably take place. Synovial changes are generally interpreted as largely secondary to the degeneration of the articular cartilage14±16 and not pathogenetically involved to a relevant extent in the disease process. However, the synovial capsule and, in particular, the synovial lining cells represent an important portion of the joint as an organ. Also, thickening of the collagen plate within the joint capsule, a typical feature in many osteoarthritic patients at least in the late stages reduces significantly the movement properties of their joints. Thus, capsular fibrosis is centrally responsible for joint stiffening, which is, after pain, the second biggest symptomatic issue of osteoarthritic joint degeneration. Overall, four types of synovial reaction pattern can be distinguished:17 presumably, in most cases the earliest event is hyperplastic synoviopathy, which is mainly characterized by synovial hyperplasia (i.e. villus formation) as well as proliferation18,19 and activation of the synovial surface cells (i.e. synovial lining layer, synoviocytes). At the end, detritus-rich synovitis is often observed, which is characterized by a lot of cartilage and bone debris as well as fibrinous exudate and some granulating inflammation. Few granulocytes might be observed as well as a giant cell rich foreign body reaction. This is at this stage also mostly combined with fibrous thickening of the joint capsule, a feature which in some cases also dominates in earlier stages of the disease and is then called fibrous OA synoviopathy (this might include hypertrophic changes as well). A forth pattern of synovial reaction in OA is inflammatory OA synoviopathy, which is characterized by significant lymphoplasmacellular infiltrates resembling that one found in rheumatoid conditions (but less pronounced). Spotty lymphocyte aggregates might also occur in other variants. Granulocytes are not part of the

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spectrum of OA synoviopathy (except minor infiltrates already mentioned in detritus-rich synoviopathy). The subchondral bone Another important tissue often neglected in osteoarthritis research is the subchondral bone,20 although it is unclear yet whether pathological changes within the subchondral bone tissue (e.g. sclerosis) can precede changes in the articular cartilage (e.g. bone mass as risk factor of osteoarthritis21) or subchondral bone changes are secondary adaptation processes following changes in the biomechanical properties of the cartilage.22 Significant changes in terms of increased thickness of the subchondral bone plate as well as underlying trabecules are already apparent in the early stages of the disease (Fig. 6.3c,f). Thus, active new bone formation is found at multiple foci in early- to mid-stage patients.23 In later stages, severe bone remodelling processes take place, in particular in areas of advanced destruction of the overlying articular cartilage. Apart from extensive bone sclerosis, significant aseptic bone necrosis is a common feature of advanced osteoarthritic joint degeneration. In areas of total cartilage destruction (i.e. eburnated bone plate) synovial fluid gets access to the bone marrow and induces fibrocytic and even chondrometaplastic changes of mesenchymal precursor cells. This leads to the characteristic `cartilage-nodules' or `tufts', which are frequently found in these areas in late stage disease. At least in moderate to advanced lesions, the changes in the subchondral bone represent one tissue responsible for the osteoarthritic joint pain.24,25

6.3.2

Post-traumatic cartilage lesions ± intra-articular bleeding

Trauma, in particular microtrauma, is thought to be one core causing event for the development of osteoarthritis. Obviously, people undertaking sporting activity and also heavy occupational loading are at high risk of developing general joint cartilage degeneration. Major traumatic events, such as intraarticular fracturing and share traumata leading to cartilage flakes, are major issues both in terms of symptoms as well as long-term outcome. They might require direct surgical or arthroscopical intervention. Also post-traumatic bleeding into the joint cavity might negatively influence cartilage and chondrocyte26,27 integrity, though this clearly depends on the extent and its duration. Such events are particularly a problem if there exists an increased bleeding susceptibility such as in haemophilic disease. In this condition, significant joint damage including the articular cartilage occurs, though part of it is thought to be mediated through synovial changes rather than a direct effect on the articular cartilage and the cells.28 Nevertheless, blood has been shown to damage chondrocytes in vitro29 and in vivo.30

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163

Osteochondrosis dissecans

In general, osteochondrosis dissecans is a condition in which ± as indicated by the name ± an osteochondral flake is torn off the joint surface. This, in principle, can occur in any joint, but is most frequently encountered in the medial femoral condyle. Pathogenetically, the necrosis of the subchondral bone compartment is the core event, which then leads to the weakening of the subchondral bone plate strength with subsequent dissociation from the remaining bone. The overlying articular cartilage might be rather vital and largely intact or may show alternatively moderate to severe degenerative alterations. As the definition, the subchondral bone part of an osteochondral flake, however, is fully necrotic. As a consequence of the destruction of part of the articular surface, often a more extended or generalized joint destruction occurs and thus, osteochondrosis dissecans is a classical cause of secondary osteoarthritis. The underlying condition of osteochondrosis dissecans is so far largely unknown: one general speculation is an articular trauma leading to a vascular occlusion and thus subchondral bone necrosis. However, there might be many other factors involved, including a familial predisposition as described at least for some cases.31 Whatever osteochondrosis dissecans finally is, it is a condition affecting primarily the bone and only on a second level the articular cartilage and the joint as such. This, however, occurs in a dramatic way in most cases.

6.3.4

Crystal deposition disease

There are two major forms of crystal deposition disease in the articulating joints: deposition of urate crystals (i.e. gout) and the deposition of pyrophosphate crystals (pseudogout, chondrocalcinosis). Both can cause significant symptomatic disease, but gout is clearly usually symptomatic and pseudogout in most cases a clinically silent process mostly associated with (osteoarthritic) cartilage degeneration. Gout Gout, a severely symptomatic disease mainly affecting the joints, has been known for a long time. It mostly affects older men and is characterized mainly by very painful synovitis or inflammation in the periarticular soft tissues, though any organ except the brain might be involved in gouty `tophus' formation. Gout occurs in repetitive phases (gouty `attacks') and often first affects the feet (often the first metatarsophalangeal joint). Typically, the urate levels are increased in the serum. The precipitation of urate crystals in the soft tissue is often following alcoholic excess or calory-rich food intake. However, the exact reason of the urate deposition, e.g. preferentially in certain joints and less often in others is not yet known. Histologically, gouty tophi are characterized by needle-shaped crystals showing a typical negative birefringence in polarized light microscopy

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6.4 Urate crystal deposition (gout) is characterized by more or less extensive deposition of strongly birefringent crystalline material within an amorphous matrix (a) hematoxyline±eosine, (b) polarized light microscopy (wonderful crystalline structure from different area)). (c, d) calcium pyrophosphate crystallopathy (chondrocalcinosis) shows the deposition of hardly birefringent short needle-like crystalline material (c, hematoxylin±eosine; d, detail photographed with polarized light microscopy).

(Fig. 6.4b). Sodium urate crystals are embedded into an amorphous eosinophilic mass. The area of crystal deposition is surrounded by a more or less dense mononuclear (histiocytic) infiltrate with multi-nuclear giant cells of the foreign body type scattered in between. The strongly birefringent crystals also make this condition easily distinguishable from the small, hardly birefringent crystals present in chondrocalcinosis. Of note, during conventional histological processing most urate crystals are dissolved in ethanol and only the weakly eosinophilic small to medium sized areas surrounded by the histiocytic and giant cells remain, a feature diagnostic for this condition (but not to be confused with small foreign body granulomas after intra-articular injections). Though gout is a condition mostly affecting the peri-articular soft tissues, urate crystal deposits can be occasionally found also in articular cartilage. Epidemiological evidence suggests also that osteoarthritic degenerative joint disease is one potential risk factor for gouty arthritis.32 Pseudogout In contrast to gout, pseudogout (chondrocalcinosis, calcium pyrophosphate dehydrate crystal deposition disease) primarily affects the articular cartilage, in which radiologically calcium deposition can be detected in plain X-rays. Besides the articular cartilage also the synovial membrane/capsule, ligaments, tendons,

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menisci and within the hip joint the limbi are affected. Usually, the calcium pyrophosphate crystals are deposited without significant (inflammatory) reaction by the surrounding tissue, but in cases of symptomatic pseudogout arthritis a mononuclear infiltrate with accompanying giant cell reaction similar to gout can be observed around the deposits. The incidence of chondrocalcinosis in the elderly is high, reaching 25% in 80-year-olds (for review, see Wise33). In polarized light microscopy the calcium pyrophosphate crystals are very small, hardly birefringent needle-like structures that are difficult to see (Fig. 6.4d). In contrast to the urate crystals, calcium pyrophosphate crystals are less water soluble and, thus, remain mostly within the tissue during histological processing. Overall, the cause and the relevance of chondrocalcinosis remain unclear: clearly, it is well correlated with the degeneration of the articular cartilage and it is probably related to a metabolic disbalance within the cells and the tissue, most likely the articular cartilage and the chondrocytes.

6.4

In vivo cartilage repair

At the margins of joints, in particular in osteoarthritic joint disease, frequently (osteo)cartilaginous outgrowths appear ((chondro-)osteophytes). They are best considered as a process of secondary chondroneogenesis in the adult.34 Osteophytes derive from mesenchymal precursor cells within periosteal or synovial tissue and often merge with or overgrow the original articular cartilage.35,36 Thus, in this process, mesenchymal precursor cells differentiate into chondrocytes. A similar, but less structured process is observed in the areas of the eburnated bone, in which the articular cartilage is completely torn off. Here, mesenchymal multipotential stem cells of the bone marrow undergo also chondrogenic differentiation: metaplastic cartilage in forms of nodules or `tufts' is found either within the bone marrow or at the naked bone surface.37 Osteophytes could be considered as endogenous repair attempts in degenerating joints and might be a physiological response to mechanical overloading by increasing the articulating joint surface. Even if their supportive effect within the joints is doubtful, their chondrogenic potential is of interest, especially having exogenous (therapeutic) repair strategies in mind. Central for the basic understanding of osteophytic tissue is the analysis of the developmental steps during osteophyte formation. Thus, although it is clear that osteophyte development is a continuous process and many osteophytes show different stages in various portions at the same time, one can define basic steps based on the cellular phenotype and the matrix composition of the predominating tissue38 (Table 6.1 and Fig. 6.5). Initially, mesenchymal precursor cells derived either from periosteum or synovium initiate chondrogenic differentiation (stages I and II). This results in fibrocartilage composed of both fibrous and cartilaginous matrix components. In early osteophytes, endochondral ossification is initiated. The deepest cell layer becomes hypertrophic and

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Table 6.1 Staging of osteophyte development according to Gelse et al.38 Stage 0 (normal)

Normal periosteum

Stage I

· Slight thickening of the periosteum · Incipient formation of fibrocartilage (some round cells, some metachromatic tissue staining of the extracellular matrix) · No/slight active bone formation Molecular markers: · Focal collagen type II expression · No collagen type X

Stage II

· Pronounced thickening of the periosteal layers · Well-established formation of fibrocartilage (many round cells, strong metachromatic tissue staining of the extracellular matrix) · Some/moderate bone formation Molecular markers: · Distinct collagen type II expression · No collagen type X

Stage III

· Pronounced thickening of the periosteal layers · Well-established formation of fibrocartilage (many round cells, strong metachromatic tissue staining of the extracellular matrix, formation of lacunae) · Strong active bone formation Molecular markers: · Distinct collagen type II expression · Collagen type X expression in basal areas

Stage IV

· Significant thickening of the periosteal layer · Apparent formation of fibrocartilage with partial hyalinization of the extracellular matrix (chondrocyte-like cells in lacunae, strong metachromatic tissue staining of the extracellular matrix) · Some active bone formation Molecular markers: · Ubiquitous presence of collagen type II · Collagen type X in basal areas · Collagen type VI within the extracellular matrix accentuated pericellularly

resembles the lowest cells found in the growth plate (stage III).39 Mature osteophytes are characterized by the predominance of a hyaline cartilage-like extracellular matrix (stage IV). At a first glance, mature osteophytes can, macroscopically and histologically, easily be mistaken for original articular cartilage. Indeed, this misconception reflects to some degree the fact that

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6.5 Osteophyte development can be subdivided into five stages with different structural organization although many osteophytes show different stages simultaneously in different areas. Stage I (early chondrophytes) shows first chondrocytic differentiation of previously undifferentiated mesenchymal precursor cells (b,g,k). Stage II (chondrophytes) shows extensive areas of newly formed cartilage, but no (endochondral) bone formation is observed (c,h,k). Stage III (early osteophytes) shows an arrangement as the fetal growth plate cartilage (d,i,k) whereas stage IV (mature osteophytes) shows a structure most resembling hyaline articular cartilage physiologically covering the joint surfaces (e,j,k). Normal periosteum is shown in a and f. (a±e, hematoxylin eosin staining; f±j, toluidine blue staining).

chondrocytic cells in osteophytes are able to construct an extracellular matrix containing all the typical components of hyaline articular cartilage such as collagen types II, IX and XI as well as aggrecan.34,38 The zonal distribution also resembles that found in adult articular cartilage with collagen type VI concentrated in the pericellular matrix.40 Although hyaline zones in osteophytes resemble articular cartilage in terms of structural composition, there are, never-

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theless, certain differences such as a more random cellular arrangement, the lack of a distinct tidemark, and a missing linear subchondral bone plate. Furthermore, the proper alignment of the described matrix components, which is obligatory for the cartilage to be able to resist high mechanical forces, has not yet been investigated at the ultrastructural level.

6.5

Grading/scoring systems for cartilage degeneration

Overall, the classification of osteoarthritic cartilage degeneration is rather complex, as all patients present with at least to some extent different histories, symptoms and morphological changes. Common to all of them is some sort of structural joint (cartilage) damage, pain and limitation in joint movement. Fortunately, for most instances an exact classification of the destructive process is of limited clinical and scientific use. Obviously, many other tissues than the articular cartilage are involved in this process, but traditionally, the cartilage has been used in order to score OA severity (at least as long as structural changes are evaluated: in general, the process of joint destruction can be always evaluated for the pathogenesis (`typing'), for its extent (`staging') and for the degree of the most extensive focal damage (`grading'). `Typing' is mostly related to `primary', i.e. idiopathic, and `secondary', i.e. `caused by . . .' OA. Primary OA is most common. Whereas the addition `primary' suggest it to be without any obvious cause, still minor pre-existing conditions also exist in this condition (i.e. `pre-conditions' or `risk factors'). The major causes leading to secondary osteoarthritic joint degeneration are listed in Table 6.2. `Grading' and `staging' have been much more under debate, also Table 6.2 Typing of joint destruction Primary

No causative reason known

Secondary

· Rheumatic disease · Overload causing excessive wear (work, sport, varus or valgus deformity) · Instability (e.g. meniscus lesions) · Trauma · Intra-articular infections · Articular gout · Psoriatic arthritis · Bone infarciation · Endocrine disorders (e.g. hyperparathyroidism) · Neuropathy (e.g. Charcot's joint) · Paget's disease · Haemophilia

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regarding the basic meaning of both words. This author suggests the use of `grading' for the evaluation of histological changes at one site of analysis, whereas `staging' should refer to the overall disease process (in analogy to `grading' and `staging' in tumour pathology). Both represent an attempt of scoring processes, which is suggested to be used as general term for such activities. The grading system most often used (partly in minor modifications) is that proposed by Mankin and coworkers in 19719 (Table 6.3; Fig. 6.6). Despite repetitive criticisms that the Mankin score shows a high interindividual variability,41 this might be related to the training status of the people doing the scoring. However, clearly some of the subcategories of the MankinÂs score do not belong to primary cartilage degeneration, but describe features observed in secondary cartilage formation (i.e. osteophyte formation: see Table 6.3) and should be excluded in future scoring attempts. A staging system largely used in Germany, but still in principle up to date is that of Otte42 (Table 6.4; Fig. 6.6). Whereas Mankin addresses the piece of cartilage under the microscope, Otte looks at the whole joint surface mostly macroscopically (but if needed the worst lesion can be evaluated histologically). At the site of the highest cartilage damage, grading according to Mankin and staging according to Otte are closely Table 6.3 Grading of osteoarthritis according to Mankin et al.9 Feature

Score

Histological feature

Cartilage structure

0 1 2 3 4 5

Normal Superficial fibrillation Pannus and superficial fibrillation* Fissures to the middle zone Fissures to the deep zone Fissures to the calcified zone

Chondrocytes

0 1 2 3

Normal Diffuse hypercellularity Cell clusters Hypocellularity

Safranin-O staining

0 1 2 3 4 5

Normal Slight reduction Moderate reduction Severe reduction No staining Total disorganization*

Tidemark

0 1

Intact Tidemark penetrated by vessels**

* Should be removed (relates to osteophyte formation). ** Might best be supplemented with: `or duplicated tidemark').

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6.6 The grading system according to Mankin et al.9 compared with the staging according to Otte.42

Table 6.4 Staging of joint destruction according to Otte42 Grade

Morphology

0 I II

Normal Superficial fibrillation, no cartilage loss Cartilage lesions (without full thickness defects) (deep fibrillation, fissures to middle zone and/or partial cartilage matrix loss) Cartilage lesions (without full thickness defects) (fissures to deep zone and partial cartilage matrix loss) Complete cartilage loss (at least focally)

III IV

correlated. Clearly, Otte is too rough for scientific purposes and a new staging system has recently proposed by Pritzker and colleagues43 (Table 6.5), but in clinical terms this provides in most instances a rough suitable classification of the condition without adding and requiring too much unneeded information. Doubtless, along with new scientific insights and with more extensive and specified medical options we will need more elaborated `grading' and `scoring' systems and this will be a major task in the near future.

6.6

Grading/scoring of cartilage repair

Many approaches (for review see Nesic et al.44) have been followed in order to promote external cartilage repair either by implanting autologous chondrocytes or chondrocyte precursor cells. Whatever therapeutic method is used, the

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Table 6.5 Grading of osteoarthritis according to Pritzker et al.43 Grade

Histological properties

0

Matrix: surface intact (normal architecture) Cells: intact, appropiate orientation

1

Matrix: superficial zone intact, oedema and/or superficial fibrillation (abrasion), focal superficial matrix condensation Cells: cell death, proliferation (cluster formation), hypertrophy

2

As above: Matrix: discontinuity at superficial zone (deep fibrillation)  loss of PG-staining in upper third of cartilage  focal perichondral increased PG-stain in middle zone  disorientation of chondron collums

3

As above: Matrix: vertical fissures into middle zone and branched fissures  loss of PG-staining into lower two-thirds of cartilage  new collagen formation Cells: cell death, regeneration, hypertrophy in cartilage domains adjacent to fissures

4

As above: Cartilage matrix loss with delamination of superficial zone Excavation with matrix loss from superficial to middle zone  formation of cysts in the middle layer

5

Complete matrix loss with denudation of the sclerotic subchondral bone or fibrocartilage  microfracture with repair limited to bone surface

6

Bone remodelling (more than osteophyte formation only) with microfracture, fibrocartilage and osseous repair above the previous surface

important issue, in terms of outcome measurement, is not only clinical symptom evaluation but also the question what tissue is formed in terms of composition and function. Both biochemical composition and biomechanical function are closely related as the newly formed tissue has to fulfil the biomechanical needs of a specific connective tissue, i.e. articular cartilage, which it has to substitute. Thus, although the final functional outcome remains the main criterion for the success of a procedure, the tissue type formed appears to be the major prerequisite for the final success. Basically, three types or levels of repair tissue (besides the complete absence of repair tissue at all) can be distinguished:45 fibrous tissue, fibrocartilage and hyaline-like repair cartilage of varying resemblance to original articular cartilage.46 All these types of tissue resemble the different stages of osteophyte development described above,38 making this in vivo phenomenon so interesting for cartilage repair biology.

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Fibrous repair tissue can be regarded as a failed repair attempt as it is unlikely to represent a successful long-term restoration of the joint function. Cells produce a matrix with a poor content of glycosaminoglycans and with abundance of type I collagen.47,48 Type II collagen is not present or represents only a very minor fraction. Since this repair tissue lacks the unique properties of articular cartilage, which are required for a successful participation in the articulating processes of joints, fibrous repair tissue fails when exposed to mechanical load.46 Fibrocartilage is the most common form of repair tissue achieved. In terms of structure and composition, fibrocartilage has an intermediate position in between fibrous and hyaline-like cartilaginous tissue. The content of glycosaminoglycans does not reach the abundance found in hyaline articular cartilage, but is increased compared with fibrous tissue. The matrix of fibrocartilage is composed of both type I collagen, typical for fibrous tissue, and type II collagen, typical for hyaline cartilage.46,49,50 Thus, although at first sight the joint surface and the macroscopic integrity of the cartilage appear to be largely restored, fibrocartilage does not possess the biomechanical properties of articular cartilage, which are needed for tolerating long-term constant mechanical loading and movement. Therefore, therapeutic attempts leading to fibrocartilagenous repair tissue are also prone to suffer from long-term deterioration with fibrillation, swelling, loss of cells and finally the loss of the repair tissue itself.47±49 Under certain conditions, a rather complete chondrogenic differentiation and remodelling process was reported that generated repair cartilage which shared great similarity with normal articular cartilage.50±52 Macroscopically, glossy surfaces of the restored defects suggested highly effective repair yielding the restoration of tissue resembling healthy articular cartilage. Histochemical analysis confirmed an abundance of water-binding glycosaminoglycans within the extracellular matrix of this repair tissue. The cells displayed a spherical shape typical for chondrocytes and were embedded in lacunar spaces. The extracellular matrix of these repair tissues was shown to contain types II, IX and XI collagens. The absence of type I collagen indicated a rather complete transformation or differentiation of the implanted cells into a functional chondrocytic phenotype forming hyaline cartilage-like repair tissue.47,51,53,54 Despite the similarities of this repair tissue to normal articular cartilage, which are apparent at first glance, there might still be subtle differences: in normal articular cartilage, the structural organization of the collagen network has a typical zonal pattern. In repair cartilage the fibres appear to be more randomly distributed and the cellular density and cellular arrangement often differ significantly from those of normal articular cartilage.46,55±57 Additionally, repair cartilage often lacks a clear tidemark which separates the upper portions of articular cartilage from the underlying calcified cartilage and bone.52,54 Consensus criteria for the evaluation of tissue repair outcome (on the histopathological level) were issued by the ICRS (International Cartilage Repair

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Table 6.6 The ICRS visual histological assessment score for assessing repair cartilage quality.45 The observer should evaluate all criteria separately without adding them up at the end I. Surface smooth/continuous discontinuities/irregularities

3 0

II. Matrix hyaline mixture: hyaline/fibrocartilage fibrocartilage fibrous tissue

3 2 1 0

III. Cell distribution columnar mixed/columnar-clusters clusters individual cells/disorganized

3 2 1 0

IV. Cell population viability predominantly viable partially viable <10% viable

3 1 0

V. Subchondral bone normal increased remodelling bone necrosis/granulation tissue detached/fracture/callus at base

3 2 1 0

VI. Cartilage mineralization (calcified cartilage) normal abnormal/inappropriate location

3 0

Society) in 2003,45 referring to basic histopathological criteria of cartilage integrity and resemblance to hyaline articular cartilage tissue (Table 6.6). Other sources available are the O'Driscoll score58 and its modifications.

6.7

Sources of further information and advice

Bullough and Vigorita's Orthopaedic Pathology ± Peter Bullough: The pathology of OA joint tissues, together with many other conditions, is well described in this excellent textbook on orthopaedic pathology. The concepts are clear and comprehensive and the documentations are excellent, both in terms of schematic representations and documentation of macroscopic and histopathological features. Peter Bullough shares his rich, long experience in looking at clinical and research specimens. After a short explanation of the physiology and anatomy of the joint tissues, he explains the major joint pathologies including

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also the inflammatory conditions not described in detail in this chapter (concentrating on the cartilage driven/concentrated diseases). Also, other conditions such as loose joint bodies as well as pathologies related to ligaments, synovium and other tissues are picked up in this comprehensive representation of joint histology and pathology. Osteoarthritis ± RW Moskowitz, RD Altman, MC Hochberg, JA Buckwalter, VM Goldberg: This book represents an up-to-date review volume on all major topics of osteoarthritic joint degeneration including the pathology and histopathology of this condition. The book includes state-of-the-art reviews to the epidemiology, etiopathogenesis, cell biology and biochemistry and genetics of OA, all aspects of diagnosis and treatment options. This work provides thus for the interested reader a quick and extensive reference to all areas of modern OA research and clinical practice. Besides general aspects relevant for all degenerative joint diseases, also separate chapters are picking up specific questions related to the affected joint systems (hip, knee, shoulder, hands, etc.).

6.8

Future trends

Better understanding of the biochemistry of the articular cartilage matrix as well as further elucidation of the cell biology of the articular chondrocytes will provide more in-depth insights into the disease process and render new, perhaps more clearly defined targets for drug development in the future. In this respect, functional genomics of osteoarthritis,59,60 not discussed in this chapter, offers a great opportunity to draw molecular portraits of the involved cells and the disease process as such and represents, thus, a very promising tool. We will refine our understanding by dissecting out different disease entities from, for example, the `melting pot' OA and learn more about the more individualized pathology of this condition.

6.9 1 2 3 4

References Heinegard, D. K.; Pimentel, E. R. Cartilage matrix proteins. In Articular Cartilage and Osteoarthritis; Kuettner, K., Schleyerbach, R., Peyron, J. G., Hascall, V. C., Eds.; Raven Press: New York, 1992; Chapter 7. Dolan, A. L.; Hart, D. J.; Doyle, D. V.; Grahame, R.; Spector, T. D. The relationship of joint hypermobility, bone mineral density, and osteoarthritis in the general population: the Chingford Study. J. Rheumatol. 2003, 30, 799±803. Daneshpouy, M.; Socie, G.; Lemann, M.; Rivet, J.; Gluckman, E.; Janin, A. Activated eosinophils in upper gastrointestinal tract of patients with graft-versushost disease. Blood 2002, 99, 3033±3040. Katzenstein, P. L.; Malemud, C. J.; Pathria, M. N.; Carter, J. R.; Sheon, R. P.; Moskowitz, R. W. Early-onset primary osteoarthritis and mild chondrodysplasia. Arthritis Rheum 1990, 33±5, 674±684.

Understanding osteoarthritis and other cartilage diseases

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5 Eckstein, F.; Mosher, J.; Hunter, D. Imaging of knee osteoarthritis ± data beyond the beauty. Current Opinion in Rheumatology 2007 19, 435±443. 6 Burstein, D.; Gray, M. New MRI techniques for imaging cartilage. J Bone Joint Surg. Am 2003, 85-A Suppl 2, 70±77. 7 Aigner, T.; Kim, A. H. Apoptosis and cellular vitality ± issues in osteoarthritic cartilage degeneration. Arthritis Rheum 2002, 46, 1986±1996. 8 Aigner, T.; Kim, H. A.; Roach, H. I. Apoptosis in osteoarthritis. Rheum. Dis. Clin. North Am. 2004, 30, 639±653. 9 Mankin, H. J.; Dorfman, H.; Lippiello, L.; Zarins, A. Biochemical and metabolic abnormalities in articular cartilage from osteoarthritic human hips. J Bone Joint Surg 1971, 53-A, 523±537. 10 Vignon, E.; Arlot, M.; Patricot, L. M.; Vignon, G. The cell densitiy of human femoral head cartilage. Clin. Orthop. Relat. Res. 1976, 121, 303±308. 11 Bullough, P. G. The pathology of osteoarthritis. In Osteoarthritis; Moskowitz, R. W., Howell, D. S., Goldberg, V. M., Mankin, H. J., Eds.; Saunders: Philadelphia, 1992; Chapter 3. 12 Aigner, T.; Vornehm, S. I.; Zeiler, G.; Dudhia, J.; von der Mark, K.; Bayliss, M. T. Suppression of cartilage matrix gene expression in upper zone chondrocytes of osteoarthritic cartilage. Arthritis Rheum 1997, 40, 562±569. 13 Martin, J. A.; Buckwalter, J. A. Telomere erosion and senescence in human articular cartilage chondrocytes. J Gerontol. A Biol Sci Med. Sci 2001, 56, B172±B179. 14 Fassbender, H. G. Inflammatory reactions in arthritis. In Immunopharmacology of Joints and Connective Tissue; Davies, M. E., Dingle, J. T., Eds.; Academic Press: London, 1994; Chapter 9. 15 Peyron, J. Inflammation in osteoarthritis: review of its clinical picture, disease progress, subsets and pathophysiology. Osteoarthritis Symposium 1981, 115±116. 16 Gardner, D. L. The nature and causes of osteoarthrosis. British Medical Journal 1983, 286, 418±424. 17 Oehler, S.; Neureiter, D.; Meyer-Scholten, C.; Aigner, T. Subtyping of osteoarthritic synoviopathy. Clin. Exp. Rheumatol. 2002, 20, 633±640. 18 Mohr, W.; Beneke, G.; Mohing, W. Proliferation of synovial lining cells and fibroblasts. Ann. Rheum. Dis. 1975, 34, 219±224. 19 Revell, P. A.; Mapp, P. I.; Lalor, P. A.; Hall, P. A. Proliferative activity of cells in the synovium as demonstrated by a monoclonal antibody, Ki-67. Rheumatol. Int. 1987, 7, 183±186. 20 Burr, D. B. The importance of subchondral bone in osteoarthrosis. Curr. Opin. Rheumatol. 1998, 10, 256±262. 21 Bergink, A. P.; Uitterlinden, A. G.; van Leeuwen, J. P.; Hofman, A.; Verhaar, J. A.; Pols, H. A. Bone mineral density and vertebral fracture history are associated with incident and progressive radiographic knee osteoarthritis in elderly men and women: the Rotterdam Study. Bone 2005, 37, 446±456. 22 Felson, D. T.; Neogi, T. Osteoarthritis: is it a disease of cartilage or of bone? Arthritis Rheum 2004, 50, 341±344. 23 Amir, G.; Pirie, C. J.; Rashad, S.; Revell, P. A. Remodelling of subchondral bone in osteoarthritis: a histomorphometric study. J. Clin. Pathol. 1992, 45, 990±992. 24 Dieppe, P. Subchondral bone should be the main target for the treatment of pain and disease progression in osteoarthritis. Osteoarthritis Cartilage 1999, 7, 325±326. 25 Felson, D. T.; Chaisson, C. E.; Hill, C. L.; Totterman, S. M.; Gale, M. E.; Skinner, K. M.; Kazis, L.; Gale, D. R. The association of bone marrow lesions with pain in knee osteoarthritis. Ann. Intern. Med. 2001, 134, 541±549.

176 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Regenerative medicine and biomaterials in connective tissue repair Hooiveld, M.; Roosendaal, G.; Vianen, M.; van den Berg, M.; Bijlsma, J.; Lafeber, F. Blood-induced joint damage: long-term effects in vitro and in vivo. J. Rheumatol. 2003, 30, 339±344. Roosendaal, G.; Vianen, M. E.; Marx, J. J. M.; van den Berg, H. M.; Lafeber, F. P. J. G.; Bijlsma, J. W. J. Blood-induced joint damage. Arthritis Rheum 1999, 42, 1025± 1032. Roosendaal, G.; Lafeber, F. P. Blood-induced joint damage in hemophilia. Semin. Thromb. Hemost. 2003, 29, 37±42. Hooiveld, M.; Roosendaal, G.; Wenting, M.; van den Berg, M.; Bijlsma, J.; Lafeber, F. Short-term exposure of cartilage to blood results in chondrocyte apoptosis. Am J Pathol. 2003, 162, 943±951. Roosendaal, G.; TeKoppele, J. M.; Vianen, M. E.; van den Berg, H. M.; Lafeber, F. P. J. G.; Bijlsma, J. W. J. Blood-induced joint damage ± a canine in vivo study. Arthritis Rheum 1999, 42, 1033±1039. Livesley, P. J.; Milligan, G. F. Osteochondritis dissecans patellae. Is there a genetic predisposition? Int Orthop 1992, 16, 126±129. Roddy, E.; Zhang, W.; Doherty, M. Are joints affected by gout also affected by osteoarthritis? Ann. Rheum. Dis. 2007, 66, 1374±1377. Wise, C. M. Crystal-associated arthritis in the elderly. Rheum. Dis. Clin North Am 2007, 33, 33±55. Aigner, T.; Dietz, U.; Stoss, H.; von der Mark, K. Differential expression of collagen types I, II, III, and X in human osteophytes. Lab Invest 1995, 73, 236±243. Resnick, D. Osteophytosis of the femoral head and neck. Arthritis Rheum 1983, 267, 908±913. Jeffery, A. K. Osteophytes and the osteoarthritic femoral head. J Bone Joint Surg 1975, 57-B, 314±324. Milgram, J. W. Morphologic alterations of the subchondral bone in advanced degenerative arthritis. Journal of Orthopaedics and Related Research 1983, 173, 293±312. Gelse, K.; Soeder, S.; Eger, W.; Diemtar, T.; Aigner, T. Osteophyte development ± molecular characterization of differentiation stages. Osteoarthritis Cartilage 2003, 11, 141±148. Reichenberger, E.; Aigner, T.; von der Mark, K.; StoÈss, H.; Bertling, W. In situ hybridization studies on the expression of type X collagen in fetal human cartilage. Dev. Biol 1991, 148, 562±572. Hambach, L.; Neureiter, D.; Zeiler, G.; Kirchner, T.; Aigner, T. Severe disturbance of the distribution and expression of type VI collagen chains in osteoarthritic articular cartilage. Arthritis Rheum 1998, 41, 986±996. van der Sluijs, J. A.; Geesink, R. G. T.; van der Linden, A. J.; Bulstra, S. K.; Kuyer, R.; Drukker, J. The reliability of the Mankin score for osteoarthritis. J Orthop. Res. 1992, 10, 58±91. Otte, P. Die konservative Behandlung der HuÈft- und Kniearthrose und ihre Gefahren. Deutsche medizinische Jahresschrift 1969, 20, 604±609. Pritzker, K. P.; Gay, S.; Jimenez, S. A.; Ostergaard, K.; Pelletier, J. P.; Revell, P. A.; Salter, D.; van den Berg, W. B. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage 2006, 14, 13±29. Nesic, D.; Whiteside, R.; Brittberg, M.; Wendt, D.; Martin, I.; Mainil-Varlet, P. Cartilage tissue engineering for degenerative joint disease. Adv. Drug Deliv. Rev. 2006, 58, 300±322. Mainil-Varlet, P.; Aigner, T.; Brittberg, M.; Bullough, P.; Hollander, A.; Hunziker,

Understanding osteoarthritis and other cartilage diseases

46 47 48 49 50 51 52 53 54 55 56 57 58

59 60

177

E.; Kandel, R.; Nehrer, S.; Pritzker, K.; Roberts, S.; Stauffer, E. Histological assessment of cartilage repair: a report by the Histology Endpoint Committee of the International Cartilage Repair Society (ICRS). J Bone Joint Surg. Am 2003, 85-A Suppl 2, 45±57. Buckwalter, J. A.; Mow, V. C. Cartilage repair in osteoarthritis. In Osteoarthritis; Moskowitz, R. W., Howell, D. S., Goldberg, V. M., Mankin, H. J., Eds.; Saunders: Philadelphia, 1992. Sellers, R. S.; Peluso, D.; Morris, E. A. The effect of recombinant human bone morphogenetic protein-2 (RhBMP-2) on the healing of full-thickness defects of articular cartilage. J Bone Joint Surg 1997, 79-A, 1452±1463. Shapiro, F.; Koide, S.; Glimcher, M. J. Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. J Bone Joint Surg 1993, 75-A, 532±553. Nehrer, S.; Spector, M.; Minas, T. Histologic analysis of tissue after failed cartilage repair procedures. Clin. Orthop. Relat. Res. 1999, 149±162. Roberts, S.; Hollander, A. P.; Caterson, B.; Menage, J.; Richardson, J. B. Matrix turnover in human cartilage repair tissue in autologous chondrocyte implantation. Arthritis Rheum 2001, 44, 2586±2598. Brittberg, M.; Nilsson, A.; Lindahl, A.; Ohlsson, C.; Peterson, L. Rabbit articular cartilage defects treated with autologous cultured chondrocytes. Clin. Orthop. Relat. Res. 1996, 326, 270±283. Breinan, H. A.; Minas, T.; Hsu, H. P.; Nehrer, S.; Shortkroff, S.; Spector, M. Autologous chondrocyte implantation in a canine model: change in composition of reparative tissue with time. J Orthop. Res. 2001, 19, 482±492. Mainil-Varlet, P.; Rieser, F.; Grogan, S.; Mueller, W.; Saager, C.; Jakob, R. P. Articular cartilage repair using a tissue-engineered cartilage-like implant: an animal study. Osteoarthritis Cartilage 2001, 9 Suppl A, S6±15. Rahfoth, B.; Weisser, J.; Sternkopf, F.; Aigner, T.; von der Mark, K. Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits. Osteoarthritis Cartilage 1998, 6, 50±65. Whipple, R. R.; Gibbs, M. C.; Lai, W. M. Biphasic properties of repaired cartilage at the articular surface. Trans Orthop Res Soc 1985, 10, 340. Campell, C. J. The healing of cartilage defects. Clin. Orthop. Relat. Res. 1969, 64, 45±63. DePalma, A. F.; McKeever, C. D.; Subin, D. K. Process of repair of articular cartilage demonstrated by histology and autoradiography with tritiated thymidine. Clin. Orthop. Relat. Res. 1966, 48, 229±242. ODriscoll, S. W.; Keeley, F. W.; Salter, R. B. Durability of regenerated articular cartilage produced by free autogenous periosteal grafts in major full-thickness defects in joint surfaces under the influence of continuous passive motion. J Bone Joint Surg 1988, 70-A, 595±606. Aigner, T.; Zien, A.; Bartnik, E.; Zimmer, R. Functional genomics of osteoarthritis. Pharmacogenomics 2002, 3, 635±650. Aigner, T.; Bartnik, E.; Sohler, F.; Zimmer, R. Functional genomics of osteoarthritis: on the way to evaluate disease hypotheses. Clin. Orthop. Relat. Res. 2004, 1, S138± S143.

7

Using animal models of cartilage repair to screen new clinical techniques C . W . M c I L W R A I T H , Colorado State University, USA

Abstract: The repair of articular cartilage defects is often inadequate and regeneration is not achieved. Animal models of articular cartilage defects are critical preclinical means of assessing repair techniques. This chapter reviews models for articular cartilage repair with emphasis on equine models that have been recently recognized to have specific advantages for translation into human articular cartilage resurfacing. Advantages include the ability to make specific chondral defects with and without calcified cartilage layer present, more repair tissue for analysis, the ability to monitor patients clinically, with diagnostic imaging and also arthroscopically, the fact that horses get similar clinical diseases and post-operative exercise can be controlled. Critically sized defect models have been developed on both the equine femoral trochlea and medial femoral condyles. Key words: full-thickness articular defects, calcified cartilage removal, arthroscopic creation and assessment, clinical assessment, range of outcome parameters.

7.1

Introduction

From a clinical point of view there are two distinct goals of cartilage repair: restoration of joint function (which includes pain relief) and prevention, or at least delay, of the onset of osteoarthritis (O'Driscoll, 2001). These goals can be potentially achieved through replacement of damaged or lost articular cartilage with a substance capable of functioning under normal physiologic environments for an extended period, but the limitations of this repair process have long been recognized (Hunter, 1743; McIlwraith and Nixon, 1996; Poole, 2003) Regeneration is not achieved. Methods of assessing putative repair techniques have not been developed in vitro and, therefore, screening of potential procedures for human clinical use is done by preclinical studies using animal models of articular cartilage defects (An and Friedman, 1999). It has been stated that the key issue in the selection of the appropriate model is to match the model to the question being investigated and the hypothesis being tested (O'Driscoll, 2001). The research must consider each animal model(s) most accurately represent the human condition being investigated and to what extent might results obtained from these models be extrapolated to

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humans (Hunziker, 1999). The obvious questions with regard to joint defect repair are: (1) `which animal model(s) most accurately represent(s) the critical chondral defect in humans?' and (2) `to what extent can preclinical research results in this model be extrapolated to humans?' (Arnoczky, 1990). Articular cartilage lesions encountered within the human joint typically arise as a consequence of trauma (usually a sports injury) or during the course of diseases such as osteoarthritis and osteochondritis dissecans and rarely encroach significantly beyond the cartilage±bone interface into the subchondral bone compartment (Hunziker, 1999). Repair strategies should focus on re-establishing the articular cartilage compartment rather than the bony one. This chapter reviews models for articular cartilage repair with an emphasis on equine chondral defect models that have been recently recognized to have specific advantages for translation into human articular cartilage resurfacing (Hendrickson et al., 1994, Frisbie et al., 1999, 2003, 2005, 2006b, 2008; Nixon et al., 1999, 2005; Goodrich et al., 2007; McIlwraith and Rodkey, 2008).

7.2

Review of models in non-equine species

Non-equine models of cartilage repair have been previously tabulated (An and Friedman, 1999). Because of their size and relatively small expense (compared with other species), rabbits have been commonly used in articular cartilage defects studies. However, emulating a human clinical defect and thickness and volume is virtually impossible (Hunziker, 1999), as defects are typically are at least 3 mm deep (Shapiro et al., 1993; Sellers et al., 1997, 2000; Wakitani et al., 1994). Hunziker (1999) explains how, if a 3 mm deep lesion in rabbit articular cartilage was created (the majority of which would be in bone), 93±95% of the volume of this defect would be ensheathed by bone, bone marrow space and vasculature (yielding an abundance of different cell types, growth factors, and signaling substances and only 5±7% of the defect volume would abut on cartilage). In some instances, a specific question can be answered with the rabbit model such as studies of donor cell fate (Ostrander et al., 2001) or when defects are filled with a plug (Campbell, 1969; Makino et al., 2001). In a classic study on continuous passive motion (CPM) full-thickness defects that were 1 mm in diameter and 4 mm deep at four different locations were created (and therefore were principally in bone), marked differences in healing of the defects at 3 weeks in adult rabbits was demonstrated using CPM (Salter et al., 1980). In one study evaluating cartilage chondrocyte transplantation in the rabbit, a 3 mm diameter chondral defect closely emulating human defects was made creating a core using a sharpened, stainless steel punch and curetting down `while not violating, the subchondral plate', but histological examination revealed failure to completely remove calcified cartilage (Grande et al., 1989) and this is one of the caveats also noted with similar defects in the dog (Breinan et al., 2001a,b).

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The need to try to limit defects to the chondral component in the rabbit has been recognized and the technique of differentially removing calcified cartilage has been described (Hoemann et al., 2007). Clinical defects were made in the center of the trochlear groove to a depth of around 200 m by scraping with 1.5± 2.75 mm flat surgical blades (the articular cartilage in this region is approximately 300 m) and other defects removed calcified cartilage. It seems that the observation of bleeding was the means of determining the depth and follow-up histology suggested that the defect might have removed the subchondral bone plate. When the calcified cartilage was removed, the authors reported extensive subchondral bone resorption and fibrous tissue repair and this is also seen as a consequence when debridement continues beyond the subchondral bone plate and into cancellous bone in the horse (Frisbie et al., 1999). In horses, it has been found that unless there is careful monitoring of the depth and the use of arthroscopy to differentiate calcified cartilage from subchondral bone, then calcified cartilage will remain (discussed below). In another study in rabbits where two 7 mm full-thickness defects were made using a curette, the authors noted that care was taken to minimize disruption of the underlying subchondral bone, which differed from cartilage in appearance, consistency and resistance to curetting (Kuo et al., 2006) and a histological appearance of the defects supported this. The thickness of cartilage in sheep is also an issue when it comes to restricting defects to the chondral zone. Studies reported in sheep have typically involved cylindrical defects penetrating deeply into the bone as used in the testing of osteochondral plug grafts (Schachar et al., 1999). Because of the thickness of articular cartilage in the goat (0.7±1.5 mm as reported by Frisbie et al., 2006a), a chondral defect that was created by Driesang and Hunziker (2000) which was 5 mm in width, 10 mm in breadth and 0.5 mm in depth could be considered as an appropriate chondral defect model. Other studies in goats have typically used deep osteochondral defects in the evaluation of osteochondral plug grafts (Lane et al., 2001). Jackson et al. (2001) have also described the spontaneous repair of full-thickness 6 mm diameter and 6 mm depth defects in goats, but progressive subchondral bone resorption and cyst formation occurred. In a more recent study in assessing matrix-induced autologous chondrocyte implantation in sheep, the authors described creation of a `standardized partial thickness' (1.5 mm) trochlear and medial condyle defects using a 6 mm diameter chondral punch (Jones et al., 2008). In view of the fact that the thickness of the articular cartilage is 0.4±0.5 mm for sheep (Frisbie et al., 2006a), it is felt that there would be a significant bony component to these defects. In another recent study in goats, engineered cartilage implants were placed in 6 mm diameter superficial osteochondral defects (about 0.8 mm in depth). The authors noted that this defect is characterized by the removal of hyaline cartilage until a thin, discontinuous layer of mostly calcified cartilage, whereby small bleeding points can be observed. A custom-built instrument using a drill with flat tip and cutting

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case was used to generate the defect (Brehm et al., 2006). On histologic examination, however, the pictures implied a defect that went deeper into the bone. An osteochondral defect model was developed in the pig to test repair with a biphasic osteochondral composite (Jiang et al., 2007). While the depth of the defect was 8 mm, it would appear that this was an appropriate model for this question in that the implant was a biphasic cylindrical scaffold with 1.5 mm depth being the chondral phase and the remainder being the more rigid osseous phase.

7.3

Early equine models of cartilage repair

Early studies of cartilage repair in the horse most commonly involved surgically created defects in the carpus. Other studies have been done in the femoropatellar and femorotibial joints (stifle) as well as the tarsocrural articulation, and these will also be discussed.

7.3.1

Articular defects in the carpus

The first description of creation of defects in the horse was by Riddle (1970). He surgically produced both superficial and full-thickness defects in the articular cartilage on the proximal articular surface of the third carpal bone in four horses and six ponies. He reported superficial defects had not healed significantly after 8 months and that full-thickness defects `healed' through metaplasia of granulation tissue that arose at the articular margin and in the subchondral bone spaces below. At 1 month full-thickness defects were covered with granulation tissue, which underwent metaplastic change to form fibrocartilage by 4 months and imperfect hyaline cartilage by 6 months. Each defect was 1.5 cm2 in the horses and 1.0 cm2 in the ponies. There was no definition of the differential removal of cartilage to create full-thickness and partial thickness defects respectively. A second study by Grant (1975) created 8 mm defects with a trephine in the medial aspect of the antebrachiocarpal joint and 4 mm defects were made on the distal aspect of the radial carpal bone and proximal surface of the third carpal bone in the middle carpal joint. The depth was not defined and there were no illustrations. The healing of full-thickness defects with radiation therapy was compared with no therapy in the study. The authors noted that better repair tissue was developed away from the synovial membrane. They also noted that the average thickness of the articular cartilage was 1.076 mm in thickness. The authors noted that specimens examined at 17 at 28 weeks had a more cartilaginous-appearing replacement tissue with fewer synovial adhesions than those examined at 54 and 67 weeks after surgery and, based on this, suggested that a 12 month post-surgical rest from training did not appear to have any greater benefit than a 3±4 month rest. They considered the results to provide

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indirect evidence that at 4 months a defect could be healed as perfectly as possible, whereas by 12 months the replacement tissue may begin to degenerate. It is also to be noted that full-thickness 4 mm defects on the proximal third carpal bone did not completely fill at 17 or 67 weeks after surgery. It had been suggested by other authors (Vachon et al., 1986) that this may be due to an inability of such a defect to repair itself which could be related to the high density of the subchondral bone and the high impact loading forces withstood by the repair tissue at this location (the author also feels that the relatively low cartilage thickness can be a factor as well). With regard to marginal versus central location of defects, the work of Hurtig et al. (1988) also suggested that synovial adhesions and reactive periochondrium interfere with healing at the cranial rim of the third carpal bone, and, in turn, cause subchondral bone resorption and disruption of cartilage adjacent to the lesion. It was felt that small, central lesions heal better than marginal lesions. In another study in ponies, repair tissue and marginal articular defects on the distal radial and distal intermediate carpal bone consisted predominately of fibrous tissue when evaluated at 8 weeks (Barr et al., 1994b). The repair tissue in three ponies with central defects had substantial quantity of fibrocartilage and there was Safranin O staining. However, biochemical data (uronic acid and type II collagen production) showed no significant difference between the repair tissue for marginal and central defects, but central defects tended to cause more marked functional disturbances based on force plate data (Barr et al., 1994a,b). However, because of the early cessation of this study (11 weeks) conclusions are difficult to make, but certainly at this time, significant differences in healing between marginal and central defects were not demonstrated. Circular defects on the radial facet of the third carpal bone have been used to evaluate subchondral bone drilling as a treatment technique. Both full-thickness and partial thickness defects in horses were used to evaluate the healing response in association with subchondral drilling (Vachon et al., 1986; Shamis et al., 1989). Subchondral bone drilling of full-thickness cartilage defects was followed by fibrocartilagenous repair tissue of superior quality and quantity to the fibrous tissue of non-drilled defects but satisfactory functional healing was not achieved (Vachon et al., 1986). In both this and another study (Sullins et al., 1985) it was noted that calcified cartilage removal was inconsistent. If calcified cartilage is not debrided it is presumed that the defect is not full-thickness and this clouds interpretation of the data. Complete removal of calcified cartilage in experimental defects in the carpus without excessive removal of subchondral bone is difficult (however, it can be done consistently in defects on the femoral condyles and femoral trochlear ridges). Subchondral drilling did not significantly improve partial thickness cartilage healing (Shamis et al., 1989). There were increased amounts of fibrocartilage associated with the drill holes at 21 weeks, but no effect on healing of the defects. Another study with 1 cm articular defects penetrating the calcified cartilage in one limb and the subchondral bone

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plate in the opposite limb was done on the radial facet of the third carpal bones (Hanie et al., 1992). Evaluation at 6 months showed that deeper defects contained thicker, whiter tissue, but both joints contained generalized degenerative change. Defects extending through calcified cartilage were filled deeply with fibrocartilage and superficially by fibrous connective tissue, whereas defects extending through subchondral bone were consistently filled with hyaline-like cartilage in the depth of the lesion, fibrocartilage in the intermediate layer and fibrous connective tissue superficially (Hanie et al., 1992). This led to speculation that penetration of the subchondral bone plate provided better healing, but the author feels that removal of subchondral bone plate should be avoided. While work in the author's laboratory demonstrated good healing generally of defects penetrating the subchondral bone plate (Vachon et al., 1991a,b; Howard et al., 1994), instances of subchondral bone resorption and some cystic formation were seen in some defects. In these studies, a 1 cm2 full-thickness defect were created using arthrotomy on the distal radial carpal bone. At 4 months full-thickness articular defects in horses contained a healing tissue consisting of fibrous tissue and fibrocartilage morphologically at 4 months (Vachon et al., 1991a) (Fig. 7.1a) and type I collagen biochemically (Vachon et al., 1991b). However, the percentages of type II collagen improved considerably by 12 months (79.4% type II collagen at 12 months compared with no detectable type II collagen at 4 months) (Howard et al., 1994) and hyaline-like cartilage was present in the deeper layers at 12 months (Fig. 7.1b). However there is considerable deficiency in the glycosaminoglycan (GAG) content of the repair tissue (hexosamine level of 20:6  1:85 mg/g dry weight) (DW) compared with normal articular cartilage (41:8  4:3 mg/kg dry weight) (Howard et al., 1994). In addition to experimental work, arthroscopic observations of the healing of clinical articular defects confirmed the failure of these defects to heal effectively on the carpal articular surfaces (McIlwraith and Vachon 1988; McIlwraith et al., 2005). Because of this inadequate healing response, it is suggested that debridement of partial thickness to full-thickness defects and aggressive debridement into the bone is contra-indicated. This opinion is also influenced by the observation that superficial defects are not necessarily progressive and do not necessarily compromise joint function. The author has abandoned the use of carpal models compared with stifle models because of these variable healing responses (see below). Another study in the equine carpus looked at the effect of lesion size on lesions of the radial and third carpal bone. Small lesions were 5 mm2 and large lesions were 15 mm2 and they were created on the central weight-bearing areas of the radial and third carpal bone, 1.5 cm from the cranial rim of the joint (Hurtig et al., 1988). Horses were euthanized at 1, 2.5, 4, 5 and 9 months respectively after surgery. At 5 months small radiocarpal bone lesions were reduced to a ripple at the articular surface with their larger counterparts having dense white connective tissue filling the defect, but the lesions original outlined

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7.1 Photomicrographs of repair tissue in full-thickness articular cartilage defects in equine radial carpal bones: (a) at 4 months and (b) at 12 months. There is a mixture of fibrous (superficially) and fibrocartilagenous (deeper) tissue in the defect.

was still evident (Hurtig et al., 1988). Matrix flow contributed to the healing of the small defects.

7.3.2

Articular defects in the femoropatellar and femorotibial (stifle) joints

Convery et al. (1972) reported on full-thickness osteochondral defects of graduated diameters created on the medial femoral condyle of mature Shetland ponies using an arthrotomy. Three defects 9, 15 and 21 mm in diameter were placed in the center of the weight-bearing surface of the medial femoral condyle. In addition, a 3 mm defect was placed immediately above the large defect (nonweight-bearing area). The subchondral plate in all of the defects was completely removed to expose the underlying cancellous bone. The animals were sacrificed after 3, 6 and 9 months and it was found that the 3 mm defects were completely repaired after 3 months and were extremely difficult to locate after 9 months. Conversely, none of the defects 9 mm or greater was completely repaired. Microscopic evidence of degeneration was present in the articular cartilage of the tibia opposite the large femoral defects in all of the nine animals. Degenerative changes sufficient to be apparent grossly were present in four specimens. The repair tissue partially replacing the defects was a variable mixture of fibrous tissue,

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fibrocartilage, hyper-cellular cartilage and (occasionally) bone. Hexosamine content in the early 3 month defects was much less than found in the control cartilage. After 6 and 9 months there was an apparent increase in hexosamine that approximated, but did not consistently reach control defects (Convery et al., 1972). Hurtig et al. (1988) also compared large (15 mm2) and small (5 mm2) fullthickness defects at the junction of the lateral trochlear ridge and trochlear groove (in an area contacted by the patella) of the femoropatellar joint and also on the axial side of the trochlear ridge in an area not contacted by the patella. The horses were euthanized in groups at 2, 1, 2.5, 4, 5 and 9 months later (Hurtig et al., 1988). Structural repair occurred in most small defects at the end of 9 months by combination of matrix flow and extrinsic repair mechanisms. Statistically better healing occurred in small weight-bearing lesions, compared to large or non-weight-bearing lesions. Matrix flow contributed to the repair process in all stifle lesions, reducing the area by 50±90%. This response was more pronounced than in defects of the same size in the carpus. In another study, osteochondral grafts were press-fitted into 13 mm drill holes on the medial trochlear ridge of the stifle (Desjardins et al., 1991a,b).

7.3.3

Articular defects in tarsus

More recently, in a study in horses evaluating autologous chondrocyte transplantation (ACT), 10 mm diameter circular articular cartilage defects were created on the distal (non-weight-bearing) region of the lateral trochlear ridge of the talus in the tarsocrural joint. Periosteum was sutured into these defects and autologous chondrocytes (10  5 000 000 cells in 0.3 ml volume) injected beneath the periosteum. Fibrin glue was then applied to the surgical sutures to provide additional sealing. Compared with untreated defects, ACT resulted in significantly improved filling with a well integrated neocartilage and comparable expression of cartilage specific markers (Litzke et al., 2004). In an earlier study in equine tarsocrural joints it was demonstrated that osteochondral defects 6.5 mm in diameter in the non-weight-bearing area of the distal lateral trochlear ridge of the talus healed with fibrocartilagenous tissue at a faster rate and more completely than those on the weight-bearing proximomedial trochlear ridge of the talus (Fischer et al., 1986).

7.4

Current models of cartilage repair in the equine femoropatellar and femorotibial joints

Most recent publications on cartilage repair with equine models have been using defects on either the medial femoral condyle or femoral trochlea in the horse, with work coming from the author's laboratory, as well as the laboratory of Nixon. There are a number of advantages to using equine models in studying cartilage repair and these will be detailed below.

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Comparison of articular cartilage thickness in rabbit, dog, sheep, goat, horse and human

As a prelude to discussing equine stifle models, a recent study of articular cartilage thickness in the stifle of animal species used in human preclinical studies compared to articular cartilage thickness in the human knee has been reported (Frisbie et al., 2006a). Histological measurements of the thickness of non-calcified and calcified cartilage, as well as the subchondral bone plate in three locations on the femoral trochlea and two locations on the medial femoral condyles of the species used in preclinical studies of articular cartilage and compared with those of the human knee. Cadaver specimens were obtained of six human knees, as well as six equine, six goat, six dog, six sheep and six rabbit stifle joints (the animal equivalent of the human knee). Specimens were taken from five locations as illustrated in Fig. 7.2. After histopathological processing, the thickness of non-calcified and calcified cartilage layers, as well as the subchondral bone plate was measured. Average articular cartilage thickness over five locations were 2.2±2.5 mm for human, 0.3 mm for rabbit, 0.4±0.5 mm for sheep, 0.6±1.3 mm for dog, 0.7±1.5 mm for goat and 1.5±2.0 mm for horse. It was considered that the horse provided the closest approximation to humans in terms of articular cartilage thickness and that this was considered relevant to preclinical studies of cartilage healing. Individual measurements for noncalcified cartilage, calcified cartilage and subchondral bone plate are presented in Fig. 7.3.

7.4.2

Cartilage defects on the medial femoral condyle (weight-bearing) and its validation

This model was initially developed to evaluate the effect of subchondral bone microfracture on articular cartilage repair (Frisbie et al., 1999). It has since been used to look at early events in cartilage repair after subchondral bone microfracture (Frisbie et al., 2003), as well as the effect of removal or retention of calcified cartilage (Frisbie et al., 2006b) and the value of augmentive gene therapy on the repair of full-thickness defects that have been microfractured (Morisset et al., 2007). Initial work creating the model involved debriding cadaver condyles under arthroscopic visualization and following up with histological examination to confirm the depth of debridement through calcified cartilage and through subchondral bone plate respectively. An instrument to evaluate depth was developed and the difference between the arthroscopic appearances of a defect with calcified cartilage retained versus removed defined. The initial in vivo study involved creating 1 mm2 defects on the central weight-bearing portion of the medial femoral condyle under arthroscopic visualization and involving removal of calcified cartilage. The technique involves a lateral arthroscopic portal

7.2 Location of collection sites for osteochondral blocks to measure articular cartilage (non-calcified and calcified layers) and subchondral bone thickness in human and animal specimens. The numbers 1±5 represent collection sites for articular cartilage thickness with 1 and 3 being upper and lower medial femoral trochlear ridge and 2 being lateral femoral trochlear ridge; 4 and 5 are two locations on the weight bearing area of the medial femoral condyle. Reproduced with permission from Frisbie et al., `A comparative study of articular cartilage thickness in the stifle of animal species used in human pre-clinical studies compared to articular cartilage thickness in the human knee'. Vet Comp Orthop Traumatol 2006; 19: 142±6 (Figure 1).

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7.3 Plot of thickness for (a) non-calcified cartilage, (b) calcified cartilage and (c) subchondral bone plate between species. Different letters indicate statistical differences between bars. PMT (proximal medial trochlea), LT (lateral trochlea), DMT (distal medial trochlea), PMC (proximal medial condyle), DMC (distal medial condyle). Reproduced with permission from Frisbie et al., `A comparative study of articular cartilage thickness in the stifle of animal species used in human pre-clinical studies compared to articular cartilage thickness in the human knee'. Vet Comp Orthop Traumatol 2006; 19: 142±6 (Figures 3, 4, and 5).

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7.3 Continued

(McIlwraith et al., 2005) and a cranial instrument portal. After demarcating the 1 cm2 area, the cartilage is removed using a curette. All debris is flushed from the joint at the end of the procedure. In the initial study the microfracture technique with holes 3±5 mm apart was done on one defect and the other defect served as an untreated control (Frisbie et al., 1999). In a second study, a significant increase in gene expression of type II collagen at 8 weeks was demonstrated (Frisbie et al., 2003). Both type II collagen and aggrecan mRNA (reverse transcriptase polymerase chain reaction, RT-PCR) expression increased at 2, 4, 6 and 8-week periods, but aggrecan expression was not significantly up-regulated by microfracture. Five horses were evaluated after 4 months and five horses after 12 months. On gross examination a greater volume of repair tissue filled treated defects (74%) compared with control defects (45%) (Fig. 7.4). Histomorphometry also confirmed more repair tissue filling treated defects, but no difference on the relative amount of tissue types was observed (Fig. 7.4). There was an increased percentage of type II collagen in treated defects compared with control defects and evidence of earlier bone remodeling in treated defects. Another study was carried out in which half the 1 cm defects had the calcified cartilage retained and in the other half the calcified cartilage was removed (Frisbie et al., 2006b). Marked differences were observed in the quality of repair, as well as the integration of the repair tissue when calcified cartilage was removed to examine the effect of calcified cartilage retention (Fig. 7.5). More recently another study has been done with the same model (calcified cartilage removed and microfractured) demonstrating the value of gene therapy with adenoviral-IL-1ra/IGF-1 administration. Joints treated with gene therapy had a

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7.4 Gross pathologic comparison (a) and histopathologic comparison (b) at 12 months between microfractured full-thickness cartilage defects and control full-thickness articular cartilage defects. Reproduced with permission from Frisbie et al., `Arthroscopic subchondral bone plate microfracture technique augments healing of large chondral defects in the radial carpal bone and medial femoral condyle of horses'. Vet Surg 1999; 28: 242±55 (Figures 3 and 4).

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7.5 Histologic comparison of articular defects that had been microfractured, but in which calcified cartilage has been removed (a) and retained (b). Arrows indicate good integration and tidemark reformation in (a) and arrows indicate poor attachment to (original) calcified cartilage in (b). Reproduced with permission from Frisbie et al., `Effects of calcified cartilage on healing of chondral defects treated with microfracture in horses'. Am J Sports Med 2006; 11: 1824±31 (Figure 6).

significant increase in both type II collagen content, as well as aggrecan content compared with control, microfractured defects (Morisset et al., 2007). In all these cases the treatment has immediately followed creation of the defects. Post-operative management is simple because arthroscopic techniques were used on the horses. Hand walking is commenced 2 weeks after surgery and at 4 months an exercise regime of 2 minutes trot, 2 minutes gallop, and 2 minutes trot on a high-speed treadmill is commenced. In this fashion, between 4 and 12 months the repair technique is subjected to athletic exercise. Outcome parameters used with this model include: clinical examination for lameness and synovial effusion, as well as response to flexion, pre-and posttreatment radiographs, magnetic resonance imaging (MRI), synovial fluid and serum biomarkers, routine synovial analysis. sequential arthroscopies in some instances, gross post-mortem examination, histopathologic, histochemical and immunohistological analysis, biochemical analysis for type II collagen/type I collagen as well as aggrecan content, as well as glycosaminoglycan content and real-time PCR evaluation for mRNA expression in the tissue.

7.4.3

Models of articular cartilage repair in the equine trochlea

The critically sized defect model on the lateral trochlear ridge of the femur created arthroscopically was developed by Alan Nixon. This model was first reported as a 12 mm diameter defect in 1994 (Hendrickson et al., 1994) and a modified 15 mm defect creation reported in 1995 by Sams and Nixon. This latter model has been reported in a number of studies (Sams and Nixon, 1995; Nixon et al., 1999; Fortier et al., 2001, 2002; Hidaka et al., 2003; Strauss et al., 2005; Wilke et al., 2007).

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The initial study with a 12 mm defect looked at chondrocyte±fibrin matrix transplants and showed a beneficial effect (Hendrickson et al., 1994). The currently used model was first reported in 1995 by Sams and Nixon and involved a lateral arthroscopic approach (McIlwraith et al., 2005) with creation of an arthroscopic portal approximately 4 cm distal to the apex of the patella and into the joint between the middle and lateral patellar ligament to locate the central aspect of the lateral trochlea of the femur (Sams and Nixon, 1995). An 18±20 mm incision is then made through the skin, subcutaneous tissue and joint capsule at the point indicated by the spinal needle and a 16 mm internal diameter cannula placed into the joint, centered on the lateral trochlear ridge of the femur, 2 cm distal to the apex of the patella. A modified spayed bit 16 mm in diameter is used to remove a full-thickness layer of cartilage and 1 mm of subchondral bone. Different numbers of horses have been used in these studies with the single 15 mm defect model, including twelve horses (Sams and Nixon, 1995), six horses (Nixon et al., 1999), eight horses (Fortier et al., 2001, 2002), ten horses (Hidaka et al., 2003) and six horses (Wilke et al., 2007). In the initial study with the 15 mm defect model, six horses were euthanized at 4 months and six at 8 months; when only six horses were used, they were all euthanized at 6 months (Nixon et al., 1999); when eight horses were used, two horses each were evaluated at 2, 4, 8 and 16 weeks after surgery (this was an evaluation of IGF-1 gene expression patterns during spontaneous repair of articular cartilage injury) (Fortier et al., 2001) and in the second study using eight horses (Fortier et al., 2002) all horses were euthanized at 8 months. In the study of Hidaka et al. (2003) in which ten horses was used, biopsies were taken at 4 weeks and termination was at 8 months. Outcome parameters include: lameness examination, gross pathological examination, thin section high-detail radiograph, histology, synovial fluid analysis, histochemistry, type I and type II collagen immunohistochemistry, collagen typing using cyanogen bromide cleavage and peptide separation by polyacrylamide gel electrophoresis, types I and II in situ hybridization and matrix biochemical determinations. Specific gene expressions studies of IGF-1 have also been done (Fortier et al., 2001). In a more recent study MRI evaluation, as well as compressive strength has been done. In the last study reported with this model, the importance of relatively longterm assessment is emphasized (Wilke et al., 2007). In this study the effect of mesenchymal stem cell implantation was evaluated and although chondrogenesis appeared to be enhanced at arthroscopic assessment at 1 month, there was no difference between treated and control defects at 8 months. At Colorado State University we had initially developed small 4 mm defects on the medial trochlear ridge of the femur (we were able to create five defects), but have since evolved into creating two 15 mm defects on the medial trochlear ridge of the femur (Fig. 7.6). As with our model on the medial femoral condyle of the femur, it is possible to create defects with calcified cartilage retained or calcified cartilage removed.

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7.6 Creation of two 15 mm defects on the medial trochlear ridge of the femur (a). Experimental implant using staples (b).

Recently we have reported the evaluation of autologous chondrocyte transplantation via a collagen membrane in equine articular cartilage defects using this model (Frisbie et al., 2008). Fifteen horses were used in this study. Two 15 mm diameter defects were created in each horse and these defects received one of three treatments: group 1 ˆ empty cartilage defect (ECD), group 2 ˆ autologous chondrocyte implantation (ACI) attached with three polydioxanone/polyglactin (PD/PGA) staples and group 3 ˆ collagen membrane alone (CMA) (no cells) attached with three PD/PGA staples. All defects had the calcified cartilage retained (this was requested by the US Food and Drug Administration (FDA) to ensure evaluation of the treatment providing the repair). The experimental design is illustrated in Fig. 7.7. Evaluation parameters in this model include daily observations, lameness evaluations, high speed exercise from 4 to 18 months, post-mortem tissue examination and histology and immunohistochemistry. Arthroscopic evaluations at 4, 8, 12 and 18 months demonstrated that CMA was significantly worse compared with ACI or ECD treatments, with ACI having the best overall subjective grade (Fig. 7.8). Overall raw histological scores demonstrated a significant improvement with ACI compared with either CMA or ECD treated defects and ACI defects had significantly more immunohistochemical staining for aggrecan than CMA or ECD treated defects (with significantly more type II collagen in ACI and ECD compared to CMA defects) at 12 and 18 months. The histologic comparison of repair tissue at 18 months is demonstrated in Fig. 7.9.

7.4.4

Advantages of equine models

Based on the studies presented above, the horse provides the closest approximation to humans in terms of articular cartilage thickness. The defect on the equine medial femoral condyle illustrates that it is possible to emulate a medial femoral condylar lesions in humans and that it is possible to selectively leave the entire calcified cartilage layer or, on the other hand, completely remove it with certainty. As seen with the trochlear ridge model, it is possible to create large multiple defects in the horse and this approach has allowed creation of five

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7.7 Diagram of tissue harvest site (a) which was followed by processing and construction creation at Verigen. Four weeks later the opposite joint had two 15 mm defects created (b) down to the level of the calcified cartilage (c). The construct was stapled in place using three absorbable PDS/PGA staples (d and e). Reproduced with permission from Frisbie et al., `Evaluation of autologous chondrocyte transplantation via a collagen membrane in equine articular defects ± results at 12 and 18 months'. Osteoarthritis and Cartilage 2008; 16: 667±79 (Figure 5).

4 mm defects in this location (and allow preliminary assessment of different techniques), as well as create critically sized lesions that are 15 mm in diameter. Other advantages of the equine model include an ability to use the arthroscope to create lesions and to do re-examinations. This latter strength has been used in more recent definitive studies, minimizing the need for large horse numbers. The advantages over other species also include more repair tissue for analysis, the ability to monitor patients clinically, as well as with diagnostic imaging, thereby allowing practical assessment of clinical response to repair techniques. Horses also get similar clinical disease to humans. The ability to have controlled exercise with horses is an advantage, both in the early rehabilitation stage and later to test the ability of the repair to cope with athletic exercise.

7.5

Current status of animal models of cartilage repair

There is no perfect model for evaluating objectively the repair in human articular defects. However, the need for preclinical studies using animal models in evaluating a new technique for repair is important and mandated by licensing bodies.

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7.8 Plot of overall arthroscopic grade and treatment group in study of autologous chondrocyte implantation (ACT) compared with scaffold alone (CMA) and empty cartilage defect (ECD). Different letters indicate a significant difference between groups averaged over time. Lower panel shows arthroscopic view of representative repair tissue from each treatment group of study horses at 3 and 12 month time points. Reproduced with permission from Frisbie et al., `Evaluation of autologous chondrocyte transplantation via a collagen membrane in equine articular defects ± results at 12 and 18 months'. Osteoarthritis and Cartilage 2008; 16: 667±79 (Figure 7).

The author feels there has been a positive evolution of model selection from its being based on cost and convenience to more critically evaluating how well an animal model simulates the human situation. There has been a notable recognition in that a chondral defect simulates the human situation better than a hole drilled into the bone and having a major bone component to the defect. Even in the small joints (and thin cartilage) of rabbits, good attempts at chondral defects have been made. It is recognized that some laboratories are content with their small animal model and that these models will continue to be used. On the other hand, it needs to be recognized that complete removal of calcified cartilage

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7.9 Histologic photomicrographs of repair tissue at 18 months for ACI, CMA and ECD. Arrow indicate defect margins on the 2 magnification and each insert shows a 10 magnification. Reproduced with permission from Frisbie et al., `Evaluation of autologous chondrocyte transplantation via a collagen membrane in equine articular defects ± results at 12 and 18 months'. Osteoarthritis and Cartilage 2008; 16: 667±79 (Figure 9).

is critical and retention of as much of the subchondral bone component equally important. The horse is a large animal and requires special care and expertise. However, because of the ability to do follow-up arthroscopic surgeries and the need for fewer animals for statistical power, the economics are not necessarily disparate from what can be gained with smaller laboratory animals. It is also important to recognize the need for long-term studies because of experience with failure between 8 and 12 months, both in quality of the tissue as well as integration. Lastly, it should also be remembered that we need to strive for the closest approximation between preclinical research results in a given model and its extrapolation to the human situation (Arnoczky, 1990).

7.6

References and further reading

An YH and Friedman RJ (1999), `Animal models of articular cartilage defect', in An YH and Friedman RJ (eds) Animal Models in Orthopaedic Research, CRC Press, 309± 325. Arnoczky SP (1990), `Animal models for knee ligament research' in Knee Ligaments: Structure, Function, Injury, and Repair, Raven Press, 401±417. Aston JE, Bentley G (1985), `Repair of articular surfaces by allografts of articular and growth-plate cartilage', J Bone Joint Surg 68-B 29±35. Barr ARS, Duance VC, Wooton SF, et al. (1994a), `Quantitative analysis of cyanogen/ bromide-cleaved peptides for the assessment of type I:II collagen ratios in equine articular repair tissues', Equine Vet J 26 29±32.

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Barr ARS, Wotton SF, Dow SM, et al. (1994b), `Effect of central or marginal location on the healing of osteochondral defects in the equine carpus', Equine Vet J 26 33±39. Brehm W, Aklin B, Yamashchita T, et al., (2006), `Repair of superficial osteochondral defects with an autologous scaffold-free cartilage construct in a caprine model: Implantation method and short-term results', Osteo Cart 14 1214±1226. Breinan HA, Minas T, Hsu HP, et al. (2001a), `Autologous chondrocyte implantation in a canine model: Change in composition of reparative tissue with time', J Orthop Res 19 482±492. Breinan HA, Hsu HP, Spector M (2001b), `Chondral defects in animal models. Effects of selective repair procedures in canines', Clin Orthop 391 S219±S230. Campbell CJ (1969), `The healing of cartilage defects', Clin Orthop 64 45±63. Convery FR, Akerson WH, Keowan GH (1972), `Repair of large osteochondral defects. An experimental study in horses', Clin Orthop 82 253±262. Desjardins MR, Hurtig MB, Palmer NC (1991a), `Incorporation of fresh and cryopreserved bone in osteochondral autografts in the horse', Vet Surg 20 446±452. Desjardins MR, Hurtig MB, Palmer NC (1991b), `Heterotopic transfer of fresh and cryopreserved autogenous articular cartilage in the horse', Vet Surg 20 434±435. Dreisang IMK, Hunziker EB (2000), `Delamination rates of tissue flaps used in articular cartilage repair', J Orthop Res 18 909±911. Fischer TA, Stover SM, Pool RR (1986), `Healing of full thickness articular cartilage defects in the horse. A comparison of weight bearing to non weight bearing areas (abstract)', Vet Surg 15 120. Fortier LA, Valkman CE, Sandell LJ, Ratcliffe A, Nixon AJ (2001), `Insulin-like growth factor-1 gene expression patterns during spontaneous repair of acute articular cartilage injuries', J Orthop Res 19 720±728. Fortier LA, Mohammed HO, Lust G, Nixon AJ (2002), `Insulin-like growth factor-1 inhances cell based repair of articular cartilage', J Bone Joint Surg Br 84 B276± 288. French DA, Barber SM, Leach DH, Doige CE (1989), `The effect of exercise on the healing of articular cartilage defects in the equine carpus', Vet Surg 18 312±321. Frisbie DD, Trotter GW, Powers BE, et al. (1999), `Arthroscopic subchondral bone plate microfracture technique augments healing of large chondral defects in the radial carpal bone and medial femoral condyles of horses', Vet Surg 28 242±255. Frisbie DD, Oxford JT, Southwood L, Trotter GW, Rodkey WG, Steadman JR, Goodnight JL, McIlwraith CW (2003), `Early events in cartilage repair after subchondral bone microfracture', Clin Orthop 407 215±227. Frisbie DD, Lu Y, Calhoun HA, et al. (2005), `In vivo evaluation of autologous cartilage resurfacing techniques in a long-term equine model', 51st Annu Meet Orthop Res Soc 1355. Frisbie DD, Cross MW, McIlwraith CW (2006a), `A comparative study of articular cartilage thickness in the stifle of animal species used in human pre-clinical studies compared to articular cartilage thickness in the human knee', Vet Comp Orthop Traumatol 19 142±146. Frisbie DD, Morisset S, Ho CP, Rodkey WG, Steadman JR, McIlwraith CW (2006b), `Effects of calcified cartilage on healing of chondral defects treated with microfracture in horse', Am J Sports Med 11 1824±1831. Frisbie DD, Bowman SM, Calhoun HA, DiCarlo EF, Kawcak CE, McIlwraith CW (2008), `Evaluation of autologous chondrocyte transplantation via a collagen membrane in equine articular defects ± results at 12 and 18 months', Osteoarthritis and Cart 16 667±679.

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Goodrich LR, Hidaka C, Robbins PD, Evans CH, Nixon AJ (2007), `Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model', J Bone Joint Surg Br 89 672±685. Grande DA, Pittman MI, Peterson L, et al. (1989), `The repair of experimentally produced defects in rabbit articular cartilage by autologous chondrocyte transplantation', J Orthop Res 7 208±218. Grant BD (1975), `Repair mechanisms of osteochondral defects in equidae: a comparative study of untreated x-irradiated defects. Proceedings AAEP 21 95±114. Hanie EA, Sullins KE, Powers BE, Nelson P (1992), `Healing of full-thickness cartilage compared with full-thickness and subchondral bone defects in equine third carpal bones', Equine Vet J 24 382±386. Hendrickson DA, Nixon AJ, Grande DA, Todhunter RJ, Minor RM, Erb H, Lust G (1994), `Chondrocyte±fibrin matrix transplants for resurfacing extensive articular cartilage defects', J Orthop Res 12 485±497. Hidaka C, Goodrich LR. Chen CT, Warren RF, Crystal RG, Nixon AJ (2003), `Acceleration of cartilage repair by genetically modified chondrocytes over expressing bone morphogenetic protein-7', J Orthop Res 21 573±583. Hoemann CD, Sun J. McKee MD, Chevrier A, Rossomacha E, Rivard G-E, Hurtig M, Buschmann MD (2007), `Chitosan-glycerol phosphate/blood implants elicit hyaline cartilage repair integrated with porous subchondral bone in microdrilled rabbit defects', Osteo Cart 15 78±89. Howard RD, McIlwraith CW, Powers BE, et al. (1994), `Long-term fate and effects of athlete exercise on sternal cartilage autografts used for repair of large osteochondral defects in the horse', Am J Vet Res 55 1158±1167. Hunter W (1743), `Of the structure and disease of articulating cartilages', Philos Cal Trans, 514±521. Hurtig MB, Fretz PB, Doige CE, et al. (1988), `Effects of lesions size and location on equine articular cartilage repair', Can Vet J Res 52 137±146. Hunziker EB (1999), `Biologic repair of articular cartilage. Defect models in experimental models and matrix requirements', Clin Orthop 367 S135±S146. Jackson DW, Lalor PA, Aberman HM, Simon TM (2001), `Spontaneous repair of fullthickness defects of articular cartilage in a goat model: a preliminary study', J Bone Joint Surg 83-A 53±64. Jiang C-C, Chiang H, Liao C-J, Lin Y-J, Kuo T-F, Shiea C-S, Huang Y-Y, Tuan RC (2007), `Repair of porcine articular cartilage defect with a biphasic osteochondral composite', J Orthop Res 25 1277±1290. Jones CW, Willers C, Keogh CA, Smolskin D, Fick D, Yates PJ, Kirk TB, Zheng MH (2008), `Matrix-induced autologous chondrocyte implantation in sheep: Objective assessments, including confocal microscopy', J Orthop Res 26 292±303. Kuo AC, Rodrigo JJ, Reddi AH, Curtiss S, Grotkopp E, Guchiu M (2006) `Microfracture and bone morphogenic protein 7 (BMP-7) synergistically stimulate articular cartilage repair', Osteo Cart 14 1126±1135. Lane JG, Tontz WL, Ball ST (2001), `A biomorphologic, biochemical and biomechanical assessment of short-term effects of osteochondral autograft plug transfer in an animal model', J Arthroscop Surg 17 856±863. Litzke L-F, Wagner E, Baungaertner W, Hetzel U, JosmivocÂ-Alasevic O, Libera J (2004), `Repair of extensive articular cartilage defects in horses by autologous chondrocyte transplantation', Annals Biomed Engineering 32 57±69. Makino T, Fujioka H, Kurosaka M (2001), `Histologic analysis of implanted cartilage in an exact-fit osteochondral transplantation model', J Arth Surg 17 747±751.

Using animal models to screen new clinical techniques

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McIlwraith CW and Vachon A (1988), `Review of pathogenesis and treatment of degenerative joint disease', Equine Vet J S6 1±10. McIlwraith CW and Nixon AJ (1996), `Joint resurfacing: attempts at repairing articular cartilage defects', in McIlwraith CW and Trotter GW, Joint Disease in the Horse, WB Saunders, 317±334. McIlwraith CW and Rodkey WR (2008), `The horse-human relationship: Research and the future', in Feagin JA, Steadman JR, The Crucial Principles and Care of the Knee, Wolters, Klumer/Lippincott, Williams and Wilkins, Philadelphia, 221±227. McIlwraith CW, Nixon AJ, Wright IM et al. (2005), Diagnostic and Surgical Arthroscopy in the Horse, 3rd edn. Elsevier Mosby. Morisset S, Frisbie DD, Robbins PD, Nixon AJ, McIlwraith CW (2007), `Il-1ra/IFG-1 gene therapy modulates repair of microfractured chondral defects', Clin Orthop Relat Res 462 221±228. Nixon AJ, Fortier LA, Williams J, et al. (1999), `Enhanced repair of extensive articular defects by insulin-like growth factor-1 laden fibrin composites', J Orthop Res 17 475±487. Nixon AJ, Houpt JL, Frisbie DD, et al. (2005), `Gene mediated restoration of cartilage matrix by combination insulin-like growth factor-1/interleukin receptor -1 antagonist', Gene Therapy 12 177±186. O'Driscoll SW (2001), `Pre-clinical cartilage repair. Current status and future perspectives', Clin Orthop 391S 387S±401S. Ostrander RV, Goomer RS, Tontz WL (2001), `Donor cell fate in tissue engineering for articular cartilage repair', Clin Orthop 389 228±237. Poole AR (2003), `What type of cartilage repair are we attempting to attain?' J Bone Joint Surg 85-A Suppl 2 40±44. Riddle WE (1970), `Healing of articular cartilage in the horse', J Am Vet Med Assoc 157 1471±1479. Salter RB, Simmonds DF, Malcolm BW, et al. (1980), `The surgical effects of continuous passive motion on the healing of full-thickness defects in articular cartilage', J Bone Joint Surg 62-A 1232±1251. Sams AE, Nixon AJ (1995), `Chondrocyte-laden collagen scaffolds for resurfacing extensive articular cartilage defects', Osteo Cart 3 47±59. Schachar NS, Novak K, Hurtig M (1999), `Transplantation of cryopreserved osteochondral dowel allografts for repair of focal articular defects in an ovine model', J Orthop Res 17 909±919. Sellers RS, Peluso D, Morris EA (1997), `The effect of recombinant human bone morphogenic protein-2 (rhBMP-2) on the healing of full-thickness defects of articular cartilage', J Bone Joint Surg 79-A 1452±1463. Sellers RS, Zhang R, Glasson SS, et al. (2000), `Repair of articular cartilage defects one year after treatment with recombinant human bone morphogenic protein-2 (rhBMP2)', J Bone Joint Surg 82-A 151±160. Shamis LD, Bramlage LR, Gabel AA, Weisbrode S (1989), `Effects of subchondral drilling on repair of partial thickness cartilage defects of third carpal bones in horses', Am J Vet Res 50 290±295. Shapiro F, Koide S, Glimcher MJ (1993), `Cell origin and differentiation in the fullthickness defects of articular cartilage', J Bone Joint Surg 75-A 532±553. Strauss EJ, Goodrich LR, Chen CT, Hidaka C, Nixon AJ (2005), `Biochemical and biomechanical properties of lesion an adjacent articular cartilage after chondral defect repair in an equine model', Am J Sports Med 33 1647±1653. Sullins KE, McIlwraith CW, Powers BE, Norrdin RW (1985), `The evaluation of

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periosteal graphs for articular cartilage repair in horses' (abstract). Vet Surg 14 66. Vachon A, Bramlage L, Gabel A, Weisbrode S (1986), `Evaluation of the repair process of cartilage defects in the equine third carpal bone with and without subchondral bone perforation', Am J Vet Res 47 2637±2345. Vachon A, McIlwraith CW, Trotter GW, Norrdin RW, Powers BE (1989), `Neochondrogenesis in free intra-articular, periosteal and perichondrial autografts in horses', Am J Vet Res 50 1787±1794. Vachon A, Keeley FW, McIlwraith CW, Chapman P (1990), `Biochemical analysis of normal articular cartilage in horses', Am J Vet Res 51 1905±1911. Vachon A, McIlwraith CW, Keeley FW (1991a), `Biochemical study of repair of induced osteochondral defects of the distal portion of the radial carpal bone in horses by use of periosteal autografts', Am J Vet Res 52 328±332. Vachon AM, McIlwraith CW, Trotter GW (1991b), `Morphologic study of induced osteochondral defects of the distal portion of the radial carpal bone in horses by use of glued periosteal autografts', Am J Vet Res 52 317±327. Vachon AM, McIlwraith CW, Powers BE, McFadden PR, Amiel D (1992), `Morphologic and biochemical study of sternal cartilage autografts for resurfacing induced osteochondral defects in horses', Am J Vet Res 53 1038±1047. Wakitani S, Goto T, Pineda SJ, et al. (1994), `Mesenchymal cell-based repair of large full-thickness defects of articular cartilage', J Bone Joint Surg 76-A 579±592. Wilke MM, Nydam DV, Nixon AJ (2007), `Enhanced early chondrogenesis in articular defects following arthroscopic mesenchymal stem cell implantation in an equine model', J Orthop Res 25 913±925.

8

Cartilage tissue repair: autologous osteochondral mosaicplasty L . H A N G O D Y , Uzsoki Hospital, Hungary, G . K I S H , Saint George Medical, USA, T . K O R E N Y , PeÂcs Medical School, Hungary, L . R . H A N G O D Y , Semmelweis Medical School, Â D I S , Debrecen Medical School, Hungary Hungary and L . M O

Abstract: This chapter reviews the role of autologous osteochondral grafting techniques in orthopaedic surgery, particularly the mosaic-like transplantation of small cyclindrical grafts, known as the mosaicplasty technique. A review is given about the research behind the development of the technique and guidelines are provided for successful surgical practice. Comparative evaluations of mosaicplasty versus other resurfacing techniques are also introduced. Key words: autologous osteochondral transplantation, mosaicplasty, fullthickness defect, autograft transfer, hyaline cartilage.

8.1

Introduction

This chapter reviews the role of grafting techniques in orthopaedic surgery, particularly the mosaic-like transplantation of small cyclindrical grafts, known as the mosaicplasty technique, used in autologous osteochondral transplantation. The chapter reviews the research behind the development of the technique, provides guidelines on good surgical practice and the clinical evidence supporting its use. Treatment of full-thickness defects of the weight-bearing gliding surfaces is a frequent problem in orthopaedic practice. Focal chondral and osteochondral defects of loading surfaces often cause several problems for the patient, such as pain, swelling, clicking and instability, and may lead to early degenerative changes. Several treatment options involving surgical resurfacing are available to treat such defects, but clinical outcomes of these procedures are controversial, and none of them represents a long-term solution.1,2 Traditional resurfacing techniques, such as debridement, subchondral penetration, microfracture technique and abrasion arthroplasty, have been shown to have limited value because of the poor biomechanical characteristics of the ingrown repair tissue.3 In the past two decades, numerous investigators have developed new techniques to provide hyaline or hyaline-like repair for articular defects. These

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recently introduced treatment options include periosteal and perichondrial grafts, autologous chondrocyte transplantation, morselized autologous osteochondral mixture, osteochondral allografts, biomaterials and autologous osteochondral transplantation.4±20 Most of these techniques are supported by experimental data, but only autologous chondrocyte transplantation and autologous osteochondral transplantation have been used extensively in clinical practice. The concept of transplanting an autogenous osteochondral graft resurfacing option is not new. Previous experimental and clinical experience with autogenous osteochondral grafting has demonstrated that the transplanted hyaline cartilage had a good rate of survival.7,10,17,20 Despite this observation, two problems with the process have been noted: the donor cartilage must be taken from surfaces that bear less weight, which limits the procurement field, and the use of large grafts can cause incongruity at the recipient site, which permanently alters the biomechanics of the joint.21±26 In our preclinical animal studies performed in 1991 and 1992 it was proved that the use of small-sized multiple cylindrical grafts rather than a single large block graft allows more tissue to be transplanted while preserving donor-site integrity, and the mosaic-like implanting fashion permits progressive contouring of the new surface.27±34 According to the basic thesis, mosaic-like transplantation of multiple, smallsized, cylindrical osteochondral grafts harvested from the relatively less weightbearing periphery of the patellofemoral joint is able to provide a congruent resurfaced area (see Fig. 8.1).27,30,35,36 The transplanted hyaline cartilage survives the procedure and results in a more durable surface compared with fibrous repair tissue.7,10,17,20 Donor-site repair will be developed via natural healing processes, resulting in filling up the tunnels with cancellous bone and covering the surface by reparative fibrocartilage from marrow-derived cells.22,24,30,32,37 Filling of the defect by bigger-sized rings results in a covering rate of only about 70±80% but means fewer contacting surfaces between local and transplanted cartilage at the same time. Using smaller grafts can improve the coverage by up to 100%, but involves more interfaces in the replaced area which are potentially vulnerable to infection.33

8.2

The development of the mosaicplasty resurfacing technique: animal and other studies

In the 1990s, several series of animal and cadaveric studies were carried out to develop the mosaicplasty resurfacing technique. As a first approach, cadaver studies were performed to develop an instrumentation providing optimal technical conditions for harvest and mosaic-like transplantation of small cylindrical osteochondral grafts. The first version of instruments was introduced

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8.1 Mini-arthrotomy mosaicplasty ± implantation of five 8.5 mm grafts into the medial femoral condyle.

in 1992, but after further research and development a second generation of technical tools were elaborated to provide standard conditions for arthroscopic implantation as well.27,30,36,38 Since 1991 several series of animal trials were performed to evaluate the mosaicplasty concept and identify an ideal donor site, graft dimension, optimized filling rate and congruity. German shepherd dogs and horses were subjects of these experimental work, but later clinical experiences of veterinary practice of horse mosaicplasties were also used to improve technical details and rehabilitation aspects.4,27,30,36,37,39 These experiments confirmed the following important observations: consistent survival of the transplanted hyaline cartilage; integration of the transplanted grafts to the host tissue; fibrocartilage formation between the transplanted grafts promoted by abrasion arthroplasty or sharp curettage of the bony base of the defect; deep matrix integration between the hyaline cap of the grafts and the host cartilage as well as the intermediaer fibrocartilage due to the defect site preparation; cancellous bone filling and fibrocartilage covering of the donor tunnels providing an acceptable gliding surface for these reduced weightbearing areas. These observations were supported by further, repeated experiments.4,27,30,36,37,39 Biomechanical testing of press fit implanted grafts was also carried out by our research group and by independent experiments. The precise determination of pull-out forces helped find the ideal press fit as an important factor of the accelerated rehabilitation process.9,40,41 On the basis of the reproducible experimental confirmation of the mosaicplasty concept, clinical application began in 1992.27,29,30,33,37,39,42 During the following years, the clinical results reported by various authors matched the

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results seen in animals, and since 1995 the procedure has been used with equal success in numerous institutions throughout the world.26,38,43±55

8.3

Surgical technique: pre-operative planning

Localized full-thickness cartilaginous lesions are usually defined at arthroscopy, but pre-operative clinical investigation may also give some useful information. Anamnestic data and actual clinical findings (e.g., tenderness in the medial joint space, swelling, clicking) may support the presence of a cartilage defect, but there are no specific signs for determining the exact type and location of existing chondral damage. Osteochondral lesions usually result in more expressed complaints. In spite of the fact that small or medium-sized chondral and osteochondral damage does not give a characteristic picture, the clinical examination is of great importance, as it is always necessary to eliminate the actual biomechanical problems. Stability of the affected knee, femorotibial alignment and patellofemoral traction conditions should be cleared at the clinical examination. Standard and standing X-ray examinations are basic elements of preoperative diagnostics. Computed tomography (CT) may inform about the subchondral bony condition; ultrasound investigation or special sequences of magnetic resonance imaging (MRI) can give useful information about the location and extension of the chondral defects, but severity cannot always be determined exactly. The last step is usually the arthroscopic examination, which should determine the exact location and stage of the damage, evaluate the quality of the donor area, and check all the other intra-articular conditions. Pre-operative preparations should include antibiotic prophylaxis. General or local anaesthesia with tourniquet control is recommended.28,31,56,57

8.4

Surgical instruments and choice of surgical technique

The patient is positioned supine with the knee capable of 120ë flexion. The contralateral extremity is placed in a stirrup. Standard arthroscopic instrumentation and the MosaicPlasty Complete System (Smith & Nephew, Inc., Endoscopy Division, Andover, MA) are required. Beside these reusable instruments disposable chisels, drillbits and tamps are also available to provide ideal conditions for precise graft harvest and tunnel preparation (Dispoposplasty System ± Smith & Nephew, Inc., Endoscopy Division, Andover, MA). A fluid management system may support the procedure. Choosing a procedure (arthroscopic or mini-arthrotomy) depends on the type, size and exact location of the defect determined during arthroscopy. As placing the grafts perpendicular to the surface is paramount to the success of the operation, the first task is to determine whether an arthroscopic or open pro-

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8.2 Open mosaicplasty ± resurfacement of a trochlear defect.

cedure is required. Although certain trochlear defects can be resurfaced arthroscopically, patellotrochlear and tibial lesions always require an open procedure (see. Fig. 8.2).58,59 Most of the femoral condylar defects can be managed arthroscopically (see Fig. 8.3). As for most of these lesions, central anterior medial and central anterior lateral portals will allow correct perpendicular access. An open procedure may be chosen in the learning curve or when an arthroscopic approach is not practical due to size or location of the lesion. Arthroscopic or open mosaicplasties have the same steps and technique.

8.3 Arthroscopic mosaicplasty ± resurfacement of the medial femoral condyle by two 8.5 mm grafts and one 6.5 mm graft.

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8.5

Arthroscopic mosaicplasty

8.5.1

Portal selection

As emphasized, perpendicular access to the lesion is critical to proper insertion of the grafts. Care is needed in making the viewing and working portals. A 1.2 mm K-wire or 18-gauge spinal needle must be used initially to locate the portal sites (see Fig. 8.4). It should be noted that these portals tend to be more central than the standard portals owing to the inward curve of the condyles. For osteochondritis dissecans on the medial femoral condyle the approach should be from the lateral side. Sometimes the standard lateral portal is too oblique, therefore the central patellar tendon portal is used giving good access to the inner positions of both the medial and the lateral femoral condyles.

8.5.2

Defect preparation

A full-radius resector or curette and a knife blade are used to bring the edges of the defect back to good hyaline cartilage at a right angle. The base of the lesion is cleaned with an arthroscopic burr (Abrader, Acromionizer) or half-round rasp to viable subchondral bone. Abrasion arthroplasty of the defect site promotes fibrocartilage grouting from the bony base. Because tapping the cutting edge of the guide into the bony base and removal of it can mark the defect site, the drill guide is used to determine the number and size (2.7, 3.5, 4.5, 6.5 and 8.5 mm in diameter) of grafts needed.11,22,37,56,60±62 Filling of the defect by same-sized contacting rings allows a filling rate of about 70±80%, but use of additional sizes to cover the dead spaces and cutting the grafts into each other can improve the coverage by up to 90±100% (see Fig. 8.5).27 Finally, the depth of the defect is measured with the laser marks of the dilator.

8.4 Determination of the perpendicular access to the damaged area by a spinal needle during arthroscopic mosaicplasty.

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8.5 Miniarthrotomy mosaicplasty ± 100% filling rate by cutting the graft into each other.

8.5.3

Graft harvest

The medial femoral condyle periphery of the patellofemoral joint above the line of the notch is the most preferred harvest site (see Fig. 8.6). The lateral femoral condyle above the sulcus terminalis and, in exceptional cases, the notch area can serve as additional donor areas. Grafts harvested from the notch area are less favourable features, as they have concave cartilage caps and less elastic underlying bone. In the case of arthroscopic mosaicplasty the medial patellofemoral periphery has easier access than the lateral one as fluid distension can promote lateral positioning of the patella and may provide easier perpendicular positioning for the harvesting chisel.56,61,62 The best view for harvesting grafts is obtained by introducing the scope through the standard contralateral portal. Extend the knee and use the standard ipsilateral portal to check the perpendicular access to the donor site. The extended position should provide perpendicular access to the most superior donor hole. Gradual flexion allows the harvest of additional grafts from the lower portions of the patellofemoral periphery. If the standard portals do not

8.6 Harvest site on the medial femoral condyle during miniarthrotomy mosaicplasty.

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allow a perpendicular approach, use a spinal needle or a K-wire to determine the location of additional harvesting portals. Once the necessary portal has been determined, introduce the proper-sized tube chisel filled with the appropriate harvesting tamp. Once the site has been clearly identified, the chisel is located perpendicular to the articular surface and driven by a hammer to the appropriate depth. The minimal length of the graft should be at least two times its diameter, but, as a rule, take 15 mm long grafts to resurface chondral lesions and 25 mm long plugs for osteochondral defects. It is important to hold the chisel firmly to avoid its shifting at the cartilage±bone interface, producing a crooked graft. By flexing the knee, lower sites can be obtained. The lower limit is the level of the top of the intercondylar notch (sulcus terminalis). Insert the appropriate harvesting tamp into the cross-hole in the tubular chisel and use it as a lever. The chisel should be toggled, not rotated, causing the graft to break free at the chisel tip. Eject the grafts from the chisel by sliding the appropriately sized chisel guard over the cutting end. Use the tamp to push out the graft onto gauze in a saline-wetted basin.11,56,60±62 The donor site holes will eventually be filled up with initial repair tissue by bleeding mediated mesenchymal stem cell invasion in a few hours. Proper rehabilitation during the first post-operative weeks may support a transformation of the primary repair tissue into cancellous bone and fibrocartilage as final coverage. During the learning curve, the grafts can also be obtained through a mini-arthrotomy (1.5 to 2.0 cm).

8.5.4

Implantation of the grafts: `drill±dilate±deliver' (three-dimensional grafting)

Drill The knee should be flexed and good distension established. Reintroduce the drill guide using the dilator as an obturator. Place these tools perpendicularly to the defected surface. By rotating the arthroscope, the drill guide and the perpendicularity of the laser mark can be seen from different angles, ensuring proper orientation. Tap the cutting edge of the guide into the subchondral bone. Insert the appropriately sized drill bit and drill to the desired depth. Generally, a recipient hole a few millimetres deeper than the length of the graft is desirable to minimize high intraosseal pressure. Reduce the inflow to minimize leakage. Finally, remove the drill bit. Dilate Insert again the conical-shaped dilator into the drill guide. Tap it to the desired depth, depending on the actual features of the recipient bone. Stiff bone needs more dilation than normal or soft bone. Hold the drill guide firmly and remove the dilator from the hole.

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Deliver Adjust the delivery tamp by turning the handle to initially allow the graft to sit slightly higher than the depth of the defect. This will minimize the likelihood of overpenetrating the graft. Stop the inflow to eliminate the graft being forced out of the tube by fluid flow. Deliver the graft under direct visualization into the recipient hole through the drill guide with the delivery tamp. Insert the graft deeper by turning the delivery tamp handle counterclockwise. The graft should match the original articular surface. Remove the drill guide to inspect the graft. If the graft is protruding, reinsert the drill guide and tap the graft down gently with the tamp of the appropriate size. Insert subsequent grafts in a similar manner by placing the drill guide immediately adjacent to the previously placed grafts. Such step-by-step graft implantation has several advantages. Dilation of the actual recipient hole allows an easy graft insertion (low insertion force on the hyaline cap), but dilation of the next hole affects surrounding bone to the previously implanted grafts, which can result in a very safe press fit fixation. Finally, when all of the holes are filled by the grafts, move the knee throughout its range of motion, provoking varus or valgus stress, depending on the site of the resurfaced area. Close the portals and introduce suction drainage into the joint through a superior portal. Use an elastic bandage to fix the appropriate dressing.11,12,23,60

8.5.5

Open mosaicplasty, post-operative management and rehabilitation

If an arthroscopic approach is impractical, it may be necessary to create a medial or lateral anterior sagittal incision or an oblique incision to perform a miniarthrotomy mosaicplasty. Patellotrochlear and tibial implantations may require an extended anteromedial approach.58,59 Further steps and technique of the implantation are identical with the arthroscopic procedure. Post-operatively, the drain should be removed at 24 hours. Appropriate pain and cool therapy as well as nonsteroidal anti-inflammatory drugs can reduce the complaints of the patient. Post-operative thrombosis prophylaxis is recommended. Autologous osteochondral mosaicplasty permits an immediate full range of motion (ROM), but requires 2 weeks non-weight bearing and a further 2±3 weeks of partial weight bearing (30±40 kg) after the operation. The initial non-weight-bearing phase is recommended to prevent graft subsidence during osseous integration. This period may be supported by controlled passive motion (CPM) to promote cartilage metabolism and moderate soft-tissue oedema around the joint. Partial weight bearing supports fibrocartilage repair round implanted cylindrical plugs, further enhancing secure graft incorporation. Normal daily activity can be achieved in 8±10 weeks. High-demand sporting activity should be delayed till after 5±6 months. This protocol can be modified

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easily in accordance with established guidelines for concurrent anterior cruciate ligament (ACL) reconstruction, high tibial osteotomy, meniscus reinsertion, meniscus resection, etc. (see Table 8.1).10,33,41,63

Table 8.1 Mosaicplasty rehabilitation protocola GENERAL VIEWPOINTS Immobilization No immobilization!b Ambulationc Two-crutch ambulation, non-weight-bearing Two-crutch ambulation, partial loading (30±40 kg s) Discontinue crutches, full weight-bearing

Immediate 2±4 weeks 4±5 weeks

Functional exercises Form walking, gait evaluation Step-up Step-down

4±5 weeks 4±5 weeks 5±6 weeks

Range of motion Early range of motion encouraged CPM in case of extended lesions 2±4 cm2 (in painless range) Full extension, flexion as tolerated Stationary bicycle

Immediate (first week) Immediate 3 weeks

Strength return Quadriceps Open chain exercises, leg raises Concentric contraction to full extension Concentric contraction against resistance Isometric exercises in different angles Excentric exercises against resistance Hamstrings Isometric exercises in different angles Concentric and excentric strengthening ± against resistance Closed chain exercisesd Pushing a soft rubber-ball with foot Closed chain exercises with half weight-bearing with full weight-bearing Stationary bicycle with resistance Stairmaster Proprioception return Balance exercises standing on both feet standing on one foot (hard ground) standing on one foot (trampoline or aerostep)

Immediate 1 week (or earlier if tolerated) 2 week Immediate 3±4 weeks Immediate 1±2 weeks 3±4 weeks Immediate 2±3 weeks 5±6 weeks 2±4 weeks (if 90ë knee flexion achieved) 6±8 weeks 5±6 weeks 6±8 weeks 8±10 weeks

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Table 8.1 Continued Return to activity Jogging Straight line running Directional changes Shear forces Sport-specific adaptations Sports activity

10 weeks 3 months 4±5 months 5 monthse 5 months 5±6 monthsf

SPECIAL VIEWPOINTS Weight bearing at different defects of knee Femur or tibia condyle, chondral defect, d < 15 mm non-weight-bearing partial weight bearing Femur or tibia condyle, chondral defect, d  15 mm non-weight-bearing partial weight bearing Femur or tibia condyle, osteochondral defect non-weight-bearing partial weight bearing Patellar defect, d < 15 mm partial weight bearing Patellar defect, d  15 mm partial weight bearing

1 week 1±3 weeks 2 weeks 2±4 weeks 3 weeks 3±5 weeks 2 weeks 3 weeks

Quadriceps strengthening and patellar mobilization ± differences at patellar defects: Vastus medialis strengthening! Isometric exercises in extension Immediate Patellar mobilization Immediate! Isometric exercises in different angles 1 week Open chain exercises 2 weeks ± against resistance 3±4 weeks Excentric exercises against resistance 4±5 weeks Closed chain exercises 2±3 weeks The treatment of underlying causes can also modify the rehabilitation programme. The most frequent combinations at knee applications are the following LCA-reconstruction combined with mosaicplasty 2±4 weeks non-weight-bearing (up to the mosaicplasty) 2 more weeks partial weight-bearing 5±90ë ROM for 4 weeks Mainly closed chain exercises for quadriceps strengthening Hamstring strengthening in open and closed chain Proprioceptive training! Meniscus reinsertion combined with mosaicplasty 4 weeks non-weight-bearing 2 more weeks partial weight-bearing 5±45ë ROM for 4 weeks

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Table 8.1 Continued Retinaculum patellae reconstruction combined with mosaicplasty 2±4 weeks non-weight-bearing (up to the mosaicplasty) 2 more weeks partial weight-bearing 0±45ë ROM for 4 weeks HTO combined with mosaicplasty Weight-bearing (for 4 weeks only with crutches and only in extension) is up to the mosaicplasty, pain, and degree of the correction of the varus (lower correction ± non-weight-bearing, overcorrection ± early weight-bearing) a

Uzsoki Hospital and Sanitas Private Clinic, Budapest, Hungary. The main point of the rehabilitation is to ensure the early motion of treated joint to promote appropriate nutrition of transplanted cartilage. Cool therapy can be used during the first week to avoid post-operative bleeding and decrease post-operative pain. In a case of a concomitant procedure requiring external fixation of the affected joint (e.g. meniscus reinsertion), limitation of ROM for a short period by bracing can be allowed. c Extent, type (chondral or osteochondral) and location of the defect may modify weight bearing. d Partial loading promotes to transform connecting tissue (between transplanted plugs) into fibrocartilage, so these exercises are mainly important, extremely in the half-weight bearing period. On the other hand, with some closed chain exercises (e.g. cycling) it is possible to ensure cyclic loading, which makes the fluid- and nutrition-transport much more efficient between synovialfluid and hyaline cartilage. e Approximately 4±5 months are needed to form a composite hyaline-like surface on transplanted area, which tolerates shear forces. f Depending on depth and extent of the defect. If strength, power, endurance, balance and flexibility are not satisfying, sport activity is allowed only later. b

8.5.6

Pitfalls and complications in surgery

One of the most common problems is neglecting the main instructions of the protocol. Perpendicular harvest and implantation of the grafts are crucial for successful transplantation. Oblique harvest and insertion may result in steps on the surface. Careful control from different angles by the arthroscope can eliminate such problems. Another frequent mistake is to implant a graft deeper than the desired level. First of all, appropriate use of the delivery tamp can help avoid too deep an insertion. If the graft has been inserted too deeply, the following steps are recommended. Insert the drill guide next to the previously implanted graft. Drill the appropriate recipient hole. Remove the guide and use the arthroscopic probe to lift the previously implanted graft to the proper level. The recipient hole adjacent to the implanted graft should provide enough room for such manipulation. As soon as the expected graft level has been achieved, continue the recommended protocol for the rest of the insertions. Dilation of the adjacent tunnel will provide perfect press fit fixation of the previously implanted graft. Septic or thromboembolic complications may result in a negative influence on the clinical outcome. Correct aseptic conditions, one-shot antibiotics, and thrombosis prophylaxis can decrease the chance of these complications.

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According to 15 years of follow-up, long-term donor site morbidity does not occur frequently. Patellofemoral complaints, such as pain or swelling after strenuous physical activity, follow the mosaicplasty procedure in fewer than 3% of cases. However, excessive post-operative bleeding occurred in 8%. Precise post-operative drainage, cool therapy and elastic bandages can diminish the chance of this complication.57

8.5.7

Clinical results from mosaicplasty procedures

Clinical results from the authors' institution Between 6 February 1992 and 31 August 2006, 1097 mosaicplasties were performed at the authors' institution: 789 implantations on femoral condyles, 147 in the patellofemoral joint, 31 on the tibia condyles, 98 on talar domes, 8 on the capitulum humeri, 3 on humeral heads and 11 femoral heads. Two-thirds of the cases were operated on because of a localized Grade III or Grade IV cartilage lesion, whereas the rest of the patients underwent surgery due to osteochondral defects. In 81% of the patients, concomitant surgical interventions were also carried out, which influenced the clinical results of the mosaicplasty procedures. The majority of these concomitant procedures were ACL reconstructions, realignment osteotomies, meniscus surgery and patellofemoral realignment procedures.24,57,64,65 The results of these resurfacing procedures were evaluated at regular intervals by standardized clinical scores, X-rays, in selected cases by MRI, and in certain cases second-look arthroscopy, histological evaluation of biopsy materials and cartilage stiffness measurement. Femoral, tibial and patellar implantations were evaluated by the modified Hospital for Special Surgery (HSS), modified Cincinnati, Lysholm and International Cartilage Repair Society (ICRS) scoring systems, while possible donor-site disturbances and morbidity were evaluated by the Bandi scoring system.12,61,64 Patients with talar lesions were subjected to Hannover ankle evaluations.28,33,35,61,66 During the above-mentioned period, 98 second-look arthroscopies were done to check the quality of the resurfaced area and to check the morphological features of the donor sites. The indications for these second-look arthroscopies were persistent or recurrent pain, swelling or postoperative intra-articular bleeding in 31 patients (2 months to 11 years) and a second trauma in 26 patients (1±9 years). In 41 patients second-look arthroscopies were indicated at 4±7 months post-operatively to evaluate the quality of the resurfaced area and to determine the earliest date to return to the professional sports activity.63 The cartilage stiffness of 25 patients was also measured by the Artscan 1000 computerized indentometric device (Artscan Oy, Helsinki, Finland) at 10 N pressure during control arthroscopy.31,57,63,64,67 Analysis of clinical scores has shown good to excellent results in 92% of patients with femoral condylar implantations, 87% of tibial resurfacings, 74% of

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8.7 Control arthroscopy of a 3-year-old mosaicplasty on the medial femoral condyle.

patellar and/or trochlear mosaicplasties and in 93% of talar procedures. Moderate and severe donor-site disturbances were present in 3% of patients according to the Bandi score (evaluations were done in a 1±10 year interval).63 Good gliding surfaces, histologically proven survival of the transplanted hyaline cartilage and acceptable fibrocartilage covering of the donor sites were found in 81 of the 98 control arthroscopies (see Fig. 8.7). Slight or severe degenerative changes were seen at the recipient and/or donor sites in 17 cases (6 chondral lesions and 11 osteochondritis dissecans). Stiffness measurement results of the resurfaced areas in 80% of the cases were similar to the surrounding, healthy hyaline cartilage.31,67 Post-operative complications were four deep infections and 56 painful haemarthroses. Arthroscopic or open debridement resolved all deep infections and 12 cases of haemorrhage also required arthroscopic or open debridement. The remaining patients with haemarthroses were treated by aspiration and cryotherapy. Four patients had minor thromboembolic complications.63 Four arthroscopic resurfacing techniques were compared in a multicentric, prospective study involving 413 patients. Three techniques providing fibrocartilage type cartilage repair (Pridie drilling, abrasion arthroplasty and microfracture) were compared with mosaicplasty, resulting in hyaline cartilage type resurfacing. According to this study, hyaline-like resurfacing gives a significantly better clinical outcome than the other techniques, especially long term, i.e. 3, 4 and 5 years post-operatively.32 The durability of the mosaicplasty results was investigated in a different study, which evaluated the clinical outcome of patients who were followed up for more than three years. In the senior author's institute between 6 February 1992 and 31 August 1996, 126 mosaicplasties were performed and 113 of them were available for late follow-up examinations.24 Two-thirds of these patients were operated on because of full-thickness cartilage defects, and one-third had osteochondral destructions. According to the modified HSS score of these patients (3±6 years' follow-up), good to excellent clinical intermediate-term outcomes were achieved in 91% of cases.65

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An integral part of the current work on autogenous osteochondral mosaicplasty remains the assessment of donor-site morbidity. A recent biomechanical study demonstrated relatively high loading forces in the donor area but stated that, to date, there has been no evidence that graft harvest would result in degenerative changes.68 In our entire study group, only transient symptoms could be attributed to the donor sites. The patients with talar, capitellar, femoral head and humeral head lesions who had knee surgery only for procurement of the osteochondral plugs, served as the donor-site controls. Those patients, with a rare exception, had no long-term symptoms in the knee. The symptoms in the knee resolved within 6 weeks in 95% of patients with a talar lesion and were completely resolved at 1 year in 98% of them.61,69 We think that full recovery of the donor site is due to the peripheral position chosen for the donor area and the small size and proper spacing of the individual grafts. These elements allow the joint to reconstitute structurally and to accept the relatively low loads in these parts of the knee. In spite of relatively low rate of donor site morbidity, donor tunnels may cause more disturbances in the early post-operative period. In our series, we observed 36 painful intra-articular bleedings following mosaicplasty procedures.63 In several series of animal trials, donor site filling by different biodegradable plugs was studied (see Fig. 8.8). These donor site plugs were implanted to prevent excessive post-operative bleeding and those same plugs could also work as scaffold for repair tissue formation in the tunnels. While polyglyconate, polylactate, hydroxilapatite, polycaprolactone and carbon rods gave poor histological results, compressed collagen plugs resulted in good outcomes.60 As a special evaluation, autogenous osteochondral mosaicplasties were evaluated retrospectively in 93 professional athletes. According to the location of the defects, 51 medial condylar, 15 lateral condylar, 1 lateral tibial condylar, 10 patellofemoral, 14 talar and 2 capitellum humeri implantations were evaluated. The average age of the patients was 26 (14±39) and the male : female

8.8 Filling of the donor tunnels by biodegradable donor site plugs during arthroscopic mosaicplasty.

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ratio was 55 : 38. Forty-two full-thickness osteochondral defects and 51 fullthickness chondral lesions were followed up. In 43% of the patients, mild or moderate osteoarthritis signs were observed pre-operatively. Evaluation of mosaicplasties in competitive athletes was determined according to complications and return to the sports activity. There were no septic or thromboembolic complications in this group. By the return to the sports activity, the following outcomes were observed: 64% of the patients returned to the same level of sports activity (five of the patients participated in the Olympic Games in 1996, 2000 and 2004); 19% were able to return a lower level of sports activity including hobby sport and 17% of the surgical patients had to discontinue any kind of sports activity.61 Long-term results of femoral condylar implantations were evaluated in a separate study. This report reviews the results of 551 patients including 93 osteoarthritic cases treated between February 1992 and October 2001. The period of follow-up ranged from 1 to 9 years with an average of 5.7 years. These patients were assessed by clinical evaluation and scoring with the modified HSS and Bandi scoring systems. Whereas the average HSS score achieved 92 in regular cases, this score was only 77 in osteoarthritic knees. According to the Bandi scoring system, 7% early (post-operative haemarthros) and 3% long-term morbidity occurred at the ipsilateral knee donor site regularly; however, these ratios were 8% and 15% respectively in osteoarthritis.57 Clinical outcomes from other sources During recent years several independent centres have published retrospective or comparative studies about the clinical outcome of the autologous osteochondral mosaicplasty technique. Most of them confirmed our clinical experiences regarding this technique of ostechondral autograft transfer. Christel et al.,44 Marcacci et al.51 and Traub et al.55 in their retrospective evaluations published around 90% good and excellent clinical outcome of knee mosaicplasty cases. Several other prospective studies confirmed similar clinical outcomes. Marcacci et al.52 in a 2-year follow-up publication, Chow et al.,70 Gudas et al.36 and Solheim et al.54 reported the same clinical efficacy. In addition to several confirming papers, Bentley et al.71 published a comparative study stating a moderate deterioration of mosaicplasty results compared with autologous chondrocyte implantation outcome. However his study contains several concerns regarding indication (defect size) and rehabilitation aspects. These problems were criticized by Kish and Hangody72 in peer reviewed form. Unlike Bentley's observations Horas et al.45 reported outstanding clinical results of mosaicplasty in a comparative, prospective study of mosaicplasty versus autologous chondrocyte transplantation. In addition, the investigators reported that the defects treated with autologous chondrocyte implantation were primarily filled with fibrocartilage rather than hyaline cartilage.

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Clinical scores as well as several other types of investigations were also used for evaluations. Ripoli et al.26 and Recht et al.73 referred to their magnetic resonance (MR) findings to evaluate mosaicplasy outcomes. As a very critical, but invasive way of follow-up, histological evaluations were also done to check the survival of transplanted hyaline cartilage tissue. Barber and Chow74 and Leo et al.75 reported histological results to support a survival of transplanted hyaline cartilage tissue. They found a consistent survival of the transplanted hyaline cartilage in their samples. Beside clinical evaluations biomechanical tests and basic science background of mosaicplasty were also extensively studied. As one of the first references of osteochondral autograft transfer, Pap and Krompecher76 published their experimental and clinical experiences. Diduch et al.40 in their biomechanical report, Duchow et al.21 and KordaÂs et al.41 in their paper discuss important details of pressfit implantation. Makino et al.77,78 published two papers about press-fitting questions and relationship between histological results and graft sizing parameters. Beside biomechanical information repeated animal trial and cadaver study results were also reported. After several rabbit, dog, sheep and goat experiments Hurtig et al.46 reported about big animal model ± they introduced their positive experiences with mosaicplasty on horses. Successful clinical experiences of mosaicplasty outside the knee were also reported by several authors. Hoser et al.79 (2004) published a special technique of computer-assisted mosaicplasty for talar osteochondritis dissecans. Nakagawa et al.59 published trochlear results, Matsusue et al.58 reported successful tibial outcomes.

8.5.8

The role of mosaicplasty in treatment compared with other procedures

Microfracture Patients with small to moderate-sized focal lesions (1±5 cm2) having low activity level or moderate expectations may be treated with marrow stimulating techniques, such as microfracture. This technique is also recommended as a treatment for extended lesions in the presence of degenerative changes. The basis of this treatment option is to stimulate fibrocartilage ingrowth into the chondral defect to cover the underlying bone.80±82 This procedure can be done after failed debridement and lavage or other cartilage-specific surgical procedures.83 This technique may provide only fibrocartilage coverage of the defect, but extended use of the microfracture technique is based on the high frequency of degenerative problems. Osteochondral autograft transplantation Autogenous osteochondral mosaicplasty was developed to treat relatively small and medium-sized focal chondral and osteochondral defects of the weight-bearing

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surfaces of the femoral condyles and the patellofemoral joint. Encouraged by successful follow-up results in these joints, indications were extended to other articular surfaces including talar, tibial, caput and capitulum humeri, and recently, femoral head lesions.13,28,35,37,41,43,61,84 Donor-site availability and other technical considerations have limited the optimal extent of defect coverage to 1±4 cm2. Usually, both patellofemoral peripheries allow graft harvest for 3±4 cm2 defects. Defects up to 8±9 cm2 can be covered with mosaicplasty as a salvage procedure, but such extension of the indication can result in a higher rate of donorsite morbidity.33,62 Age, with decreased repair capacity, also seems to be a limiting factor. The recommended upper limit for this procedure is 50, which reflects the clinical experiences with single block osteochondral transfer.29,54,67,76 Theoretical contra-indications for mosaicplasty include infection, tumours and generalized or rheumatoid arthritis because of the biochemical alterations that may occur in the involved joint's milieu under these conditions. According to the regular indications of the mosaicplasty procedure, osteoarthritis is a contra-indication, but in certain motivated patient groups, mosaicplasty is considered as salvage intervention.57,61 Disadvantages include donor site morbidity, technical difficulty in matching the contour of the defective articular surface to the donor plug, and the risk of cartilage or bone collapse.83 Osteochondral allograft transplantation Fresh osteochondral allograft transplantation entails the implantation of a cadaveric osteochondral graft into the cartilage defect.8,11,12,85,86 A small arthrotomy is made to expose the cartilage defect. An osteochondral allograft plug is harvested to match the defect's size and contour and then press fit to achieve stability. This procedure is used for medium to large articular cartilage defects (3 cm2 up to an entire hemicondyle) in elder high-demand patients who may have associated bone loss (>6±8 mm).87±89 Most commonly used for defects involving the femoral condyles, but osteochondral allografts may also be used for patella, trochlea or tibial plateau lesions. The major advantage of osteochondral allografts is the ability to replace large osteochondral defects in a single-stage procedure. Additionally, the articular cartilage defect is replaced with articular cartilage rather than fibrocartilage.90 The disadvantages include graft availability, technical difficulty, cost, and possible disease transmission.83,91 Autologous chondrocyte implantation Based on previous results described by Chesterman and Smith,92 Green,93 Itay et al.94 and Grande et al.,95 autologous chondrocyte transplantation has been introduced into clinical practice by Peterson and co-workers.5 The procedure is a two-stage technique where during the first surgery, a small piece of cartilage is harvested arthroscopically from a non-weight-bearing portion of the patient's

Cartilage tissue repair: autologous osteochondral mosaicplasty

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knee. This cartilage is then sent to a laboratory for processing the cartilage cells. The interstitial matrix is enzymatically digested and chondrocytes are isolated and grown in tissue culture to allow them to multiply for several weeks. This results in millions of autologous cartilage cells that are suspended in a solution and shipped back to the surgeon for implantation. The second stage of the procedure involves a second surgery, an open arthrotomy to expose the lesion, which is debrided, so that the defect has intact edges of normal articular cartilage. The chondrocyte culture is then injected into the sealed pouch (1st generation) or transplanted in association with matrix scaffold (2nd generation, MACI, MACT). Third generation methods are in preclinical phase.96,97 The repair tissue that results from this procedure is hyaline-like in histology.98 Autologous chondrocyte implantation (ACI) is used for intermediate to highdemand patients who have failed arthroscopic debridement or microfracture. The technique is used for larger (2±10 cm2) symptomatic lesions involving both the femoral condyles and trochlea and the patella.5,98±102 One of the disadvantages is that an intact bone bed is required, making cartilage lesions associated with bone loss better treated by osteochondral grafting.83 Moreover, arcadic conformation of the chondrocytes existing in healthy hyaline cartilage is missing, therefore durability and mechanical stability might be problematic.45,103 Knutsen et al.104 reported on the results of a randomized controlled clinical trial comparing autologous chondrocyte implantation to microfracture in 80 patients with single chronic symptomatic cartilage defect on the femoral condyle in a stable knee without general osteoarthritis. At 2 and 5 years, there was no significant difference in the clinical and radiographic results between the two treatment groups and no correlation between the histological findings and the clinical outcome. Jakobsen et al.105 performed a literature search in which the primary aim of the investigation was to report the outcome after cartilage repair in the knee with use of different surgical cartilage repair techniques. Owing to generally low methodological quality found in the studies they concluded that caution is required when interpreting results after surgical cartilage treatment.

8.6

Conclusions

Chondral and osteochondral defects located at weight-bearing articular surfaces create a great challenge for the orthopaedic surgeon. The traditional treatment with chondral resurfacing techniques generates reparative fibrocartilage with poor biomechanical properties and the clinical outcome is sub-optimal. According to the literature, several new techniques have evolved in the last decade for the treatment of full thickness chondral defects located at weightbearing surfaces to create a hyaline or a hyaline-like gliding surface. Recommendations on which procedure to choose depends on the age, activity, failure of

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conservative therapy or established surgical interventions. Important details are presence of disabling pain, locking, degenerative arthritis, articular stability, location of damaged surface, size of the cartilage and subchondral bone defect in depth, length and area. However, resurfacing is only one element of treatment for full-thickness chondral and osteochondral defects. In every case, it is necessary to treat any accompanying joint abnormalities, otherwise early wear of transplanted cartilage or even further degeneration may develop. Accordingly, solutions for instabilities, malalignments and meniscal and ligamental tears must be incorporated in the operative and post-operative rehabilitation algorithm.3,35,36,41,56,57,106 Correction of these factors has proven useful in slowing or even preventing osteoarthritic changes.3,36,41,106 Nevertheless, a cooperative person is necessary for postoperative weight-bearing restrictions and a potential for completion of postoperative rehabilitation; moreover, their consent must be informed, with realistic expectations. To a great extent, an unfavourable outcome may arise from improper patient selection. Clarification of indication calls for better understanding of underlying pathophysiological and biomechanical alterations. According to our encouraging results in increasingly large series supported by similar findings in other centres, it seems that autologous osteochondral mosaicplasty may be a viable alternative treatment of localized full-thickness cartilage damage of weightbearing surfaces of the knee and other synovial joints.

8.7

References

1 Grana W. A healing of articular cartilage. Am J Knee Surg. 2000. 13: 29±32. 2 Levy AS, Lohnes J, Sculley S, LeCroy M, Garrett W. Chondral delamination of the knee in soccer players. Am. J. Sports Med. 1996; 24(5): 634±639. 3 Buckwalter JA, Mankin HJ. Articular cartilage restoration. Arthritis Rheumatism. 1998; 41: 1331±1342. 4 Blevins FT, Steadman JR, Rodrigo JJ, Silliman J. Treatment of articular cartilage defects in athletes: an analysis of functional outcome and lesion appearance. Orthopedics. 1998; 21(7): 761±767. 5 Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med. 1994; 331: 889±895. 6 Bruns J, Kersten P, Lierse W, Silbermann M. Autologous rib perichondrial grafts in experimentally induced osteochondral lesions in the sheep-knee joint: morphological results. Virchows Arch A Pathol Anat Histopathol. 1992; 421: 1±8. 7 Campanacci M, Cervellati C, Donati U. Autogenous patella as replacement for a resected femoral or tibial condyle. A report of 19 cases. J Bone Joint Surg Br. 1985; 67: 557±563. 8 Convery FR, Akeson WH, Keown GH. The repair of large osteochondral defects. An experimental study in horses. Clin Orthop. 1972; 82: 253±262. 9 Coutts RD, Woo SL, Amiel D, von Schroeder HP, Kwan MK. Rib perichondrial

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10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

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autografts in full-thickness articular cartilage defects in rabbits. Clin Orthop. 1992; 275: 263±273. Fabbricciani C, Schiavone Panni A, Delcogliano A, et al. Osteochondral autograft in the treatment of osteochondritis dissecans of the knee. In: American Orthopedic Society for Sports Medicine Annual Meeting. Orlando, FL; 1994: 78±79. Garrett JC. Treatment of osteochondral defects of the distal femur with fresh osteochondral allografts: a preliminary report. Arthroscopy. 1986; 2: 222±226. Gross AE, Silverstein EA, Falk J, Falk R, Langer F. The allotransplantation of partial joints in the treatment of osteoarthritis of the knee. Clin Orthop. 1975; 108: 7±14. Homminga GN, Bulstra SK, Bouwmeester PS, van der Linden AJ. Perichondral grafting for cartilage lesions of the knee. J Bone Joint Surg Br. 1990; 72: 1003± 1007. Mahomed MN, Beaver RJ, Gross AE. The long-term success of fresh, small fragment osteochondral allografts used for intraarticular post-traumatic defects in the knee joint. Orthopedics. 1992; 15: 1191±1199. Messner K, Gillquist J. Synthetic implants for the repair of osteochondral defects of the medial femoral condyle: a biomechanical and histological evaluation in the rabbit knee. Biomaterials. 1993; 14: 513±521. Muckle DS, Minns RJ. Biological response to woven carbon fibre pads in the knee. A clinical and experimental study. J Bone Joint Surg Br. 1990; 72: 60±62. Outerbridge HK, Outerbridge AR, Outerbridge RE. The use of a lateral patellar autologous graft for the repair of a large osteochondral defect in the knee. J Bone Joint Surg Am. 1995; 77: 65±72. Ritsila VA, Santavirta S, Alhopuro S, Poussa M, Jaroma H, Rubak JM, et al. Periosteal and perichondral grafting in reconstructive surgery. Clin Orthop. 1994; 302: 259±265. Stone KR, Walgenblach. A surgical technique for articular cartilage transplantation to full-thickness cartilage defects in the knee joint. Oper Tech Orthop. 1997; 7: 305±311. Yamashita F, Sakakida K, Suzu F, Takai S. The transplantation of an auto-generic osteochondral fragment for osteochondritis dissecans of the knee. Clin Orthop. 1985; 201: 43±50. Duchow J, Hess T, Kohn D. Primary stability of press fit-implanted osteochondral grafts: influence of graft size, repeated insertion and harvesting technique. Am J Sports Med. 2000; 28: 24±27. Hangody L, Kish G, KaÂrpaÂti Z, Eberhart R. Osteochondral plugs ± autogenous osteochondral mosaicplasty for the treatment of focal chondral and osteochondral articular defects. Oper Tech Orthop. 1997; 7(4): 312±322. Hangody L, Kish G, KaÂrpaÂti Z. et al. Treatment of osteochondritis dissecans of talus: the use of the mosaicplasty technique. Foot Ankle Int. 1997; 18(10): 628±634. Hangody L, Kish G, KaÂrpaÂti Z. et al. Arthroscopic autogenous osteochondral mosaicplasty for the treatment of femoral condylar articular defects. Knee Surgy Sports Traumatol Arthrosc. 1997; 5: 262±267. Hangody L, Miniaci A, Kish G. MosaicPlastyTM Osteochondral Grafting ± technique Guide. Smith and Nephew Inc. 1997. Ripoli PL, de Prado M, Ruiz D, Salmeron J. Transplantes osteocondrales en mosaico: estudio de los resultados mediante RMN y segunda artroscopia. Cuadernos Artroscopia. 2000; 6: 11±16. In Spanish. Hangody, L. Surgical treatment of knee chondropathies I±II [thesis]. Debrecen

222

Regenerative medicine and biomaterials in connective tissue repair

Medical School, Debrecen, Hungary. 1994. In Hungarian. 28 Hangody, L, FuÈles, P. Autologous osteochondral mosaicplasty for the treatment of full thickness defects of weight bearing joints ± 10 years experimental and clinical experiences. J Bone Joint Surg. 2003; 85-A Supplement (2): 25±32. 29 Hangody L, Karpati Z. New possibilities in the management of severe circumscribed cartilage damage in the knee. Magy Traumatol Ortop Kezseb Plasztikai seb. 1994; 37: 237±243. 30 Hangody L, KaÂrpaÂti Z, ToÂth J. et al. Autogenous osteochondral grafting in the knees of German Shepherd dogs: radiographic and histological analysis. Hungarian Rev Sportsmedicine. 1994; 35: 177±183. 31 Szerb I, Hangody L, KaÂrpaÂti Z. The relationship between the stiffness and the histological degeneration of articular cartilage. Rev Osteologie. 2003; 9: 213±220. 32 Hangody L, Kish G, KaÂrpaÂti Z. Arthroscopic autogenous osteochondral mosaicplasty ± a multicentric, comparative, prospective study. Index Traumat Sport. 1998; 5: 3±9. 33 Hangody L, Feczko P, Bartha L, et al. Mosaicplasty for the treatment of articular defects of the knee and ankle. Clin Orthop. 2001; 391(Suppl): 328±336. 34 Hangody L, Kish G, KaÂrpaÂti Z. et al. Autogenous osteochondral graft technique for replacing knee cartilage defects in dogs. Orthop Int Ed. 1997; 5: 175±181. 35 Hangody L, Duska Zs, KaÂrpaÂti Z. Autologous osteochondral mosaicplasty. Tech Knee Surg. 2002; 1(1): 1±10. 36 Gudas R, Kalesinskas RJ, Kimtys V, et al. A prospective randomized clinical study of mosaic osteochondral autologous transplantation versus microfracture for the treatment of osteochondral defects in the knee joint in young athletes. Arthroscopy. 2005; 21: 1066±1075. 37 Hangody L, Kish G, KaÂrpaÂti Z, et al. Mosaicplasty for the treatment of articular cartilage defects: application in clinical practice. Orthopedics. 1998; 21: 751±756. 38 Hangody L, Kish G. Surgical treatment of osteochondritis dissecans of the talus. In: Duparc J, European Textbook on Surgical Techniques in Orthopaedics and Traumatology. Editions Scientifiques et Medicales Elsevier; 2000. 39 Bodo G, Hangody L, Szabo Zs, et al. Arthroscopic autologous osteochondral mosaicplasty for the treatment of subchondral cystic lesion in the medial femoral condyle in a horse. Acta Vet Hung. 2000; 48: 343±354. 40 Diduch RD, Chhabra A, Blessey P, Miller MD. Osteochondral autograft plug transfer: achieving perpendicularity. J Knee Surg. 2003; 16: 17±20. 41 KordaÂs G, Szabo JS, Hangody L. The effect of drill-hole length on the primary stability of osteochondral grafts in mosaicplasty. Orthopedics. 2005; 28: 401±404. 42 Bodo G, Kaposi AD, Hangody L, et al. The surgical technique and the age of the horse both influence the outcome of mosaicplasty in a cadaver equine stifle model. Acta Vet Hung. 2001; 49: 111±116. 43 Berlet GC, Mascia A, Miniaci A. Treatment of unstable osteochondritis dissecans lesions of the knee using autogenous osteochondral grafts (mosaicplasty). Arthroscopy. 1999; 15: 312±316. 44 Christel P, Versier G, Landreau P, Djian P. Les greffes osteo-chondrales selon la technique de la mosaicplasty. Maitrise Orthop. 1998; 76: 1±13. In French. 45 Horas U. et al. Autologous chondrocyte implantation and osteochondral cylinder transplantation in cartilage repair of the knee joint. J Bone Joint Surg. 2003; 85-A: 185±192. 46 Hurtig M, Pearce S, Warren S, Kalra M, Miniaci A. Arthroscopic mosaic arthroplasty in the equine third carpal bone. Vet Surg. 2001; 30: 228±239.

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47 Imhoff AB, Ottl GM, Burkart A, Traub S. Autologous osteochondral transplantation on various joints. Orthopade. 1999; 28: 33±44. In German. 48 Imhoff AB, Ottl GM. Arthroscopic and open techniques for transplantation of osteochondral autografts and allografts in various joints. Surg Technol Int. 1999; 8: 249±252. 49 Kish G, MoÂdis L, Hangody L. Osteochondral mosaicplasty for the treatment of focal chondral and osteochondral lesions of the knee and talus in the athlete. Clin Sports Med. 1999; 18: 45±61. 50 Lane JG, Tontz WL Ball ST, et al. A morphologic, biochemical and biomechanical assessment of short-term effects of osteochondral autograft plug transfer in an animal model. Arthroscopy. 2001; 17(8): 856±863. 51 Marcacci M, Kon E, Zaffagnini S, Visani A. Use of autologous grafts for reconstruction of osteochondral defects of the knee. Orthopedics. 1999; 22(6): 595± 600. 52 Marcacci M, Kon E, Zaffagnini S, Iacono F, Neri MP, Vascellari A, Visani A, Russo A. Multiple osteochondral arthroscopic grafting (mosaicplasty) for cartilage defects of the knee: prospective study results at 2-year follow-up. Arthroscopy. 2005; 21: 462±470. 53 Maynou C, Mestdagh H, Beltrand E, Petroff E, Dubois H. Long-term results of proximal osteocartilaginous autografts in extensive cartilagenous destruction of the knee. Apropos of 5 cases. Acta Orthop Belg. 1998; 64: 193±200. In French. 54 Solheim E. Mosaicplasty in articular cartilage injuries of the knee. Tidsskr Nor Laegeforen. 1999; 119: 4022±4025. In Norwegian. È ttl G. Die Technik der osteochondralen autologen 55 Traub S, Imhoff AB, O Knorpeltransplantation (OATS) zum Ersatz chondraler oder osteochondraler Defekte. Osteologie. 2000; 9: 46±55. In German. 56 Hangody L, RaÂthonyi G, Duska Zs, et al. Autologous osteochondral mosaicplasty ± surgical technique. J Bone Joint Surg. 2004; 86-A(Suppl. I): 65±72. 57 Hangody L, RaÂthonyi G. Management of osteochondral defects ± mosaicplasty technique. In: FH Fu, BD Browner, Management of Osteoarthritis of the Knee: An International Consensus. American Academy of Orthopaedic Surgeons Monograph Series No. 25. 2004: 41±48. 58 Matsusue Y, Kotake T, Nakagawa Y, et al. Arthroscopic osteochondral autograft transplantation for chondral lesion of the tibial plateau of the knee. Arthroscopy. 2001; 17(6): 653±659. 59 Nakagawa Y, Matsusue Y, Suzuki T, Kuroki H, Nakamura T. Osteochondral grafting for cartilage defects in the patellar grooves of bilateral knee joints. Arthroscopy. 2004; 20: 32±38. 60 Feczko P, Hangody L, Varga J, et al. Experimental results of donor site filling for autologous osteochondral mosaicplasty. Arthroscopy. 2003; 19(7): 755±761. 61 Hangody L, RaÂthonyi GK. Mosaicplasty in active sportsmen. SportorthopaÈdie Sporttraumatologie. 2004; 20: 159±164. 62 Hangody L, Duska Zs, KaÂrpaÂti Z. Osteochondral plug transplantation. In: D Jackson, Mastertechniques in Orthopaedics; The Knee, 2nd edn. LippincottWilliams-Wilkins; 2003: 337±352. 63 Bartha L, Vajda, A, Duska Zs, et al. Autologous osteochondral mosaicplasty grafting. J Orthop Sports Phys Therapy. 2006; 36(10): 739±750. 64 Hangody L. Treatment of symptomatic deep cartilage defects of the patella and trochlea with and without patellofemoral malalignment. Basic science and treatment. In: V Sanchis-Alfonso, Anterior Knee Pain and Femoropatellar

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65 66 67 68 69 70 71

72 73 74 75 76 77 78 79 80 81 82 83 84

Regenerative medicine and biomaterials in connective tissue repair Instability in the Active Young ± `The Black Hole of Orthopaedics', Editorial Medica Panamericana; 2003: 181±205. Hangody L, Kish G, Karpati Z, Udvarhelyi I, Szigeti I, Bely M. Mosaicplasty for the treatment of articular cartilage defects: application in clinical practice. Orthopedics. 1998; 21: 751±756. Hangody L, Kish G, MoÂdis L, et al. Mosaicplasty for the treatment of osteochondritis dissecans of the talus: two to seven year results in 36 patients. Foot Ankle Int. 2001; 22: 552±558. Szerb I, KaÂrpaÂti Z, Hangody L. In vivo arthroscopic cartilage stiffness measurement in the knee. Arthroscopy. 2006; 22: 682±683. Simonian PT, Sussmann PS, Wickiewicz TL, Paletta GA, Warren RF. Contact pressures at osteochondral donor sites in the knee. Am J Sports Med. 1998; 26: 491± 494. Hangody, L. The mosaicplasty technique for osteochondral lesions of the talus. Foot Ankle Clin N Am. 2003; 8: 259±273. Chow JCY, Hantes ME, Houle JB, Zalavras CG. Arthroscopic autogenous osteochondral transplantation for treating knee cartilage defects: a 2- to 5-year follow-up study. Arthroscopy. 2004; 20: 681±690. Bentley G, Briant L C, Carrington,W J, Akmal M, Goldberg A, Williams A M, Skinner J A, Pringle J. A prospective, randomised comparison of autologous chondrocyte implantation versus mosaicplasty for osteochondral defects in the knee. J Bone Joint Surg. 2003; 85-B: 223±230. Kish G, Hangody L. Correspondence ± a prospective, randomised comparison of autologous chondrocyte implantation versus mosaicplasty for osteochondral defects in the knee. J Bone Joint Surg. 2004; 86-B: 619±620. Recht M, White LM, Winalski CS et al. MR imaging of cartilage repair procedures. Skeletal Radiol. 2003; 32: 185±200. Barber FA, Chow JCY. Arthroscopic osteochondral transplantation: histologic results. Arthroscopy. 2001; 17(8): 832±835. Leo BM, Turner MA, Diduch DR. Split-line pattern and histologic analysis of a human osteochondral plug graft. Arthroscopy. 2004; 20 Suppl 2: 39±45. Pap K, Krompecher I. Arthroplasty of the knee ± experimental and clinical experiences. J Bone Joint Surg. 1961; 43-A: 523±537. Makino T, Fujioka H, Kurosaka M et al. Histologic analysis of the implanted cartilage in an exact-fit osteochondral transplantation model. Arthroscopy. 2001; 17: 747±751. Makino T, Fujioka H, Terukina M, Yoshija S, Matsui N, Kurosaka M. The effect of graft sizing on osteochondral transplantation. Arthroscopy. 2004; 20: 837±840. Hoser C, Bichler O, Bale R, et al. A computer assisted surgical technique for retrograde autologous osteochondral grafting in talar osteochondritis dissecans (OCD): a cadaveric study. Knee Surg Sports Traumatol Arthrosc. 2004; 12: 65±71. Steadman JR, Stereet WI. The surgical treatment of knee injuries in skiers. Med Sci Sports Exerc. 1995; 27(3): 328±335. Freedman KB, Fox JA, Cole BJ. Knee cartilage: diagnosis and decision making. In: MD Miller, BJ Cole (eds), Textbook of Arthroscopy. Saunders; 2004: 555±567. Gill TJ, MacGillivray JD. The technique of microfracture for the treatment of articular cartilage defects in the knee. Oper Tech Orthop. 2001; 11: 105±107. Detterline AJ, Goldberg S, Bach BR, Cole BJ Jr. Treatment options for articular cartilage defects of the knee. Orthop Nursing. 2005; 24(5): 361±366. Miller RH. Osteochondral tissue transfer. Am J Knee Surg. 2000; 13(1): 51±62.

Cartilage tissue repair: autologous osteochondral mosaicplasty

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85 Mankin HJ, Doppelt SH, Tomford WW. Clinical experience with allograft implantation: the first 10 years. Clin Orthop. 1983; 174: 69±78. 86 Friedlaender GE, Horowitz MC. Immune responses to osteochondral allografts: nature and significance. Orthopedics. 1992; 15: 1171±1176. 87 Aubin PP, Cheah HK, Davis AM, Gross AE. Long-term follow-up of fresh femoral osteochondral allografts for posttraumatic knee defects. Clin Orthop. 2001; 39(suppl): 318±327. 88 Bugbee WD. Fresh osteochondral allografting. Oper Tech Sports Medicine. 2000; 8: 158±162. 89 Garrett JC. Fresh osteochondral allografts for treatment of articular defects in osteochondritis dissecans of the lateral femoral condyle in adults. Clin Orthop. 1994; 303: 33±37. 90 Gross A. Fresh osteochondral allografts for posttraumatic knee defects: Surgical technique. Oper Tech Orthop. 1997; 7: 334±339. 91 Tomford WW, Springfield DS, Mankin HJ. Fresh and frozen articular cartilage allografts. Orthopedics. 1992; 15: 1183±1192. 92 Chesterman PJ, Smith AU. Homotransplantation of articular cartilage and isolated chondrocytes. An experimental study in rabbits. J Bone Joint Surg. 1968; 50B: 184±197. 93 Green WT Jr. Articular cartilage repair: behavior of rabbit chondrocytes during tissue culture a subsequent allografting. Clin Orthop. 1977; 124: 237±250. 94 Itay S, Abramovici A, Nevo Z. Use of cultured embryonal chick epiphyseal chondrocytes as grafts for defects in chick articular cartilage. Clin Orthop. 1987; 220: 284±303. 95 Grande DA, Pitman MI, Peterson L et al. The repair of experimentally produced defects in rabbit articular cartilage by autologous chondrocyte transplantation. J Orthop Res. 1989; 7: 208±218. 96 Woodfield T. 'Cartilage tissue engineering: instructing cell-based tissue repair through scaffold design'. Paper published by IsoTis. 2004. 97 Atik OS, Uslu MM, Eksioglu F. Osteochondral multiple autograft transfer (OMAT) for the treatment of cartilage defects in the knee. Bull Hospital Joint Diseases. 2005; 63: 37±40. 98 Peterson L, Brittberg M, Kiviranta I, Akerlund EL, Lindahl A. Autologous chondrocyte transplantation. Biomechanics and long-term durability. Am J Sports Med. 2002; 30: 2±12. 99 Chu CR, Convery FR, Akeson WM, Meyeis M, Amiel D. Articular cartilage transplantation. Clinical results in the knee. Clin Orthop Rel Res. 1999; 360: 159± 168. 100 Gillogly SD, Voight M, Blackburne T. Treatment of articular cartilage defects of the knee with autologous chondrocyte implantation. J Orthop Sports Phys Therapy. 1998; 28: 241±251. 101 Micheli LJ, Browne JE, Erggelet C, Fu F, Mandelbaum B, Moseley JB, et al. Autologous chondrocyte implantation of the knee: multicenter experience and minimum 3-year follow-up. Clin J Sports Med. 2001; 11: 223±228. 102 Minas T. Autologous chondrocyte implantation for focal chondral defects of the knee. Clin Orthop. 2001; 39(suppl): 349±361. 103 Sharma A, Wood LD, Richardson JB, Roberts S, Kuiper NJ. Glycosaminoglycan profiles of repair tissue formed following autologous chondrocyte implantation differ from control cartilage. Arthritis Res Ther. 2007; 9(4): R79. 104 Knutsen G, Drogset JO, Engebretsen L, Grontvedt T, Isaksen V, Ludvigsen TC,

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Roberts S, Solheim E, Strand T, Johansen O. A randomized trial comparing autologous chondrocyte implantation with microfracture. Findings at five years. J Bone Joint Surg Am. 2007; 89(10): 2105±2112. 105 Jakobsen RB, Engebretsen L, Slauterbeck JR. An analysis of the quality of cartilage repair studies. J Bone Joint Surg. 2005; 87A: 2232±2239. 106 O'Driscoll SW, Keeley FW, Salter RB. Durability of regenerated articular cartilage produced by free autogenous periosteal grafts in major full-thickness defects in joint surfaces under the influence of continuous passive motion. A follow-up report at one year. J Bone Joint Surg Am. 1988; 70: 595±606.

9

Cartilage tissue repair: autologous chondrocyte implantation M . B R I T T B E R G , University of Gothenburg, Sweden

Abstract: Articular cartilage lesions have become an important target for tissue engineering due to the poor intrinsic repair capacity of articular cartilage. The first example of clinical cartilage tissue engineering was performed in 1987 when a cartilage lesion was treated by implanting in vitro expanded autologous chondrocytes into the defect covered by periosteum, a technique now known as autologous chondrocyte implantation (ACI). Today, many modifications of the technique exist, from the first generation to now second and third generations of chondrocyte implantation. This chapter describes the basic theories behind the use of chondrocytes for cartilage repair and presents what is known about the clinical use of chondrocyte implantation. Keywords: cartilage repair, cartilage tissue engineering, cartilage regeneration, chondrocytes, autologous chondrocyte implantation, autologous chondrocyte transplantation.

9.1

Introduction

Hyaline cartilage provides the diarthrodial joint with a low friction surface, resilience and compressive stiffness and this unique tissue is under normal conditions wear-resistant. Loss of cartilage function may lead to a painful joint with a decreased mobility. There are many causes to loss of cartilage; either due to trauma or to diseases such as primary osteoarthritis (O'Connor and Brandt, 1993). As cartilage has a limited capacity for self-repair, articular cartilage lesions have become an important target for cell tissue engineering. A first example of clinical cartilage tissue engineering was performed in 1987, when a knee with an articular cartilage defect on the femoral condyle was treated by implanting the patient's own chondrocytes that had been expanded in vitro and then implanted into the defect in combination with a covering mechanical membrane ± the periosteum (Brittberg et al., 1994). This chapter describes the basic theories behind the use of chondrocytes for cartilage repair and presents what is known about the clinical use of chondrocyte implantation including a discussion of future research.

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Chondrogeneic cell implantation

In order to learn more about how cartilage functions and how cartilage develops, studies on the skeletal development during embryonic life could be helpful. During the initial 12-week period of embryonic life the formation of the skeleton begins. The process starts with a mesenchymal cell aggregation which is fundamental for the cartilage differentiation in the developing limb with the formation of a prechondrogeneic cell condensation, a blastema. In in vitro studies it has been shown that mesenchymal cell aggregates must achieve a threshold size before chondrogenesis can proceed (Brittberg 1996). To start chondrogenesis in a repair of a cartilaginous tissue you will subsequently need a large number of chondrogeneic cells and implant those cells in high densities. In an injured mesenchymal tissue there is a cell density-dependent differentiation in progress. The initial number of cells that can take part in the repair events is of crucial importance (Caplan et al., 1997; Goldberg and Caplan, 1994). But the repair of an injured mesenchymal tissue is dependent also on the local availability of cells with the ability to regenerate a new tissue. The goal for the orthopaedic surgeon is to try to deliver as many of the acquired cell types as possible into the cartilage lesion in order to achieve some sort of repair. All current cartilage repair methods depend on an introduction of chondrogeneic cells into the defect area. Which cells should be used and from what donor tissue they are to be harvested can be discussed but, in general, there are more chondrogenic cells in younger patients.

9.3

Articular or other types of chondrocytes, allogeneic or autologous chondrocytes?

Exogenous sources of chondrogeneic cells have been studied by many. An exogenous source is attractive as those cells could be cheaper and more easily attainable `off the shelf'. Importantly, Nevo et al. (1998) studied mesenchymal cells as chondrogeneic progenitors and found problems such as a delayed pace of endochondral ossification in the deep zones of the subchondral region of the study defects, or ossification above the tidemark, within the superficial cartilaginous articular regions. However, the articular chondrocytes are responsible for the unique features of articular cartilage; therefore it seems rational to use true committed chondrocytes to repair a cartilaginous defect (Brittberg 1996). Pure chondrocytes, epiphyseal or mature, allergenic or autologous as well as other types of mesenchymal cells have been used. In 1965 Smith perfected the isolation of chondrocytes, and chondrocytes were thereafter able to be grown using standard culture methods. Chestermann and Smith (1968) isolated chondrocytes from rabbit cartilage and those cells were then transplanted into cartilage defects in the humerus of adult sister rabbits. The defects healed but no hyaline cartilage was found. Bentley and

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Greer (1971) transplanted isolated articular cartilage and epiphyseal chondrocytes from young rabbits to cartilage defects on adult rabbits of the same inbreed. A nice repair with hyaline-like neocartilage was described in more than 50% of the defects. Green (1977) reported a significant better repair after heterologous chondrocyte transplantation of chondral lesions of the rabbit knee. Autoradiography confirmed that the implanted isolated chondrocytes were responsible for the repair and three generations of transplanted chondrocytes could be identified by the decrease in intensity in nuclear labelling. Bentley et al. (1979) transplanted isolated epiphyseal chondrocytes as allografts into rabbit knee tibial drill holes. Cells were pipetted into the defects with a success rate of 47% of the defects repaired. Aston and Bentley (1986) compared allografts of intact cartilage, isolated chondrocytes and cultured chondrocytes from the epiphyseal growthplate and articular surface from immature rabbits inserted into full thickness defects in mature rabbits. Both intact articular cartilage as well as intact epiphyseal growth-plates induced significantly better repair than controls. Cultured chondrocytes also produced a significantly better repair than when the defects were left ungrafted in arthritic joints. Yoshihashi (1983) studied isolated articular chondrocytes in comparison with normal articular cartilage and used chondrocytes that were released by enzymatic digestion from slices of articular cartilage taken from 8-week-old white rabbits. The isolated cells were inoculated in a large number into a 0.28 cm2 stainless cylinder on a Millipore filter. After 12 hours these chondrocytes were layered by gravity onto the Millipore filter and were cultured in the same medium during 7 days. Subsequently the cell aggregate was transferred to an organ culture system and was fed every other day. Aggregates of cells were sampled at 1, 2, 4, 8 and 12 weeks in culture for morphological and biochemical studies. The results obtained were as follows: after 1 week in culture, deposition of metachromatic matrix was observed under a light microscope only at the periphery of the aggregate of cells. Matrix formation in the whole aggregate occurred after 2 weeks in culture. The tissue reformed in this culture consisted of metachromatic hyaline cartilage-like matrix and chondrocytes within lacunae but for cells at the surface arranged in a tangential flattened layer. The collagen in this tissue was of type II mixed with a very small amount of type I. The tissue reconstituted in vitro by freshly isolated chondrocytes had characteristics of hyaline cartilage except over the surface. Compared with normal articular cartilage, the cells in this tissue were distributed more randomly, the intercellular hyaline matrix was poor under a light microscope, and collagen fibrils in the matrix observed under an electron microscope were much thinner than those of normal articular cartilage. This method provides a tissue culture model of cartilage organization. Wakitani and associates (1994) implanted osteochondral progenitor cells from the periosteum and bone marrow cells±mesenchymal stem cells (MSC) into the femoral condyle cartilage defects of rabbit knees. Those cells were

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preferred to chondrocytes because they were said to be capable of a broader range of chondrogenic expression and they may recapitulate the embryonic lineage transitions originally involved in the formation of joint tissue. No difference between the bone marrow cells and the periosteum-derived cells was seen and the best scores were seen at 4 weeks. At 12 weeks the subchondral bone plate was restituted. There was a gap between the host cartilage and the neocartilage but the underlying bone was almost completely united with that of the host. Goldberg and Caplan (1994) compared implantation of mature chondrocytes, so-called committed, with MSC for the repair of full thickness defects in the femoral condyles of adult rabbits. Both cell types repaired the defect with a hyaline-like neocartilage. However, the MSC repair had a more hyaline-like morphology and cartilage zonal characteristics than the repair from the committed chondrocytes. They hypothesised that the open subchondral bone releases host-derived bioactive factors such as different growth factors and cytokines that could influence the biologic properties of the MSC but not of the already differentiated chondrocytes. Robinson et al. (1989) implanted chondrocytes derived from chick embryos into defects of articular surfaces of young and old adult chickens. The embryonic chondrocytes underwent an accelerated ageing process in the old chickens, implying that the maturation stages within the implant were shorter in the old animals, leading to a shorter healing time. The above reported cell transplantations have been allografts. The chondrocyte have transplantation antigens and these cells; can theoretically participate in immunological reactions. The cartilage matrix acts as a protective barrier (Elves, 1974). Kawabe and Yoshinao (1991) studied the immune responses to reparative tissue formed by allergenic growth plate chondrocyte implants. The neocartilage yielded by implantation of these cells into cartilage defects of adult rabbits looked very good in the beginning but began to degenerate 2±3 weeks after implantation partially because of humoral immune response but also because of cell-mediated cytotoxicity. Host lymphocytes were seen around the allograft at 2±12 weeks. Noguchi and associates (1994) compared allografted chondrocytes with autografts that were cultured in a collagen gel and transplanted into osteochondral defects in knee joints of inbred rats. At 12 weeks 100% of the autografts had healed successfully while only 50% of the allografts were healed. At 26 and 52 weeks, all defects except one had healed and there was no significant difference in the success rate between the groups.

9.4

Autologous chondrocytes

Peterson et al. in 1984 and Grande et al. in 1989 presented their results in rabbit models with autologous chondrocytes grown in vitro on the healing rate of

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chondral defects not penetrating the subchondral bone plate. In defects that had received transplants, a significant amount of cartilage was reconstituted (82%) compared with ungrafted controls (18%) 8 weeks post-implantation. To determine whether any of the reconstituted cartilage resulted from the chondrocyte graft an experiment was conducted involving grafts with chondrocytes that had been labelled prior to grafting with a nuclear tracer. Autoradiography on reconstituted cartilage showed that there was labelled cells incorporated into the repair matrix. Brittberg et al. (1996) compared periosteal grafts with and without chondrocytes in rabbit patellar chondral defects. One year following implantation, periosteum and chondrocytes demonstrated an average repair area of 87% compared with the 30% repair area observed in the periosteum-alone graft. A histological grading system also demonstrated a statistically significant difference between the two graft types, with the tissue produced by chondrocytes in combination with a periosteal flap reaching much higher histological scoring compared with defects treated with only a periosteal flap. Rahfoth et al. in 1998 transplanted allograft chondrocytes embedded in agarose gel into rabbit articular cartilage defects. Defect repair was followed for 6±18 months. At 18 months in 47% of the grafts it was noted that a morphologically stable hyaline-like cartilage was developed and such an extent of recovery was never observed in controls. To monitor the persistence and the phenotype of the injected chondrocytes in the repair tissue Dell'Accio et al. in 2002 used a fluorescent labelling protocol for articular chondrocytes, which allowed cell tracking in vivo using the fluorescent dye PKH26. Their data indicate that the implanted cells can persist for at least 14 weeks in the defects, can participate in the integration with the surrounding tissues, and become structural part of the repair tissue rich in collagen type II and sulphated proteoglycans. A similar experiment was done with chondrocytes from rabbits, sheep, cattle and humans which were isolated and infected with murine leukaemia virusderived retroviruses carrying the green fluorescent protein (GFP) gene (Hirschmann et al., 2002). For in vivo follow-up of GFP expression the authors used marked allergenic chondrocyte populations grown on scaffold material and implanted them into full-thickness defects in knee joints of rabbits and observed stable GFP expression in the transplants during a 4-week time course. In a few reports at 18 months a tendency of impairment of tissue quality has been reported as well as poor integration to surrounding native cartilage. Using a dog model to study autologous chondrocyte repair on chondral repair, Breinan et al. (1994) found that at 12±18 months post-surgery there were no differences in the repair with autologous chondrocytes and periosteum versus periosteum alone but a substantial number of animals had a break through to the subchondral bone. In a later paper from the same authors (Breinan et al., 2001) the authors found in the canine model that hyaline cartilage always was found superficial to intact calcified cartilage, whereas damaged calcified cartilage was

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covered only by fibrous tissue or fibrocartilage. The authors speculate about the importance of having an intact bone plate for the development of a hyaline repair tissue. Recently using the previously established canine model for repair of articular cartilage defects, Lee et al. in 2003 evaluated the 15-week healing of chondral defects (e.g., to the tidemark) implanted with an autologous articular chondrocyte-seeded type II collagen scaffold that had been cultured in vitro for 4 weeks before implantation. The amount and composition of the reparative tissue were compared to results from the Breinan 2001 study using the same animal model in which the following groups were analysed: defects implanted with autologous chondrocyte-seeded collagen scaffolds that had been cultured in vitro for approximately 12 h before implantation, defects implanted with autologous chondrocytes alone, and untreated defects. Chondrocytes, isolated from articular cartilage harvested from the left knee joint of six adult canines, were expanded in number in monolayer for 3 weeks, seeded into porous type II collagen scaffolds, cultured for another 4 weeks in vitro and implanted into chondral defects in the trochlear groove of the right knee joints. The percentages of specific tissue types filling the defects were evaluated histomorphometrically and certain mechanical properties of the repair tissue were determined. The reparative tissue filled 88  6% of the cross-sectional area of the original defect, with hyaline cartilage accounting for 42  10% (range 7±67%) of defect area. These values were greater than those reported previously for untreated defects and defects implanted with a type II collagen scaffold seeded with autologous chondrocytes within 12 h before implantation. Most interesting, was the decreased amount of fibrous tissue filling the defects in the current study, 5  5% as compared with previous treatments. Despite this improvement, indentation testing of the repair tissue formed revealed that the compressive stiffness of the repair tissue was well below (20-fold lower stiffness) that of native articular cartilage. Long follow-ups with a reliable and maybe larger animal are still lacking. However, it appears that the above different animal models with allogenous or autologous chondrocytes used in the treatment of cartilage defects provide benefit over cartilage defects treated without cells or not treated at all at least in reports to little more than 1 year's follow-up. Preliminary, short-term proof of concept has subsequently been performed in rabbits and in goats (Brittberg et al., 1996; Dell'Accio et al., 2003). Recently Yan and Yu (2007) presented a study on full-thickness rabbit cartilage defects treated with chondrocytes, mesenchymal stromal cells, fibroblasts and umbilical cord cells. Repair with chondrocyte or MSC transplantation induced a hyaline-like cartilage repair tissue, and repair was significantly better than in tissues treated with fibroblasts and umbilical cord stem cells, as well as in the control group with no treatment at all.

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9.5

233

Human clinical use and studies with autologous chondrocyte implantation

The animal results stimulated a pilot study with autologous chondrocyte implantation in humans with the first operation performed in 1987 when a knee with an articular cartilage defect on the femoral condyle was treated by implanting the patient's own chondrocytes that had been expanded in vitro and then implanted into the defect in combination with a covering mechanical membrane ± the periosteum. This, the first generation of chondrocyte transplantations, was initially termed autologous chondrocyte transplantation (ACT). Today, the technique is called either ACT or, what I prefer, autologous chondrocyte implantation (ACI) and there exist many modifications of the technique, from the first generation to now second and third generations of chondrocyte implantation.

9.5.1

Indications for ACI

The gold standard for determining if a patient is a suitable candidate for ACI has to be done by an arthroscopic evaluation as magnetic resonance imaging (MRI) still does not have enough sensitivity or specificity for a complete cartilage injury evaluation. Location, depth and size of the lesion, the quality of the surrounding cartilage, the degree of undermining cartilage and status of opposing chondral surface can all be evaluated by the arthroscopic procedure. The ideal patient to be treated with autologous chondrocyte implantation is a symptomatic patient with a full thickness chondral or osteochondral defect surrounded by healthy, normal cartilage in an otherwise healthy knee. However, the ideal lesion is more the exception than the rule, as many lesions occur in knees with concomitant pathology and different degrees of containment.

9.5.2

ACI surgical technique (knee joint)

The operative technique for both open procedure with periosteum or collagen membrane as well as new generation techniques with transarthroscopic scaffolds are briefly presented.

9.5.3

Cartilage harvest

The steps include an initial arthroscopically harvested cartilage biopsy from which chondrocytes can be isolated by enzymatic digestion and are expanded in an in vitro culture to several times the initial number of cells. In a clinical setting today, the aim is to transplant a density of 30  106 cells/ml or at least 2  106 cells/cm2. Articular cartilage is composed of phenotypically different zones. The zones have different mechanical properties and play specific roles within functional

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cartilage tissue. In young animals there are only two zones, the superficial and the growth zone (Darling et al., 2004). Surface abrasion can be used to harvest cells from articular cartilage to produce samples in a simple, reproducible process. Using this harvesting technique, the authors could differentiate between the superficial zones from the underlying growth zone. They found that the superficial cells made up approximately 4% of the total cells obtained. Superficial and growth zone chondrocytes from articular cartilage were analysed using real-time reverse transcriptase polymerase chain reaction (RT-PCR). Expressed superficial zone protein was 3-fold greater in the superficial zone population than in the growth zone population (p < 0:01). Additionally, type II collagen was expressed 8-fold more abundantly in the growth zone than in the superficial zone (p < 0:005). There was no difference in aggrecan expression between the two zones. Regional variations among the femoral groove and medial and lateral condyles were also examined. No significant variations in superficial zonal proteins (SZP), type II collagen or aggrecan were found, which makes the pooling of zonal cells from different regions an acceptable option for tissue engineering studies. But the authors comment also that using a heterogeneous mixture of cells may inhibit the zone-specific expression of certain genes, which is anticipated to have a negative effect on the formation of a functional cartilage tissue (Darling et al., 2004). Klein and co-workers (2003) also looked upon different cartilage zones to test if subpopulations could be used to engineer cartilage constructs with features of normal stratification. Chondrocytes from the superficial and middle zones of immature bovine cartilage were cultured in alginate, released and seeded either separately or sequentially to form cartilage constructs. Constructs were cultured for 1 or 2 weeks and were evaluated for growth, compressive properties, and deposition, and localisation of matrix molecules and SZP. The cartilaginous tissue formed from superficial zone chondrocytes exhibited less matrix growth and lower compressive properties than constructs from middle zone chondrocytes, with the stratified superficial-middle constructs exhibiting intermediate properties. Expression of SZP was highest at the construct surfaces, with the localisation of SZP in superficial-middle constructs being concentrated at the superficial surface. Furthermore, Waldman et al. (2003) studied the different layers of cartilage. They isolated full-thickness (FT), mid-and-deep zone (MD) and deep-zone (DEEP) chondrocytes, placed them on the surface of porous ceramic substrates, and maintained them in culture for 8 weeks. The tissue developed from DEEP chondrocytes was thicker and had accumulated larger amounts of extracellular matrix than the tissues formed by the FT and MD chondrocytes. The tissue formed by the FT chondrocytes accumulated the greatest amount of collagen whereas the tissue formed by the MD chondrocytes accumulated significantly more proteoglycans. The MD chondrocytes produced tissue that had compressive mechanical properties up to four times greater than the cartilaginous tissues

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formed by cells from either the FT or DEEP part of the cartilage. The authors concluded that a combined population of intermediate and DEEP chondrocytes might be more suitable for the tissue engineering of articular cartilage. Dowthwaite et al. reported in 2004 on the isolation of an articular cartilage progenitor cell from the surface zone of articular cartilage using differential adhesion to fibronectin. This population of cells exhibits high affinity for fibronectin, possesses a high colony-forming efficiency and expresses the cell fate selector gene Notch. There seems to be a chondrocyte subpopulation with progenitor-like characteristics in the surface layer of cartilage, indicating that it might be sufficient to harvest the superficial layers for chondrocyte isolation and expansion. However, still clinically the chondrocytes that are harvested come most often from a full thickness biopsy through all layers down to the subchondral bone.

9.5.4

Harvest sites

The most common sites for cartilage biopsy are the superomedial edge of the femoral condyle and the lateral intercondylar notch in the same location where a notchplasty is performed during anterior cruciate ligament (ACL) reconstruction. The other recommended area is the superlateral edge of the femoral condyle that is non-articulating with the tibia or patella. An arthroscopic gouge or ring curette is used to obtain two or three small pieces of partial to full thickness cartilage with a depth the size of a fingernail clipping (200±300 mg). At the same time, 10  10 ml of autologous venous blood is collected for preparation of serum to be used together with the culture medium.

9.5.5

In vitro cell expansion

The primary goal of in vitro chondrocyte manipulation is to increase the cell number. The culture technique has been developed first for implantation of cells in suspensions, first generation of ACI with persiosteal or collagen membrane covering. However, today both second and third generation ACI exist with cells cultured on a carrier membrane (matrix-induced ACI, MACI) or with cells in a 3D scaffold. Individual (e.g. Hyalograft-C by Fidia) In common to all generations is that the chondrocytes are isolated by collagenase digestion overnight and cultured in DMEM/F12 with 10% autologous serum supplement (Fig. 9.1). Primary cultures are performed in 25 cm2 culture flasks and after 1 week cell expansion n the cells are trypsinised and passaged to 75 cm2 culture flasks at a cell density of 8000 cells/cm2. For the suspension culture, after 2 weeks of cell culture the cells are trypsinised, washed and

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9.1 Human chondrocytes in monolayer culture. Courtesy Catherine Concaro.

resuspended to a treatment density of 30 million cells/m. The cells are under sterile conditions put into a syringe to be used as a suspension injection of cells into a defect covered by a periosteal or collagen membrane. For cells on a carrier membrane like MACI, the cells are cultured for about 3±5 weeks before seeding on an I±III collagen membrane (Zhang et al., 2006). Regarding a 3D-scaffold as an example, Hyalograft can be used. Here the biopsy material is taken to the laboratory for in vitro isolation and expansion of autologous chondrocytes for a duration of 3 weeks. Finally, the cells are seeded into the HYAFF 11 scaffold (HYAFF, Fidia Advanced Biopolymers) for 1 week, where they adhere, continue to proliferate, and redifferentiate into mature chondrocytes capable of producing their own extracellular matrix (Gobbi et al., 2006). This nonwoven 3D structure consists of a network of 20 m thick fibres with interstices of variable sizes, which constitute an optimal physical support to allow cell-to-cell contact, cluster formation, and extra cellular matrix deposition (Gobbi et al., 2006).

9.5.6

The chondrocyte implantation

With the initial technique (Brittberg et al., 1994), implantation consists of an arthrotomy, defect preparation, periosteal flap harvest, fixation of periosteal flap to defect, suturing the periosteal flap or collagen membrane (Fig. 9.2), securing a watertight seal with fibrin glue, implanting the chondrocytes (Fig. 9.3) and wound closure. However, today the periosteum has been more or less replaced by a resorbable membrane such as collagen I/III membrane ChondroGide (Geistlich, Wollhausen, Switzerland) or Restore (De Puy, Warzaw, Indiana, USA). Such techniques are second-generation ACI. Third-generation ACI are cell-seeded membranes such as matrix-induced ACI (MACI from Genzyme Biosurgery, Boston, MA, USA) and different 3D matrices with cultured chondrocytes such as

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9.2 Cartilage defect on a femoral condyle treated by debridement and on top of a sutured collagen membrane.

with hyaluronic acid (Hyalograft-C, Fidia, Italy). Both the MACI and Haylograft-C have facilitated the procedure as those methods can be used transarthroscopically.

9.5.7

Coexisting knee pathology

Cartilage lesions are often seen combined with other injuries such as menisci, cruciate ligaments and patellar instabilities, and as with any cartilage repair method, good results should not be expected if coexisting knee pathology is not carefully taken care of. Biomechanical misalignment and ligamentous insufficiency can lead to excessive forces and abnormal compressive loads that can destroy the induced repair tissue. Therefore, it is critical that any associated knee pathology responsible for or contributing to the chondral defect is identified and corrected prior to or in conjunction with the cells and scaffold being implanted.

9.3 A membrane covered cartilage defect and a concomitant opening wedge osteotomy with plate fixation has been done. Chondrocytes in suspension are implanted into the defect.

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Biomechanical malalignment

If the mechanical axis on long-standing X-rays passes through the compartment in which the chondral injury is located, an unloading osteotomy is recommended to shift abnormal forces away from that compartment. Unloading osteotomies should also be considered when the lesions on the condyles are large even without a malalignment or as an alternate use, protect weight-bearing with the use of a custom-made unloader brace. It is also important to evaluate any abnormal patellar tracking and, if needed, unload the patellofemoral joint by realignments procedures shifting the distal patella tendon insertion medial/ lateral and or anteriorly.

9.5.9

Ligamentous insufficiency

Ligamentous insufficiency produces excessive shear forces in the knee, which may negatively influence the maturing process of the repair tissue. If performed concomitantly, cruciate ligament reconstruction should precede ACI and should be performed in standard fashion with the preferred technique of the surgeon and patient.

9.5.10 Meniscal deficiencies The importance of a functional and intact meniscus when considering cartilage repair cannot be overstated. Whenever possible, the meniscus should be preserved or repaired In the presence of a total meniscectomy, or when the meniscal function is equivalent, meniscus transplantation may be considered. The meniscal allograft will help reduce the concentrated forces in the involved compartment and help protect the newly formed repair tissue. When performing a meniscal allograft concomitantly with an ACI, the meniscal allograft should be placed and secured, after which the ACI can be completed.

9.5.11 Osteochondral defects Bone grafting can be done at the time of arthroscopic evaluation and chondral biopsy. Another option is to do the procedure as a one-stage procedure with ACI in combination with bone grafting via the so-called `sandwich' technique (Jones and Peterson, 2007) in which the bony defect is filled with bone grafts, and the periosteum is both on top of the bone grafts level with the subchondral bone plate and also on top of the cartilage defect with the cells in between both cell layers.

9.5.12 Post-operative rehabilitation The philosophy of a slow gradual time course of maturation of the repair is critical to successful rehabilitation of patients following ACI. If the intra-

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articular region is well-protected, the maturation of the induced tissue, an ongoing process of remodelling of the tissue, will continue safely. However, if the graft is overloaded, a thinning of the repair may occur with eventual failure. There is always a degree of individual variation to be considered, meaning that the rehabilitation programme needs to be designed according to the patient's status and needs, weight and age as well as such factors as the size and location of the lesion, and any possible concomitant operations performed. Protection of the repair tissue from excessive intra-articular forces is critical during the early post-operative period; avoiding twisting rotational shearing forces. A gradually increased weight-bearing status should be the initial steps of the rehabilitation process. Isometric quadriceps training, straight leg raises and hamstring strengthening should be introduced early and progressively advanced to resisted exercises and return to greater degrees of functional activities. From 3 weeks post-operation, progressive closed chain exercises with light resistance can be started. Open chain exercises can be initiated around the 8th week. Running is not advised until the 8th or 9th month post-ACI with high level activities being initiated at the 12th month.

9.6

Other joints besides the knee joint

The most common joint treated with ACI is the knee joint. The ankle joint, shoulder, elbow, hip and wrist (Johansen et al., 2000; Nilsson et al., 2000; Romeo et al., 2002; Peterson et al., 2003) are other locations that have been tried in smaller number of patients. With the development of arthroscopic techniques (Marcacci et al., 2002; Erggelet et al., 2003) the use of ACI will be increased also in those smaller joint compartments. The author is today doing 90% of the ACI with a transarthroscopic approach giving patients a substantial lower morbidity due to the surgery (Figs 9.4±9.7). In particular, the use of resorbable membranes instead of the periosteum will be important for those joints by which one may avoid the risk of hypertrophy. The periosteal hypertrophy could become a problem in the smaller joints.

9.7

Clinical follow-up results

Since the report from Brittberg et al. with the first 23 patients in 1994, ACI has been performed in more than 30 000 patients throughout the world. The clinical results have been reported from numerous centres worldwide. In a clinical evaluation of 244 patients with 2±10 years' follow-up (Brittberg et al., 2003), subjective and objective improvements were seen in high numbers of patients with femoral condyle lesions and osteochondritis dissecans. There was a high percentage of good to excellent results (84±90%) in patients with different types of single femoral condyle lesions while other types of lesions had a lower degree of success (mean 74%). The reported

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9.4 A 2  2 cm2 Hyalograft scaffold with cultured chondrocytes prepared for implantation. Courtesy Catherine Concaro.

9.5 Microscopic view of chondrocytes cultured in a Hyalograft scaffold. Courtesy Catherine Concaro.

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9.6 Trans-arthroscopic implantation of chondrocytes in a Hyalograft scaffold.

9.7 The Hyalograft scaffold has been implanted trans-arthroscopically into an osteochondritis dissecans defect on a femoral condyle.

histology mostly shows a mixed tissue repair of hyalinic-fibrocartilaginous appearance (Fig. 9.8). To study the long-term durability of ACI treated patients, 61 patients who had passed two years post-surgery were followed for at least 5 years up to 11 years post-surgery (mean 7.4 years) (Brittberg et al., 2003). After 2 years, 50 out of 61 patients were graded good±excellent. At the 5 to 11 years follow-up 51 of the 61 were graded good±excellent. The total failure rate was 16% (10/61) at mean 7.4 years. All ACI failures occurred in the first 2 years and patients showing good to

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9.8 A biopsy of the repair after chondrocyte implantation with periosteum. Note typical fibrous top cartilaginous layer followed by a more cartilaginous layer and finally the bone area.

excellent improvement at 2 years had a high percentage of good results at longterm follow-up (Brittberg et al., 2003). Most reports on the use of autologous chondrocyte transplantation from other centres (Minas and Chiu, 2000; Bentley et al., 2003; Gillogly, 2003) show similar figures with a high degree of success in regard to the total number of improved patients, but the criticism has been that ACT needs to be evaluated against other cartilage repair techniques in randomised trials (Lohmander, 2003).

9.8

Imaging evaluation of the cartilage repair

MRI has become the method of choice for non-invasive follow-up of patients after cartilage repair surgery. Trattnig et al. (2007) have suggested that it should be performed with cartilage sensitive sequences, including fat-suppressed proton density-weighted T2 fast spin-echo (PD/T2-FSE) and three-dimensional gradient-echo (3D GRE) sequences, which provide good signal-to-noise and contrast-to-noise ratios. Furthermore, in vivo biochemical imaging such as dGEMRIC, T2 mapping and diffusion-weighted imaging will be more used, which make functional analysis of cartilage possible. In the future, enhanced peripheral quantitative computed tomography (pQCT) may also be used for in vivo quantification of proteoglucan (PG) depletion in osteoarthritic cartilage (Kalloniemi et al., 2007) and to evaluate the repair with chondrocytes. Brown et al. (2004) studied 180 MRI examinations that were obtained in 112 patients who had cartilage-resurfacing procedures, including 86 microfractures and 35 autologous chondrocyte implantations, at a mean of 15 and 13 months after surgery, respectively. Autologous chondrocyte implantations showed consistently better fill of the defects at all times compared with microfracture. The repair cartilage over the microfracture generally was depressed with respect

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to surrounding cartilage. Propensity for bony overgrowth was most marked in the microfracture group, with loss of adjacent cartilage evident with progressive follow-up.

9.9

Randomised controlled studies

To date, seven clinical randomised trials have been published in peer-reviewed papers. Those studies could be seen as short-term and mid-term studies. Knutsen et al. (2007) studied 80 patients who needed local cartilage repair because of symptomatic lesions on the femoral condyles measuring 2±10 cm2. The patients were randomised into ACT or micro-fracture treatment. At 2 and 5 years, both groups had significant clinical improvement compared with the pre-operative status. At the 5-year follow-up interval, there were nine failures (23%) in both groups compared with two failures of the autologous chondrocyte implantation and one failure of the microfracture treatment at two years. Younger patients did better in both groups. The authors did not find a correlation between histological quality and clinical outcome. However, none of the patients with the best-quality cartilage (predominantly hyaline) at the 2-year mark had a later failure. Horas et al. (2003) performed a prospective clinical study to investigate the two-year outcomes in 40 patients with an articular cartilage lesion of the femoral condyle who had been randomly treated with either transplantation of an autologous osteochondral cylinder or implantation of autologous chondrocytes. Both treatments resulted in a decrease in symptoms. However, the improvement provided by the autologous chondrocyte implantation lagged behind that provided by the osteochondral cylinder transplantation. Bentley and associates (2003) studied a total of 100 patients with a mean age of 31.3 years (16 to 49) and with symptomatic chondral and osteochondral lesions the knee which were suitable for cartilage repair and randomised to undergo either ACT or mosaicplasty; 58 patients had ACI and 42 mosaicplasty. Most lesions were post-traumatic and the mean size of the defect was 4.66 cm2. The mean duration of symptoms was 7.2 years and the mean number of previous operations, excluding arthroscopy, was 1.5. The mean follow-up was 19 months (12 to 26). Functional assessment using the modified Cincinnati and Stanmore scores and objective clinical assessment showed that 88% had excellent or good results after ACT compared with 69% after mosaicplasty. Arthroscopy at 1 year demonstrated excellent or good repairs in 82% after ACI and in 34% after mosaicplasty. This prospective, randomised, clinical trial showed significant superiority of ACT over mosaicplasty for the repair of articular defects in the knee. Dozin et al. (2005) studied 47 patients who were randomly assigned to ACI or mosaicplasty and subjected to arthroscopic debridement of the lesion at the time of enrolment. It was notable that 14 patients (31.8%) experienced substantial improvement following the initial debridement and, being clinically

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cured, received no further treatment. Among the 23 patients (52.3%) who could effectively be evaluated, a complete recovery was observed upon clinical examination in 88% of the mosaicplasty-treated patients and in 68% of the ACItreated ones (P ˆ 0:093). Furthermore, there are two randomised study comparing two types of ACI; ACI with collagen membrane versus MACI for osteochondral defects of the knee (Bartlett et al., 2005) and a prospective, randomised study comparing periosteum covered ACI versus type I/III collagen covered (Gooding et al., 2006). There were no differences in the outcome when comparing collagen covered ACI versus MACI even though MACI was technically more attractive (Bartlett et al., 2005). In the other (Gooding et al., 2006) of those studies, there were no statistical differences between the clinical outcome of collagen covered ACI versus periosteum covered ACT at 2 years. A significant number of patients who had the periosteum covered ACI required shaving of a hypertrophied graft. The authors conclude that there is no advantage in using periosteum. However, recently a seventh well-performed randomised study was presented by Saris et al. (2008). Characterised chondrocyte implantation was compared with microfracture in patients with single grade III to IV symptomatic cartilage defects of the femoral condyles in a multicentre trial. Patients aged 18 to 50 years were randomised to characterised chondrocyte implantation (n ˆ 57) or microfracture (n ˆ 61). One year after treatment, characterised chondrocyte implantation was associated with a tissue regenerate that was superior to that after microfracture. Short-term clinical outcome was similar for both treatments. Wasiak et al. published in 2006 a Cochrane review that included four randomised controlled trials (266 participants). Their conclusions were that at present time there is no evidence of significant difference between ACI and other cartilage repair interventions. Additional good quality randomised controlled trials with long-term functional outcomes are required. The last randomised study from 2008 by Saris et al. was not evaluated by the above review and as it has a greater number of patients included in the follow-up compared with the other studies future reviews of that study's long-term results and other coming studies will be of great interest.

9.10

Chondrocyte implantation and osteoarthritis

Today, treatment of cartilage defects is often done with chondrocyte implantation. The defects are often of traumatic origin and seldom degenerative lesions. However, the increased use of tissue engineering has led to a wish to treat widespread lesions such as osteoarthritis (OA). If you want to use autologous chondrocytes, osteoarthritic chondrocytes will be used. The question is if those chondrocytes that may have a defective function could be used in such tissue engineering purposes.

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Tallheden and co-workers (2005) investigated the expansion and redifferentiation potential in vitro of chondrocytes isolated from patients undergoing total knee replacement. The results demonstrate that OA chondrocytes have a good proliferation potential and are able to redifferentiate in a three-dimensional pellet model. During the redifferentiation, the OA cells expressed increasing amounts of DNA and proteoglycans, and at day 14 the cells from all donors contained type II collagen-rich matrix. The accumulation of proteoglycans was in comparable amounts to those from ACT donors, whereas total collagen was significantly lower in all of the redifferentiated OA chondrocytes. When the OA chondrocytes were loaded into a scaffold based on hyaluronic acid, they bound to the scaffold and produced cartilage-specific matrix proteins. Thus, autologous chondrocytes are a potential source for a biological treatment of OA patients but the limited collagen synthesis of the OA chondrocytes needs to be further explained transplanted autologous bone marrow mesenchymal cells into articular cartilage defects in osteoarthritic knee joints. There are several studies showing the chondrogeneic cell therapy could be successful when treating local cartilage defects in humans. However, in OA, an entire compartment is damaged and there is now a total organ disease to be taken care of. An implantation of cells placed into a defect would be surrounded by diseased cartilage. The destructive cytokines produced by the OA-chondrocytes around the grafted area may destroy the implanted cells. Furthermore, the chondrocytes must be harvested from non-diseased cartilage. Non-diseased allograft chondrocytes or mesenchymal stem cells could be alternatives. There is no evidence to date that treating a local cartilage defect may stop or halt the progression of a local defect into OA. Furthermore, as OA is an organ disease, the subchondral bone also has to be taken care of. A combination of mesenchymal stem cells and committed chondrocytes in biphasic hydrogels may be a dual cell gel construct therapy for OA in the future.

9.11

Conclusions and future trends

Today local cartilage defects are treated. What about the future? Could we treat OA with chondrocyte implantation? What are the options? The ideal articular cartilage repair technology ought to be cheap, available off-the-shelf, deliverable arthroscopically, give rise to a durable well-integrated repair tissue, result in clinical improvement, eliminate or significantly postpone the need for arthroplasty, and be suited for both focal defects and degenerative joint disease. Techniques today available for cartilage repair are still `plug-in' methods for local cartilage repair. From those techniques there exists not yet enough data to justify regular use of them in osteoarthritic repairs. However, recent studies (Kaul et al., 2006; Goodrich et al., 2007) have shown that there is the possibility of repairing and rebuilding osteoarthric cartilage by means of resurfacing the diseased cartilage with genetically modified chondrocytes.

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However, even those modified chondrocytes would need a scaffolding material and a concomitant unloading. Despite the promising clinical results with some of the new articular cartilage repair methods, there remains a considerable distance between where the technology is and where we want it to be in clinical usage. However, with the new interdisciplinary tissue engineering groups (translational regenerative medicine) formed over the world where the cell biologists, engineers and surgeons work closely together with the construction of artificial connective, epithelial and neuronal tissues using living cells and different kinds of biomaterials I am quite confident that we will be able to produce tissues that can be used as real spare parts instead of destroyed human tissues; biomedical surgery.

9.12

Sources of further information and advice

Besides that which can be learned from the references included here are some further suggestions of recommended literature about cartilage repair and chondrocytes: Cole B, Malek M, eds. Articular Cartilage Lesions. A practical guide to assessment and treatment. Springer Verlag 2004, 95±104. Cole B, Malek M. Chondral Disease of the Knee: A case-based approach. Springer 2006. Meyer U, Wiesmann HP, Meyer T. Bone and Cartilage Engineering. Springer Verlag 2006. Van Blitterswijk, Thomsen P, Lindahl A, Hubbell J, Williams D, Cancedda R, de Bruijn J, Sohier J. Tissue Engineering. Academic Press Series in Biomedical Engineering, Elsevier 2008. Zanasi S, Brittberg M, Marcacchi M, eds. Basic Science, Clinical Repair and Reconstruction of Articular Cartilage Defects: Current status and prospects. Timeo Editore, Bologna 2006. Volume I±II.

Courses are provided by the ICRS (International Cartilage Repair Society) about cartilage repair with cell therapies.

9.13

References

Aston JE, Bentley G. Repair of articular surfaces by allografts of articular and growthplate cartilage. J Bone Joint Surg 1986; 68B: 29±34. Bartlett W, Skinner JA, Gooding CR, Carrington RW, Flanagan AM, Briggs TW, Bentley G. Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study. J Bone Joint Surg Br 2005 May; 87(5): 640±5. Bentley G, Greer RB. Homotransplantation of isolated epiphyseal and articular chondrocytes into joint surfaces. Nature 1971; 230: 385±8. Bentley G, Smith AV, Mukerjhee R. Isolated epiphyseal chondrocyte allografts into joint surfaces. Ann Rheum Dis 1979; 37: 449±58. Bentley G, Biant LC, Carrington RW, Akmal M, Goldberg A, Williams AM, Skinner JA, Pringle J. A prospective, randomised comparison of autologous chondrocyte

Cartilage tissue repair: autologous chondrocyte implantation

247

implantation versus mosaicplasty for osteochondral defects in the knee. J Bone Joint Surg Br 2003 Mar; 85(2): 223±30. Breinan HA, Minas T, Hsu H-P, Nehrer S, Sledge CB, Spector M. Effect of cultured autologous chondrocytes on repair of chondral defects in a canine model. J Bone Joint Surg 1994; 79A: 1439±51. Breinan HA, Hsu H-P, Spector M. Condral defects in animal models. Clin Orthop Rel Res 2001; 391: 219±30. Brittberg M. Cartilage repair. On cartilaginous tissue engineering with the emphasis on chondrocyte transplantation, PhD thesis, University of Gothenburg, GoÈteborg, Sweden, 1996. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. New England J Med 1994, 331: 889±95. Brittberg M, Nilsson A, Lindahl A, Ohlsson C, Peterson L. Rabbit articular cartilage defects in the knee with autologous cultured chondrocytes. Clin Orthop Rel Res 1996; 326: 270±83. Brittberg M, Peterson L, Sjogren-Jansson E, Tallheden T, Lindahl A. Articular cartilage engineering with autologous chondrocyte transplantation. A review of recent developments. J Bone Joint Surg Am 2003; 85-A Suppl 3: 109±15. Brown WE, Potter HG, Marx RG, Wickiewicz TL, Warren RF. Magnetic resonance imaging appearance of cartilage repair in the knee. Clin Orthop Rel Res 2004 May; 422: 214±23. Caplan AI, Elyaderani M, Mochizuki Y, Wakitani S, Goldberg VM. Principles of cartilage repair and regeneration. Clin Orthop Rel Res 1997; 342: 254±69. Chestermann PJ, Smith AU. Homotransplantation of articular cartilage and isolated chondrocytes. J Bone Joint Surg 1968; 50B: 184±97. Darling EM, Hu JC, Athanasiou KA. Zonal and topographical differences in articular cartilage gene expression. J Orthop Res 2004 Nov; 22(6): 1182±7. Dell'Accio F, Vanlauwe J, Bellemans J, Neys J, De Bari C, Luyten FP. Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. Osteoarthritis Cartilage 2002; 10(3): 199±206. Dell'Accio F, Vanlauwe J, Bellemans J, Neys J, De Bari C, Luyten FP. Expanded phenotypically stable chondrocytes persist in the repair tissue and contribute to cartilage matrix formation and structural integration in a goat model of autologous chondrocyte implantation. J Orthop Res 2003 Jan; 21(1): 123±31. Erratum in: J Orthop Res 2003 May; 21(3): 572. Dowthwaite GP, Bishop JC, Redman SN, Khan IM, Rooney P, Evans DJ, Haughton L, Bayram Z, Boyer S, Thomson B, Wolfe MS, Archer CW.The surface of articular cartilage contains a progenitor cell population. J Cell Sci 2004 Feb 29; 117(Pt 6): 889±97. Dozin B, Malpeli M, Cancedda R, Bruzzi P, Calcagno S, Molfetta L, Priano F, Kon E, Marcacci M. Comparative evaluation of autologous chondrocyte implantation and mosaicplasty: a multicentered randomized clinical trial. Clin J Sport Med 2005 Jul; 15(4): 2206 Elves MW. A study of the transplantation antigenes on chondrocytes from articular cartilage. J Bone Joint Surg 1974; 56B: 178±85. Erggelet C, Sittinger M, Lahm A. The arthroscopic implantation of autologous chondrocytes for the treatment of full-thickness cartilage defects of the knee joint. Arthroscopy 2003; 19(1): 108±10.

248

Regenerative medicine and biomaterials in connective tissue repair

Gillogly SD. Treatment of large full-thickness chondral defects of the knee with autologous chondrocyte implantation. Arthroscopy 2003 Dec; 19 Suppl 1: 147±53. Gobbi A, Kon E, Berruto M, Francisco R, Filardo G, Marcacci M. Patellofemoral fullthickness chondral defects treated with hyalograft-C: a clinical, arthroscopic, and histologic review. Am J Sports Med Nov 2006; 34: 1763±73. Goldberg VM, Caplan AI. `Cellular repair of articular cartilage' in KE Kuettner, VM Goldberg (eds) Osteoarthritic Disorders. Am Acad Orthop Surg, Monterrey, pp 357±64, 1994. Gooding CR, Bartlett W, Bentley G, Skinner JA, Carrington R, Flanagan A. A prospective, randomised study comparing two techniques of autologous chondrocyte implantation for osteochondral defects in the knee: periosteum covered versus type I/III collagen covered. Knee 2006 Jun; 13(3): 203±10. Goodrich LR, Hidaka C, Robbins PD, Evans CH, Nixon AJ. Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model. J Bone Joint Surg Br 2007 May; 89(5): 672±85. Grande DA, Pitman MI, Peterson L, Menche D, Klein M. The repair of experimentally produced defects in rabbit articular cartilage by autologous chondrocyte transplantation. J Orthop Res 1989; 7: 208±18. Green WT. Articular cartilage repair, behaviour of rabbit chondrocytes during tissue culture and subsequent allografting. Clin Orthop 1977; 124: 237±50. Hirschmann F, Verhoeyen E, Wirth D, Bauwens S, Hauser H, Rudert M. Vital marking of articular chondrocytes by retroviral infection using green fluorescence protein. Osteoarthritis Cartilage 2002 Feb; 10(2): 109±18. Horas U, Pelinkovic D, Herr G, Aigner T, Schnettler R. Autologous chondrocyte implantation and osteochondral cylinder transplantation in cartilage repair of the knee joint. A prospective, comparative trial. J Bone Joint Surg Am 2003 Feb; 85A(2): 185±92 Johansen O, Lindahl A, Peterson L, Olsen B, Winge JF, Knutsen G. Hip osteochondritis treated by debridement, suture of a periosteal flap and chondrocyte implantation. A case report. In: Hunziker E, Mainil-Varlet, eds. Updates in Cartilage Repair: a multimedia production on cartilage repair on 6 CD-Roms. Philadelphia: Lippincott Williams and Wilkins, 2000. Jones DG, Peterson L. Autologous chondrocyte implantation. Instr Course Lect 2007; 56: 429±45. Kallioniemi AS, Jurvelin JS, Nieminen MT, Lammi MJ, ToÈyraÈs J. Contrast agent enhanced pQCT of articular cartilage. Phys Med Biol 2007 Feb 21; 52(4): 1209±19. Kaul G, Cucchiarini M, Arntzen D, Zurakowski D, Menger MD, Kohn D, Trippel SB, Madry H. Local stimulation of articular cartilage repair by transplantation of encapsulated chondrocytes overexpressing human fibroblast growth factor 2 (FGF2) in vivo. J Gene Med 2006 Jan; 8(1): 100±11. Kawabe N, Yoshinao M. The repair of full thickness articular cartilage defects. Immune responses to reparative tissue formed by allogeneic growth plate chondrocyte implants. Clin Orthop 1991; 268: 279±93. Klein TJ, Schumacher BL, Schmidt TA, Li KW, Voegtline MS, Masuda K, Thonar EJ, Sah RL. Tissue engineering of stratified articular cartilage from chondrocyte subpopulations. Osteoarthritis Cartilage 2003 Aug; 11(8): 595±602. Knutsen G, Drogset JO, Engebretsen L, Grùntvedt T, Isaksen V, Ludvigsen TC, Roberts S, Solheim E, Strand T, Johansen O. A randomized trial comparing autologous chondrocyte implantation with microfracture. Findings at five years. J Bone Joint Surg Am 2007 Oct; 89(10): 2105±12.

Cartilage tissue repair: autologous chondrocyte implantation

249

Lee CR, Grodzinsky AJ, Hsu HP, Spector M. Effects of a cultured autologous chondrocyte-seeded type II collagen scaffold on the healing of a chondral defect in a canine model. J Orthop Res 2003; 21(2): 272±81. Lohmander LS. Tissue engineering of cartilage: do we need it, can we do it, is it good and can we prove it? Novartis Found Symp 2003; 249: 2±10; discussion 10±16, 170±4, 239±41. Review. Marcacci M, Zaffagnini S, Kon E, Visani A, Iacono F, Loreti I. Arthroscopic autologous chondrocyte transplantation: technical note. Knee Surg Sports Traumatol Arthrosc 2002 May; 10(3): 154±9. Minas T, Chiu R. Autologous chondrocyte implantation. Am J Knee Surgery 2000; 13: 41±50. Nevo Z, Robinson D, Horowitz S, Yayon A. The manipulated mesenchymal stem cells in regenerated skeletal tissues. Cell Transplant 1998; 7: 208±18. Nilsson A, Lindahl A, Peterson L, Brittberg M, Sollerman C. Autologous chondrocyte transplantation of the human wrist. In Hunziker E, Mainil-Varlet (eds). Updates in Cartilage Repair. A multimedia production on cartilage repair on 6 CD-ROMS. Philadelphia, Lippincott, Williams and Wilkins Company, 2000. Noguchi T, Oka M, Fujino M, Neo M, Yamamuro T. Repair of osteochondral defects with grafts of cultured chondrocytes. Comparison of allografts and isografts. Clin Orthop 1994; 302: 251±8. O'Connor BL, Brandt KD. Neurogenic factors in the ethiopathogenesis of osteoarthritis. Rheum Dis N Am 1993; 19: 581±604. Peterson L, Menche D, Grande D, Pitman M. Chondrocyte transplantation; an experimental model in the rabbit. In: Transactions from the 30th Annual Meeting Orthopaedic Research Society, Atlanta, February 7±9, 1984. Palatine III, Orthopaedic Research Society, 218. Petersen L, Brittberg M, Lindahl A. Autologous chondrocyte transplantation of the ankle. Foot Ankle Clin 2003 Jun; 8(2): 291±303. Rahfoth B, Weisser J, Sternkopf F, Aigner T, von der Mark K, Brauer R. Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits. Osteoarthritis Cartilage 1998; 6: 50±65. Robinson D, Halperin N, Nevo Z. Fate of allogeneic embryonal chick chondrocytes implanted orthtotopically as determined by the host's age. Mechanisms of Ageing and Development 1989; 50: 71±80. Romeo AA, Cole BJ, Mazzocca AD, Fox JA, Freeman KB, Joy E. Autologous chondrocyte repair of an articular defect in the humeral head. Arthroscopy 2002; 18: 925±9. Saris DB, Vanlauwe J, Victor J, Haspl M, Bohnsack M, Fortems Y, Vandekerckhove B, Almqvist KF, Claes T, Handelberg F, Lagae K, van der Bauwhede J, Vandenneucker H, Yang KG, Jelic M, Verdonk R, Veulemans N, Bellemans J, Luyten FP. Characterized chondrocyte implantation results in better structural repair when treating symptomatic cartilage defects of the knee in a randomized controlled trial versus microfracture. Am J Sports Med 2008 Feb; 36(2): 235±46. Smith AV. Survival of frozen chondrocytes isolated from cartilage of adult animals. Nature 1965; 205: 782±4. Tallheden T, Bengtsson C, Brantsing C, SjoÈgren-Jansson E, Carlsson L, Peterson L, Brittberg M, Lindahl A. Proliferation and differentiation potential of chondrocytes from osteoarthritic patients. Arthritis Res Ther 2005; 7(3): R560±8. Trattnig S, Millington SA, Szomolanyi P, Marlovits S. MR imaging of osteochondral grafts and autologous chondrocyte implantation. Eur Radiol 2007 Jan; 17(1): 103± 18. Review.

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Wakitani S, Goto T, Pineda SJ, Toung RG, Mansour JM, Caplan AI, Goldberg VM. Mesenchymal cell-based repair of large, full thickness defects of articular cartilage. J Bone Joint Surg 1994; 76A: 579±592. Waldman SD, Grynpas MD, Pilliar RM, Kandel RA.The use of specific chondrocyte populations to modulate the properties of tissue-engineered cartilage. J Orthop Res 2003 Jan; 21(1): 132. Wasiak J, Clar C, Villanueva E. Autologous cartilage implantation for full thickness articular cartilage defects of the knee, 2006. Review. http://www.thecochrane library.com Yan H, Yu C. Repair of full-thickness cartilage defects with cells of different origin in a rabbit model. Arthroscopy 2007 Feb; 23(2): 178±87. Yoshihashi Y. Tissue reconstitution by isolated articular chondrocytes in vitro. NipponSeikeigeka-Gakkai-Zasshi 1983; 57: 629±41. Zhang Z, Ye Q, Yang Z, Yin M, Bai J, Hou S, Gao C, Kuang Z, Pang X, Li H, Zheng M, Wood D. Matrix-induced autologous chondrocyte implantation for treatment of chondral defects of knee: a preliminary report. J Musculoskel Res 2006; 10: 95± 101.

10

Cell sheet technologies for cartilage repair M . S A T O , Tokai University School of Medicine, Japan

Abstract: This chapter outlines the principles of cell sheet technology, its clinical applications, how to repair cartilaginous defects using layered chondrocyte sheets, the properties of layered chondrocyte sheets, future trends in cartilage repair, and regulation of this area in Japan. Key words: cell sheet, temperature-responsive culture dish, articular cartilage, cultured chondrocyte, tissue engineering.

10.1

Introduction

This section outlines cell sheet technology, specifically introducing the temperature-responsive culture dish. Cell sheets can be obtained without enzymes. It is also possible to obtain layered constructs. Prof. Okano,1,2 from Tokyo Women's Medical University, developed this technology involving the building of three-dimensional tissue constructs, consisting of individual units of the cell sheets with tight junctions between the cells and extracellular matrices. A temperature-responsive culture dish is used to obtain the cell sheet. An Nisopropylacrylamide monomer solution is spread on commercial tissue culture polystyrene dishes. These dishes are then subjected to electron beam irradiation, thus resulting in polymerization and covalent binding of the isopropylacrylamide to the surface of the dish. Poly N-isopropylacrylamide (PIPAAm) is a unique polymer that exhibits thermally reversible soluble-insoluble changes in an aqueous solution in response to temperature changes across an LCST (lower critical solution temperature) of 32 ëC. Polymerized chains of acrylamide hydrate to expand in water below the LCST, while the isopropyl group dehydrates to form compact, insoluble conformations above the LCST. These dishes, therefore, reverse their hydrophobic and hydrophilic properties in response to changing temperature. When the temperature-responsive polymer (PIPAAm) is fixed to a cell-culturing dish using Bionano interface technology, the surface of the plate changes in response to temperature change across an LCST of 32 ëC. The surface becomes hydrophobic above 32 ëC, which enables cells to attach to the surface and grow. However, when the temperature is reduced to 20 ëC, the polymer surface becomes hydrophilic and the hydrated polymer chains allow the cultured cells to

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10.1 Cell detachment mechanism of temperature-responsive surface of the dish. Cultured cells by using a temperature-responsive surface could be released from the dish surface only by reducing the temperature without proteolytic enzyme. N-isopropylacrylamide monomer solution was spread onto commercial tissue culture polystyrene dishes. The surface of the temperatureresponsive culture dish is grafted with a polymer (poly-N-isopropylacrylamide) which becomes either hydrophilic or hydrophobic in a reversible manner, depending on the temperature. Based on this characteristic, the temperatureresponsive culture dish has a weakly hydrophobic surface similar to that of commercially available dishes and it can be used to culture cells in a conventional manner when the temperature is 37 ëC or higher. However, the surface of the dish becomes hydrophilic when the temperature falls below the critical solution temperature of 32 ëC. Therefore, confluent sheets of cultured cells can be spontaneously released from the hydrophilic dish surface by reducing the temperature to below 32 ëC.

detach easily (Fig. 10.1, Movie: http://www.cellseed.com/technology-e/ 003.html). Cells detach because the hydrophobic surface they are attached to disappears below the LCST of 32 ëC. Since cell-damaging enzymes are not required, cells can detach while maintaining the cell±cell junction. This enables the cultured cells to be harvested as a single `sheet'. The cell sheets are highly effective when transplanted into patients due to the tight connections between the cells. The cultured cell sheet can be easily moved and layered upon other cultured cell sheets to generate a 3-dimensional cell culture. If fibronectin is present, the multiple-layer cell sheets are easily constructed by simply laying one cell sheet on top of another. The technology is ultimately applicable to the construction of organs by layering different types of cell sheet.

10.2

Overview of present clinical applications

This section will discuss the present clinical applications of cell sheet technology (i.e., the regeneration of the cornea, myocardium, esophageal mucosa, etc.). Prof. Okano and his colleagues are working on building three-dimensional tissue constructs. Cell sheet technology has already been used for regenerative medicine in corneal3,4 and myocardial tissues.5±8 In Japan, the lack of cornea and heart donors and the immune system's reaction to a transplanted organ are recognized as serious problems. Prof. Nishida, at Tohoku University, has produced cell sheets from the cultured epithelial stem cells of cornea and oral

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mucosal cells. He has transplanted these two types of cell sheet into more than 12 patients without sutures for tissue regeneration and has had excellent clinical results. Prof. Sawa, at Osaka University, made cell sheets from cultured myoblasts of the femur of a patient with dilated cardiomyopathy who underwent surgery for the implantation of a ventricular assist device in February 2006. He transplanted 20 cell sheets into the walls of the left ventricle of the heart in May 2007. The patient recovered well and he no longer needed the ventricular assist device, which was removed in September 2007. This was the first report of successful cell sheet therapy for a patient with dilated cardiomyopathy. The cell sheet therapy can provide regenerative treatment as an alternative for patients who need cardiac transplantation. Prof. Sawa plans to treat six patients, using the same type of cell sheet, in Osaka University Hospital. Other preclinical studies using cell sheets are under way. Clinical studies of cell sheet therapy for urothelial tissue9 (Tokyo Women's Medical University), oesophageal tissue10±12 (Tokyo Women's Medical University), periodontal tissue13±15 (Tokyo Women's Medical University and Tokyo Medical and Dental University), hepatic tissue16±19 (Tokyo Women's Medical University) and articular cartilaginous tissue20,21 (Tokai University School of Medicine) are currently taking place.

10.3

Challenge for cartilage repair

This section will discuss how to repair cartilaginous defects using layered chondrocyte sheets. Articular cartilage is an avascular tissue that is nourished by synovial fluid. Adult articular cartilage shows poor self-repair after degeneration or injury and it is therefore unlikely to be restored to normal once it has been damaged. The current treatments available for cartilage defects include the application of a periosteal patch to cover the defect22 and mosaicplasty, in which an osteochondral pillar is grafted from a non-weight-bearing site.23 However, the use of periosteal patches has limitations owing to problems with ossification and the limited area that can be treated. Although the microfracture technique is widely used, in which drilling is employed to induce bone marrow cells to differentiate into chondrocytes, the cartilage obtained by this technique is fibrocartilage, with different characteristics to those of hyaline cartilage. Since promising results for the transplantation of cultured autologous chondrocytes have been reported,24 various articular cartilage regeneration techniques have been applied clinically, including the use of scaffolds such as atelocollagen25 and cell transplantation therapy with bone marrow-derived mesenchymal stem cells.26 However, current cartilage regeneration techniques are intended for the treatment of full thickness defects and there have been no reports on the clinical application of a technique for partial thickness defects in patients with early osteoarthritis. Defects in articular cartilage are classified as either full or partial thickness defects, according to whether or not they penetrate the marrow spaces of the subchondral bone. Partial thickness defects are analogous to the clefts and

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fissures that are seen in the early stages of osteoarthritis in humans. These fibrillated lesions grow larger and deeper during the course of the disease but never repair themselves spontaneously. It has also been suggested that partial thickness defects do not heal because they are walled off from the marrow and thus have no access to the macrophages, endothelial cells and mesenchymal cells that reside therein.27 Articular cartilage is composed of scattered chondrocytes embedded in an abundant extracellular matrix (ECM). The matrix is mainly composed of type II collagen and proteoglycans and is responsible for specific joint functions, including smooth movement and shock absorption. When cultured chondrocytes are employed in vitro, it is important to harvest the cells without damaging the ECM. However, current methods damage the cultured cells and disrupt the ECM because proteolytic enzymes are used when harvesting the cultured cells. To achieve the repair and regeneration of partial thickness articular cartilage defects, cultured chondrocytes can be harvested without ECM damage by using temperature-responsive culture dishes. Such cell sheets have been reported to have various advantages, including the preservation of the early phenotype and the expression of adhesion proteins on the base.1 Furthermore, these cell sheets can be layered onto each other to prepare a layered `tissue' because the ECM is preserved on the base and such three-dimensional manufactured tissues have already been used for transplantation.5 In this study, human chondrocyte sheets with the ECM were obtained using the temperature-responsive culture dish method and were then combined in layers. Following this, the `tissue' was compared with that of a single sheet and the adhesion of the sheets was examined both in vivo and ex vivo. This demonstrated the first step towards bioengineering cartilaginous tissues for the treatment of partial thickness cartilage defects using cell sheet technology (Fig. 10.2).

10.3.1 Allograft study Articular chondrocytes from Japanese white rabbits Twelve Japanese white rabbits aged 3±4 weeks and weighing between 800 and 1000 g were used as the source of articular cartilage cells. Cartilage samples were collected from the femoral compartment of the knee joint and were subjected to the same enzymatic treatment process as that used for human articular cartilage cells. Thereafter, the isolated cells were seeded and cultured in temperature-responsive culture dishes. Cell proliferations on a temperature-responsive surface Chondrocytes were digested for 1 h in Dulbecco's modified Eagle's medium/ F12 (D-MEM/F12; GIBCO, NY USA) containing 0.4% Pronase E

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10.2 Cell sheet technology for articular cartilage repair. The advantages of using such cell sheets are that they are easy to culture and they proliferate easily, they have good adhesion, and a barrier function which is also very important because it enables the protection of intra-articular catabolic factors, while also preventing proteoglycans from escaping. Furthermore, cell sheets are useful for various types of cartilageous defect in conjunction with the use of scaffold free tissue-engineered cartilage.

(Kakenseiyaku Inc.) and subsequently for 4 h in DMEM/F12 containing 0.016% Collagenase P (Roche, Mannhein, Germany). The digested tissue was passed through a cell strainer (BD FalconTM) with a pore size of 100 m. The cells were then seeded at a high density (10 000 cells/cm2) onto the surfaces of temperature-responsive culture dishes (UpCellTM, diameter: 35 mm provided by CellSeed, Tokyo, Japan) and cultured in DMEM/F12 supplemented with 20% Fetal Bovine Serum (FBS; GIBCO, NY) and 50 g/ml ascorbic acid (Wakojunyakukougyou Corp. Japan) and 1% Antibiotics-Antimycotic (GIBCO, NY) at 37 ëC in an atmosphere of 5% CO2 and 95% air for a week. Human articular cartilage cells were also seeded onto commercially available culture dishes (diameter: 35 mm, Iwaki, Japan) and cultured under the same conditions. Harvesting of cell sheets Each culture dish was removed from the incubator when the cells reached confluence and was then left to stand at about 25 ëC for 30 minutes. After the

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culture medium has been removed, the cell sheet was harvested using a polyvinylidene difluoride (PVDF) membrane according to the method reported by Yamato et al.28 In brief, the PVDF membrane was placed on the cell sheet and then the sheet was rolled up with the membrane from one corner. Cultured human chondrocytes were able to be successfully harvested as a single contiguous cell sheet using this method. Then each cell sheet was placed on top of another confluent cell sheet to create multilayered sheets. Since the multilayered sheets floated in the culture medium, a 0.4 m cell culture insert (Falcon, USA) was placed on top to prevent this and then the culture of the sheets was continued for 1 week. Three-layer sheets of cartilage cells from Japanese White rabbits were also prepared. A 0.4 m filter was also used to compress these sheets onto the culture dish and sheets were incubated for 3 weeks to prepare the multilayered sheets for transplantation. Transplantation of chondrocyte sheets The articular cartilage of the medial femoral condyle of Japanese white rabbits, weighing about 3000 g, was removed to a depth of less than 1 mm using a file to prepare a model of partial thickness cartilage damage. The damaged cartilage was covered with a three-layered chondrocyte sheet, which was stabilized with a nylon suture until the initial fixation was achieved. This was done in four knees of two rabbits as the transplantation group. At the same time, the articular cartilage of the medial femoral condyle was similarly filed, but not covered with a cell sheet, in four knees of two rabbits (the control group). The cartilage was harvested after 4 weeks, fixed in 4% PFA for one week and decalcified with KCX Decalcifying Solution (Fujisawa Pharmaceutical, Japan) for 1 week. The specimens were then embedded in paraffin, cut into sections and stained with safranin-O and toluidine blue for evaluation. Histological findings of the allografted chondrocyte sheet and injured sites The three-layered cell sheet remained well attached to tissue sections 4 weeks after transplantation. The area covered by the sheet was better stained than the area not covered with it, as observed in the previously mentioned ex vivo experiment. In the partial damaged cartilage model, the area not covered with the multilayered sheets showed progressive cartilage degeneration with fibrillation and poor staining of the matrix at 4 weeks. In contrast, the area covered with the three-layered sheets showed relatively mild degeneration and a well-stained matrix (Figs 10.3 and 10.4). This study confirmed that chondrocytes could be harvested as sheets and thus be made into multilayered `tissue' by culturing in temperature-responsive dishes and then collecting them using a temperature recovery system.

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10.3 Layered chondrocyte sheets transplantation to the cartilageous defects of the rabbit knee joints. In this rabbit animal model, the allografted cell sheet maintained its cartilageous thickness, but the group without any cell sheets showed an exposure of subchondral bone and severe osteoarthritis.

10.4 Histological findings after transplantation of layered chondrocyte sheets. A histological analysis of an in vivo study (left side: Safranin-O staining, right side: Toluidine blue staining): (a) the normal articular cartilage of the Japanese white rabbit femoral chondrocyte; (b) the partial thickness defect model; (c) the partial thickness defect models which covered the three-layered chondrocyte sheets. These showed a better stainability than those not covered by the chondrocyte sheets (d). The partial thickness defect models themselves showed progressive cartilage degeneration with fibrillation (d). The arrows demonstrate the layered chondrocyte sheets (c) (bar: 100 m).

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The importance of treatment and prophylaxes for osteoarthritis is increasing due to a progressively ageing society. However, there are only a few conservative therapies available at this time, such as non-steroidal anti-inflammatory drug (NSAID) administration and the injection of hyaluronic acid. There is still no means of preventing the future exacerbation of cartilage degeneration. Based on the results of this study, the use of bioengineered chondrocyte sheets may be potentially useful in the treatment of partial thickness defects of articular cartilage. The advantages of such cell sheets are that they are easy to culture and grow and, most importantly, they show good adhesion and barrier functions. This means that they can protect against intra-articular catabolic factors while also preventing the escape of proteoglycan from the injured site. They have a promising growth factor supply and furthermore, such cell sheets could be useful as an alternative to periosteum grafting, which is the most commonly used treatment. Good adhesion and the inhibition of cartilage degeneration at the injured sites were also confirmed even after the experimental study of allografts of layered chondrocyte sheets had been running for 2 months. The sites where the cell sheets showed adhesion were well stained with safranin-O. Therefore, it is suggested that multilayered chondrocyte sheets may serve as a barrier for preventing proteoglycan loss from damaged cartilage, while also protecting the injured site from the catabolic factors in synovial fluid. A strategy has been developed for repairing a full thickness defect of articular cartilage using the layered cell sheets because it is necessary to treat the bleeding from the bone marrow.

10.4

Properties of chondrocyte sheets

This section will discuss the properties of chondrocyte sheets. In particular, layered chondrocyte sheets contain few of the destructive factors that cause cartilage to degenerate and they also have good adhesion properties that help to both protect and repair the cartilage surface.

10.4.1 Human articular chondrocytes The cells used for this in vitro experiment included human articular chondrocytes obtained from patients who had undergone anterior cruciate ligament reconstruction and had given their informed consent at the Tokai University Oiso Hospital from December 2004 to August 2005. Chondrocytes were obtained while forming the interfoveolar ligament and they were then isolated by enzymatic treatment. Twenty-five knees of 25 patients aged 14 to 49 years (average 23 years, 19 males and 6 females) were used as the source of these cells. The chondrocytes were enzymatically dissociated and were then seeded and cultured according to the method of Sato et al.29

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10.4.2 RNA isolation and cDNA synthesis Total RNA was isolated using the RNeasy Mini kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. The RNA quality in each sample was confirmed by the A260/280 absorbance ratio and by electrophoresis on a 1.2% agarose formaldehyde gel. Approximately 1.0±2.0 g of total RNA was reverse transcribed into single strand cDNA using Moloney murine leukemia virus (MuLV) reverse transcriptase (Applied Biosystems, Foster City, CA). The reverse transcriptase (RT) reaction was carried out for 60 min at 42 ëC and then for 5 min at 95 ëC in a thermocycler.

10.4.3 Primer design and real-time polymerase chain reaction (PCR) All oligonucleotide primer sets were designed based upon the published mRNA sequence. The expected amplicon lengths ranged from 70 bp to 200 bp. The oligonucleotide primers used in this study are listed in Table 10.1. Real-time polymerase chain reaction (PCR) was carried out in a SmartCycler II (Cepheid, Sunnyvale, CA) using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). For real-time PCR, 2±2.5 l of cDNA template was used in a final volume of 25 l. The cDNA was amplified according to the following conditions: 95 ëC for 15 s and 60 ëC for 60 s for 35 to 45 amplification cycles. Fluorescence changes were monitored with SYBR Green after every cycle. A Table 10.1 List of primers used in real-time PCR Primer ID

Accession No.

Collagen Type I-F Collagen Type I-R Collagen Type II-F Collagen Type II-R Aggrecan1-F Aggrecan1-R Fibronectin1-F Fibronectin1-R MMP3-F MMP3-R MMP13-F MMP13-R TIMP1-F TIMP1-R ADAMTS5-F ADAMTS5-R GAPDH-F GAPGH-R

NM_000088

Sequence

AAG GGT GAG ACA GGC GAA CAA TTG CCA GGA GAA CCA GCA AGA NM_033150 GGA CTT TTC TTC CCT CTC T GAC CCG AAG GGT CTT ACA GGA NM_001135 TCG AGG ACA GCG AGG CC TCG AGG GTG TAG GCG TGT AGAGA NM_001030524 GCA CAG GGG AAG AAA AGG AG TTG AGT GGA TGG GAG GAG AG NM_002422 ATT CCA TGG AGC CAG GCT TTC CAT TTG GGT CAA ACT CCA ACT GTG NM_002427 TCA CGA TGG CAT TGC TGA CA AGG GCC CAT CAA ATG GGT AGA NM_003254 CAG CGT TAT GAG ATC AAG ATG GAC CA AGT GAT GTG CAA GAG TCC ATC CTG NM_007038 GAG CCA AGG GCA CTG GCT ACT A CGT CAC AGC CAG TTC TCA CACA NM_002046 GCA CCG TCA AGG CTG AGA AC ATG GTG GTG AAG ACG CCA GT

Expected size (bp) 170 113 94 189 138 77 186 120 142

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melting-curve analysis was performed (0.5 ëC/s increase from 55 to 95 ëC with continuous fluorescence readings) at the end of the cycles to ensure that single PCR products were obtained. The amplicon size and reaction specificity were confirmed by 2.5% agarose gel electrophoresis. All reactions were repeated in six separate PCR runs using RNA isolated from four sets of human samples. The results were evaluated using the SmartCycler II software program. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used to normalize the samples. To monitor crossover contamination of PCR, RNase-free water (Qiagen Inc., Valencia, CA) was included in the RNA extraction and used as a negative control. To ensure the quality of the data, a negative control was always applied in each run.

10.4.4 Analysis of gene expression Layered chondrocyte sheets were shown to adhere to porcine cartilage after a oneday organ culture. It is possible that an increase of fibronectin in the multilayered chondrocyte sheets may have been involved (Fig. 10.5). Good adhesion could thus be obtained because harvesting without enzymatic treatment makes it possible to better preserve the activity of both fibronectin and adhesion proteins such as integrin. Matrix±matrix interactions therefore play an important role in the adhesion between chondrocyte sheets and injured cartilage, and specific enzymes may modify the surface of each matrix and permit interaction between integrin proteins and fibronectin. Although this adhesive phenomenon is currently being studied using a cDNA microarray, some of the results were demonstrated here. The expression of fibronectin1, collagen type II, aggrecan 1 and TIMP1 mRNA

10.5 The expression of fibronectin. The localization of fibronectin was the surface of the layered chondrocyte sheet. The high magnification demonstrated that the expression of fibronectin localized among extracellular matrices and cell-cell junctions.

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was observed at significantly high levels, while the expression of collagen type I, MMP3 and ADAMTS5 was at significant levels in the layered chondrocyte sheets in comparison to the monolayer culture. Another interesting aspect of these chondrocyte sheets is the fact that catabolic factors, such as MMP3,30±33 MMP1332±35 and ADAMTS536,37 were observed to decrease at the time of layering, while the expression of TIMP1 with antagonistic actions against MMP3

10.6 The relative expressions of mRNA of key genes. The mRNA expression of fibronectin 1 of three-layered chondrocyte sheets was higher than monolayer culture. The mRNA expressions of aggrecan and type II collagen of chondrocyte sheets were higher than that of monolayer culture. The mRNA expression of type I collagen demonstrated a low level in the three-layered chondrocyte sheets. The expressions of MMP3 and ADAMTS5, which promote cartilage degeneration were low in the three-layered chondrocyte sheets while the mRNA expression of TIMP1, which is an antagonistic factor of MMP3, showed a significantly high level in the three-layered chondrocyte sheets. Mono: conventional monolayer culture, UpCell: monolayer culture using temperature-responsive culture dish, Layered: layered chondorocyte sheets (±-: P < 0:05).

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increased (Fig. 10.6). The layered chondrocyte sheets to be transplanted had low levels of the enzymes that degenerate cartilage and had good adhesion properties that help to both protect and repair the cartilage surface.

10.5

Future trends in cartilage repair

The development of a less invasive therapeutic approach for grafting a cell sheet to the injured site may be fundamental in expanding its use for the treatment of patients demonstrating the early stages of osteoarthritis. This section will discuss future trends in cartilage repair. In the future, it will be important to determine how long such chondrocyte sheets can adhere to and live on the grafted sites, while also clarifying the optimum conditions for the adhesion of other cell sheets, such as synovial sheets, to injured sites. Therefore, use of a combination of both chondrocyte sheets and synovial sheets may also be possible. Although further research is necessary, the use of chondrocyte sheets is useful for the treatment of partial thickness defects of articular cartilage. Scaffolds may not always be fundamental to the engineering of regenerative tissues. Today, many kinds of scaffold are used, such as atellocollagen, polyglycolide (PGA), poly-lactic-co-glycolic acid (PLGA) and poly-L-lactic acid (PLLA). These compounds are both biocompatible and biodegradable. Although they provide the advantage of initial strength against loading on the engineered tissue, they also introduce the possibility of side effects, such as a foreign-body reaction. Therefore, the long-term effectiveness of these scaffolds is questionable. Scaffold-free tissue engineering using novel technologies could be applied to cartilage repair in the near future.

10.6

Regulations regarding regenerative medicine in Japan

Finally, we will discuss some of the problems in the development of laws for regenerative medicine in Japan, which have not been improved as of September 2009. The Japanese medical system is structured to ensure the future safety and validity of both medical products and medical devices during the business development phase, in accordance with the Pharmaceutical Law. However, this Pharmaceutical Law is based on uniform manufacturing and selling practices for the general public and provides for only the two categories of medical products and medical devices. Therefore, if the concepts of the Pharmaceutical Law are applied directly to technology for regenerative medicine, which provides customized processes for autologous cells, it would result in requirements and stipulations that are far removed from the actual treatment situation, thus interfering with the spread of treatments that use the tissue engineering of autologous cells. In 2006, the Ministry of Health, Labour and Welfare presented

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a set of guidelines for clinical research using human stem cells, but much like the Pharmaceutical Law, the guidelines fail to differentiate between autologous and allogeneic cells, and the differences between cell provision to the general public and technology provision through customized processes using autologous cells are not specified. The process of applying the achievements of clinical research using autologous cells for practical and general use requires a completely different qualification system from that of the conventional Pharmaceutical Law and the guidelines for stem cells. We strongly feel that new legislation is therefore urgently required in order to quickly realize the full potential of regenerative medicine using autologous cells for patients. Unfortunately, if this process is delayed, we will face a situation in which most of these products and technologies will have to be imported, much like the medical devices and materials currently being used in the clinical field in Japan.

10.7

References

[1] T. Okano, N. Yamada, H. Sakai, and Y. Sakurai, A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(Nisopropylacrylamide), J Biomed Mater Res 27 (1993) 1243±1251. [2] T. Okano, N. Yamada, M. Okuhara, H. Sakai, and Y. Sakurai, Mechanism of cell detachment from temperature-modulated, hydrophilic-hydrophobic polymer surfaces, Biomaterials 16 (1995) 297±303. [3] K. Nishida, M. Yamato, Y. Hayashida, K. Watanabe, N. Maeda, H. Watanabe, K. Yamamoto, S. Nagai, A. Kikuchi, Y. Tano, and T. Okano, Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface, Transplantation 77 (2004) 379±385. [4] K. Nishida, M. Yamato, Y. Hayashida, K. Watanabe, K. Yamamoto, E. Adachi, S. Nagai, A. Kikuchi, N. Maeda, H. Watanabe, T. Okano, and Y. Tano, Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium, N Engl J Med 351 (2004) 1187±1196. [5] S. Sekiya, T. Shimizu, M. Yamato, A. Kikuchi, and T. Okano, Bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential, Biochem Biophys Res Commun 341 (2006) 573±582. [6] T. Shimizu, M. Yamato, Y. Isoi, T. Akutsu, T. Setomaru, K. Abe, A. Kikuchi, M. Umezu, and T. Okano, Fabrication of pulsatile cardiac tissue grafts using a novel 3dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces, Circ Res 90 (2002) e40±48. [7] T. Shimizu, M. Yamato, T. Akutsu, T. Shibata, Y. Isoi, A. Kikuchi, M. Umezu, and T. Okano, Electrically communicating three-dimensional cardiac tissue mimic fabricated by layered cultured cardiomyocyte sheets, J Biomed Mater Res 60 (2002) 110±117. [8] T. Shimizu, M. Yamato, A. Kikuchi, and T. Okano, Two-dimensional manipulation of cardiac myocyte sheets utilizing temperature-responsive culture dishes augments the pulsatile amplitude, Tissue Eng 7 (2001) 141±151. [9] Y. Shiroyanagi, M. Yamato, Y. Yamazaki, H. Toma, and T. Okano, Urothelium regeneration using viable cultured urothelial cell sheets grafted on demucosalized gastric flaps, BJU Int 93(7) (2004) 1069±1075.

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[10] D. Murakami, M. Yamato, K. Nishida, T. Ohki, R. Takagi, J. Yang, H. Namiki, and T. Okano, Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells, J Artif Organs 9(3) (2006) 185±191. [11] D. Murakami, M. Yamato, K. Nishida, T. Ohki, R. Takagi, J. Yang, H. Namiki, and T. Okano, The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets, Biomaterials 27(32) (2006) 5518±5523. [12] T. Ohki, M. Yamato, D. Murakami, R. Takagi, J. Yang, H. Namiki, T. Okano, and K. Takasaki, Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in a canine model, Gut 55(12) (2006) 1704±1710. [13] M. G. Flores, M. Hasegawa, M. Yamato, R. Takagi, T. Okano, and I. Ishikawa, Cementum-periodontal ligament complex regeneration using the cell sheet technique, J Periodontal Res 43(3) (2008) 364±371. [14] M. Hasegawa, M. Yamato, A. Kikuchi, T. Okano, and I. Ishikawa, Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model, Tissue Eng 11(3±4) (2005) 469±478. [15] T. Akizuki, S. Oda, M. Komaki, H. Tsuchioka, N. Kawakatsu, A. Kikuchi, M. Yamato, T. Okano, and I. Ishikawa, Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs, J Periodontal Res 40(3) (2005) 245±251. [16] K. Ohashi, T. Yokoyama, M. Yamato, H. Kuge, H. Kanehiro, M. Tsutsumi, T. Amanuma, H. Iwata, J. Yang, T. Okano, and Y. Nakajima, Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets. Nat Med 13(7) (2007) 880±885. [17] Y. Tsuda, A. Kikuchi, M. Yamato, A. Nakao, Y. Sakurai, M. Umezu, and T. Okano, The use of patterned dual thermoresponsive surfaces for the collective recovery as co-cultured cell sheets, Biomaterials 26(14) (2005) 1885±1893. [18] M. Yamato, O. H. Kwon, M. Hirose, A. Kikuchi, and T. Okano, Novel patterned cell coculture utilizing thermally responsive grafted polymer surfaces, J Biomed Mater Res 55(1) (2001) 137±140. [19] M. Harimoto, M. Yamato, M. Hirose, C. Takahashi, Y. Isoi, A. Kikuchi, and T. Okano, Novel approach for achieving double-layered cell sheets co-culture: overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature-responsive culture dishes, J Biomed Mater Res 62(3) (2002) 464±470. [20] N. Kaneshiro, M. Sato, M. Ishihara, G. Mitani, H. Sakai, and J. Mochida, Bioengineered chondrocyte sheets may be potentially useful for the treatment of partial thickness defects of articular cartilage, Biochem Biophys Res Commun 349(2) (2006) 723±731. [21] N. Kaneshiro, M. Sato, M. Ishihara, G. Mitani, H. Sakai, T. Kikuchi, and J. Mochida, Cultured articular chondrocytes sheets for partial thickness cartilage defects utilizing temperature-responsive culture dish, European Cells Mater 13 (2007) 87±92. [22] S. W. O'Driscoll, and R. B. Salter, The repair of major osteochondral defects in joint surfaces by neochondrogenesis with autogenous osteoperiosteal grafts stimulated by continuous passive motion. An experimental investigation in the rabbit, Clin Orthop Relat Res (1986) 131±140. [23] Y. Matsusue, T. Yamamuro, and H. Hama, Arthroscopic multiple osteochondral transplantation to the chondral defect in the knee associated with anterior cruciate

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ligament disruption, Arthroscopy 9 (1993) 318±321. [24] M. Brittberg, A. Lindahl, A. Nilsson, C. Ohlsson, O. Isaksson, and L. Peterson, Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation, N Engl J Med 331 (1994) 889±895. [25] M. Ochi, Y. Uchio, K. Kawasaki, S. Wakitani, and J. Iwasa, Transplantation of cartilage-like tissue made by tissue engineering in the treatment of cartilage defects of the knee, J Bone Joint Surg Br 84 (2002) 571±578. [26] S. Wakitani, K. Imoto, T. Yamamoto, M. Saito, N. Murata, and M. Yoneda, Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees, Osteoarthritis Cartilage 10 (2002) 199±206. [27] E. B. Hunziker, and L. C. Rosenberg, Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane, J Bone Joint Surg Am 78 (1996) 721±733. [28] M. Yamato, M. Utsumi, A. Kushida, C. Konno, A. Kikuchi, and T. Okano, Thermoresponsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature, Tissue Eng 7 (2001) 473±480. [29] M. Sato, T. Asazuma, M. Ishihara, T. Kikuchi, K. Masuoka, S. Ichimura, M. Kikuchi, A. Kurita, and K. Fujikawa, An atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS-scaffold) for the culture of annulus fibrosus cells from an intervertebral disc, J Biomed Mater Res A 64 (2003) 248±256. [30] A. Fehrenbacher, E. Steck, M. Rickert, W. Roth, and W. Richter, Rapid regulation of collagen but not metalloproteinase 1, 3, 13, 14 and tissue inhibitor of metalloproteinase 1, 2, 3 expression in response to mechanical loading of cartilage explants in vitro, Arch Biochem Biophys 410 (2003) 39±47. [31] E. J. Uitterlinden, H. Jahr, J. L. Koevoet, Y. M. Jenniskens, S. M. Bierma-Zeinstra, J. Degroot, J. A. Verhaar, H. Weinans, and G. J. van Osch, Glucosamine decreases expression of anabolic and catabolic genes in human osteoarthritic cartilage explants, Osteoarthritis Cartilage 14 (2006) 250±257. [32] M. Fichter, U. Korner, J. Schomburg, L. Jennings, A. A. Cole, and J. Mollenhauer, Collagen degradation products modulate matrix metalloproteinase expression in cultured articular chondrocytes, J Orthop Res 24 (2006) 63±70. [33] J. Flannelly, M. G. Chambers, J. Dudhia, R. M. Hembry, G. Murphy, R. M. Mason, and M. T. Bayliss, Metalloproteinase and tissue inhibitor of metalloproteinase expression in the murine STR/ort model of osteoarthritis, Osteoarthritis Cartilage 10 (2002) 722±733. [34] A. R. Klatt, G. Klinger, O. Neumuller, B. Eidenmuller, I. Wagner, T. Achenbach, T. Aigner, and E. Bartnik, TAK1 downregulation reduces IL-1beta induced expression of MMP13, MMP1 and TNF-alpha, Biomed Pharmacother 60 (2006) 55±61. [35] H. Ling, and A. D. Recklies, The chitinase 3-like protein human cartilage glycoprotein 39 inhibits cellular responses to the inflammatory cytokines interleukin-1 and tumour necrosis factor-alpha, Biochem J 380 (2004) 651±659. [36] A. M. Malfait, R. Q. Liu, K. Ijiri, S. Komiya, and M. D. Tortorella, Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage, J Biol Chem 277 (2002) 22201±22208. [37] S. S. Glasson, R. Askew, B. Sheppard, B. Carito, T. Blanchet, H. L. Ma, C. R. Flannery, D. Peluso, K. Kanki, Z. Yang, M. K. Majumdar, and E. A. Morris, Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis, Nature 434 (2005) 644±648.

11

Cell therapies for articular cartilage repair: chondrocytes and mesenchymal stem cells R . A N D R I A M A N A L I J A O N A , University of Caen, France

Abstract: This chapter discusses the difficulties of maintaining the chondrogenic phenotype in culture. The chapter first describes chondrogenesis and the changes that occur in the chondrocyte phenotype. The particular features of the chondrocyte and its environment are then described. Finally, the various in vitro methods developed to obtain differentiated chondrocytes for clinical use are reviewed. Key words: dedifferentiated and hypertrophic chondrocytes, mesenchymal stem cells, chondrogenesis, cell therapy for cartilage repair.

11.1

Introduction

Articular cartilage is a highly specialized tissue that covers the distal end of the bones. It is produced by chondrocytes and is avascular and devoid of lymph vessels and nerves. It resists compression and tension and allows bones to slide over each other during movement. Cartilage has a lower metabolic activity than tissues such as muscle or bone, and is less sensitive to attacks. Articular cartilage has a limited capacity for self-regeneration in the case of injury because of its nature and complexity. Mature chondrocytes have relatively low metabolic activity and proliferation capacity. The abundant extracellular matrix (ECM) may act as a barrier against chondrocyte migration to defects and, as the tissue is avascular, there is no direct access to progenitor cells. Self-regeneration is also limited because growth and differentiation promoting factors can only be supplied via diffusion from the synovial fluid. A few long-term treatments for damaged or failed cartilage are available today, including mosaicplasty, periosteal transplantation and autologous chondrocyte implantation (ACI) (Hangody et al., 1998; Brittberg, 1999; O'Driscoll, 1999). However, these approaches do not result in the regeneration of fully functional hyaline-like cartilage. Tissue engineering based on cell therapy is therefore one of the most promising new approaches to repairing articular cartilage. This process involves the use of various cell types, but their phenotypic state and potential to be fully chondrogenic have a profound effect on their ability to direct functional tissue restoration.

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This chapter first describes chondrogenesis and the changes that occur in the chondrocyte phenotype. I then describe the particular features and environment of the chondrocyte, and finally discuss various in vitro culture systems which may promote maintenance of the chondrogenic phenotype.

11.2

The chondrocyte: a unique cell

Chondrocytes are the only cellular constituent of cartilage and are responsible for the synthesis and degradation of the matrix components of cartilage. Each cell is isolated from its neighbours by ECM and has no direct access to the vascular system. This leaves chondrocytes under conditions of low oxygen concentration (Brighton and Heppenstall, 1971; Silver, 1975), making them dependent mainly on anaerobic metabolism. Chondrocytes can survive in oxygen tension as low as <0.1%, indicating that they are well adapted to hypoxia. Under hypoxic conditions, they produce less lactic acid and are less metabolically active than in normoxia (Rajpurohit et al., 1996). Oxygen, as well as other nutrients, must diffuse into the tissue from the synovial fluid. An oxygen gradient exists in cartilage, the superficial layers having a tension of 6% O2 and the deeper zones less than 1% O2 (Silver, 1975). There are various types of cartilage, depending on the age of the individual. Chondrocytes thus have a different phenotype depending on the growth stage or age of the individual, and also differ in size, shape and metabolic activity in different areas of cartilage. The functional specificity of the different types of cartilage depends on the degree of cell proliferation and maturation, the nature of proteins synthesized and the three-dimensional arrangement of these proteins around the cell.

11.2.1 Embryonic origin and chondrocyte differentiation Chondrocytes are derived from pluripotent mesenchymal stem cells (MSC), which are able to differentiate into chondrocytes and osteocytes. The formation of cartilage in the proximal and distal buds of the members in the chicken embryo is characterized by concentration of prechondrogenic MSCs. During this process, the cells are grouped at sites of future skeletal elements as a result of multiple growth factors and morphogens such as Wnts, the transforming growth factor superfamily (TGF s), including TGF s and bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs) (reviewed in Steinert et al., 2007). At this stage, MSCs express type I and type IIA collagens, which are nonspecific for chondrocytes (reviewed in Archer and Francis-West, 2002). Type IIA procollagen, one of the two splice forms of type II procollagen, contains an additional 69-amino acid cysteine-rich domain and is present in human prechondrogenic and newly formed cartilage tissues (Sandell et al., 1994; Valcourt et al., 2003). High expression of fibronectin, hyaluronan, CD44, neural

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cadherin and neural cell adhesion molecules has also been detected at these sites, as well as numerous intracellular signalling pathways transduced by integrins, mitogen-activated protein kinases, protein kinase C, lipid metabolites and cyclic AMP (reviewed in Steinert et al., 2007). These elements suggest that aggregation and condensation of chondrocytes are mediated by both cell±cell and cell±matrix adhesion (Kulyk et al. 1989; Oberlender and Tuan, 1994; reviewed in DeLise et al., 2000). Chondrogenic differentiation, in which prechondrocytes differentiate into mature chondrocytes, occurs under the influence of factors still poorly defined. This process is characterized by the extinction of the genes encoding type I collagen and cellular adhesion molecules, and the expression of a number of matrix proteins specific to chondrocytes, including type IX and XI collagens, and proteoglycans (PGs) such as aggrecan. The type II collagen mRNA splice form switches from type IIA to IIB procollagen, the major ECM component of cartilage (Sandell et al., 1994). Some factors involved in chondrogenic differentiation have been characterized in mice and mammals. Among them are the growth factors FGF-2, 4 and 8 (Cohn et al., 1995; reviewed in Hall and Miyake, 2000), members of the TGF and BMP families (Duprez et al. 1996; Pizette and Niswander, 2000) and transcription factors of the Sox family, Sox9, L-Sox5 and Sox6 (Lefebvre and de Combrugghe 1998, review in de Combrugghe et al., 2000). Sox9 promotes chondrocyte differentiation by activating chondrocyte-specific enhancer elements in type II, IX, XI collagens and aggrecan genes (Lefebvre et al., 2001). Sox9 deletion in undifferentiated limb bud MSCs results in a complete failure of chondrogenesis, and if the gene is inactivated following initiation of chondrogenesis (in collagen type I-expressing cells), most cells fail to undergo differentiation into chondrocytes, i.e. they do not reach the differentiation stage (Akiyama et al., 2002).

11.2.2 The maturation of differentiated chondrocytes: cell proliferation and hypertrophy The chondrocyte in growth cartilage In growth cartilage, approximately 50% of the total volume of tissue is cellular. The chondrocytes are characterized by a high potential for cell division, forming many columns of proliferative cells. The activity of proliferation is associated with a renewal of `reserve' cells by the recruitment of prechondrocytes. Chondrocytes reach their highest level of metabolic activity in this phase and secrete matrix molecules until each cell is included in a dense matrix divided into two zones: a peripheral area which encompasses and extends over the articular surface, and a central area which also serves as the centre of the endochondral ossification of the epiphysis. The tissue becomes recognizable

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under the microscope as cartilage when matrix accumulation separates the cells and they take a spherical shape. Members of the FGF family, including FGF-2, -4, -8, -9, -10 and -18, together with the FGF receptors, FGFR1, 2 and 3, coordinate patterning and cell proliferation during chondrogenesis and endochondral ossification in embryonic and postnatal growth plates. Sox9 is also expressed by proliferative chondrocytes in growth cartilage and induces the expression of components of cartilage matrix, including collagen type II in vitro (reviewed by Lefebvre and Smits, 2005). Cartilage hypertrophy and regulation Once the cartilage model is established, chondrocytes in the central region increase in size and become hypertrophic. They secrete and organize a different extracellular matrix, characterized by the synthesis of type X collagen and decreased expression of type IIB collagen (Oegema and Thompson, 1990). Type X collagen was first identified in the culture medium of chicken embryonic chondrocytes and is considered a specific marker for hypertrophic chondrocytes (Capasso et al., 1982; Schmid and Conrad, 1982). Few transcriptional determinants specifying hypertrophic chondrocytespecific type X collagen gene expression have been identified to date. One candidate is the runt domain transcription factor Runx2/Cbfa, which can directly transactivate Col10a1 promoter, both in vitro and in vivo, through putative Runx2 binding sites found in this promoter region (Kawaguchi, 2008; Higashikawa et al., 2009). Runx2 is assisted in this task by Runx3 (reviewed by Lefebvre and Smits, 2005). It has also been shown to be required for BMP-2 induced differentiation of MSCs into the osteoblast lineage, by inducing osteocalcin and type I collagen expression (reviewed in Katagiri and Takahashi, 2002; Bae et al., 2007; Javed et al., 2009). As well as Runx 2, binding of Sp-1 transcription factor on two canonical Sp1 binding sites on the Col10a1 promoter region also appears to be critical for high level expression of type X collagen in hypertrophic chondrocytes (Long et al., 1998; Magee et al., 2005). Several studies have revealed that the normal initiation and progression of chondrocyte hypertrophy depends on the balance between transcriptional activation and repression of Runx2. Myocyte enhancer factor-2 (MEF2C) acts upstream of Runx2 in the induction of chondrocyte hypertrophy, as Mef2c-null mice fail to undergo hypertrophy and to express Runx2 (Arnold et al., 2007). Other transcription factors repress Runx2 activity and delay terminal hypertrophic differentiation, and these include Sox9, Sox5 and Sox6 (Smits et al., 2004; Zhou et al., 2006), Nkx3/Bapx1, which mediates the actions of parathyroid-hormone-related protein (PTHrP) (Provot et al., 2006), and HDAC4 (Vega et al., 2004; Arnold et al., 2007). Runx2 activates Indian hedgehog (IHH) promoter and stimulates its expression (Yoshida et al., 2004). IHH is expressed

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in prehypertrophic chondrocytes in the growth plate and maintains the expression of PTHrP, which inhibits Runx2 expression, delaying chondrocyte hypertrophy (Guo et al., 2006). Therefore, Runx2 contributes to the IHH/ PTHrP negative feedback loop, and thus plays an important role in maintaining an appropriate balance between continued proliferation and progression to hypertrophy in chondrocytes in growth cartilage.

11.2.3 Hypertrophic cartilage mineralization The hypertrophic cartilage becomes vascularized and is infiltrated by osteoprogenitor cells in the process of calcification. The chondrocytes undergo apoptosis, while the osteoprogenitor cells differentiate into osteoblasts and replace the cartilage with mineralized bone tissue (reviewed in Steinert et al., 2007). Calcium deposition begins at various locations around the cells and eventually colonizes the extracellular matrix. This mineralization promotes cartilage degradation by osteoclasts, whose activities are stimulated by interaction with the calcified matrix (Cancedda et al., 1995). The growth cartilage disappears at around the age of 20 years, leaving hyaline cartilage in the adult.

11.2.4 The chondrocyte in adult cartilage Mature chondrocytes are distinguished from other cells by their spheroid shape, the synthesis of type IIB collagen, large aggregates of proteoglycans and specific non-collagenous proteins. The lack of vascularization means that mature chondrocytes have a mainly anaerobic metabolism, and receive nutrients only by diffusion from the synovial fluid. Since they are surrounded by extracellular matrix, there is little if any cell-to-cell contact. In normal adult articular cartilage, the process of cell maturation continues in chondrocytes, but has different characteristics. The supply of prechondrocytes ceases at an early age (not specified). This lack of recruitment is compensated for by a decrease in the speed and intensity of maturation, with proliferative clones of only two to three cells and expression of type X collagen in trace amounts. The percentage of renewal of matrix proteins, particularly of PGs that have a short half-life (7 to 200 days), decreases as well. The adult chondrocyte is still responsible for the maintenance of cartilage homeostasis through the synthesis and degradation of macromolecular components. Regulation of this is influenced by anabolic factors such as TGF , BMP, insulin-like growth factor IGF-1 and catabolic factors including proinflammatory cytokines interleukin-1, interleukin-6 and tumor necrosis factor-.

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271

The macromolecular network and biomechanical properties of cartilage

The role played by cartilage tissue may be attributed to its specific biomechanical properties, which result from its ECM. Articular cartilage ECM has two components: tissue fluid (nearly 70% by weight of articular cartilage) and structural macromolecules. The interaction of liquid with macromolecules gives the tissue its mechanical strength and elasticity, as well as the potential to absorb compressive, tensional and shearing forces applied externally. The framework of the macromolecules is mostly responsible for the form and stability of cartilage. The macromolecules in cartilage, collagens, proteoglycans and noncollagenous proteins make up 30% of the tissue. Collagen fibrils and PG aggregates give hyaline cartilage its unique physiological properties. Noncollagenous proteins (cartilage oligomeric matrix protein, fibronectin, tenascins, matrilins, link protein, etc.) are involved in stabilizing the macromolecular structure and chondrocyte±matrix interactions.

11.3.1 Collagens In articular cartilage, 90±95% of the collagen is type IIB. It is considered, with the aggrecan, as the main marker of the chondrocyte phenotype and is one of the fibrillar collagens. A molecule of collagen type II is composed of three chains, 1, associated to form a triple helix. After synthesis, collagen type II molecules join to form fibrils which then join by covalent bridges to form collagen fibres. These fibres are organized into a tight mesh network that extends throughout the tissue and provides the articular cartilage with its tensile stiffness and strength, and also contributes to tissue cohesion by mechanically trapping large PGs. Collagen type II interacts with chondrocytes through members of the integrin family and the non-integrin collagen receptor, discoidin domain receptor-2 (DDR2; a receptor tyrosine kinase; reviewed by Leitinger and Hohenester, 2007). It is required for cell survival, as demonstrated by the use of Col2a1-null mice (Yang et al., 1997). Other types of collagen are expressed and are minor, making up 10% of collagen tissue. The non-fibrillar collagen type VI is located in the pericellular matrix and interacts with a large number of matrix molecules (Bidanset et al., 1992; Chang et al., 1997). Molecules of collagen type IX are covalently bonded in the fibres of collagen type II (van der Rest and Mayne, 1988) and connect the mesh network of collagen fibrils to PGs. Molecules of collagen type XI are localized within the fibres of collagen type II and ensure the cohesion of the fibres. Proteoglycans PGs are molecules that consist of a protein axis binding one or more glycosaminoglycan (GAG) chains (Roughley and Lee, 1994; reviewed in

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Buckwalter and Mankin, 1998). GAG chains are long strings of negative charges (carboxylate or sulphate groups), which repel other negative charges and attract cations. By their ability to bind growth factors, these molecules regulate bioavailability, stability and biological activity, and play important roles as modulators of proliferation and cell adhesion. Extracellular PGs are mainly represented by aggrecan (Hardingham et al., 1994), which has a high molecular weight (between 1500 and 4000 kDa). One hundred aggrecan molecules can bind to a hyaluronic acid polymer, via the link protein (LP), forming a molecular aggregate with a high molecular mass. Aggrecan's biological properties are based partly on its anionic character and partly on the multiple interactive properties of its various fields with extracellular matrix (ECM) and membrane surface components. The large aggrecans mainly confer resistance to compression due to their hydrophilic nature, because they attract a large amount of water into the intra- and intermolecular space. Other PGs are well-characterized and include decorin, biglycan, fibromoduline, syndecan and betglycan (Grover and Roughley, 1995; reviewed in ReÂdini, 2001). Their roles are related to their interaction with other matrix components (fibronectin, collagen, cytokines, TGF , etc.) in regulating stability, bioavailability and biological activity (Hildebrand et al., 1994). Biomechanical properties of articular cartilage Articular cartilage has the vital role of ensuring smooth movement between articular bone pieces, keeping the friction coefficient very low, while absorbing and distributing the pressure and minimizing contact stress. Cartilage lubrication is due to the viscoelastic properties of hyaluronic acid, the major constituent of synovial fluid. Under compression forces, cartilage behaves as a viscoelastic material. Under load, there is an instantaneous deformation, followed by a deformation phase that increases with time and stabilizes. This ability of cartilage to deform is directly related to the content of PG aggregates and negative charges. The overall volume of supramolecular complexes may decrease by 80% when cartilage is subjected to a load. Lower amounts of GAGs by dry weight of cartilage were reflected in lowered compression capability (Treppo et al. 2000; Kerin et al., 2002). Conversely, collagen type II fibres give cartilage a force of cohesion which limits deformation when the cartilage is subjected to a constraint. Thus, the more numerous and organized into parallel bundles the fibres are, the greater the resistance to deformation and fracture. These factors explain the orientation and organization of collagen fibres in the surface layer. Articulation is crucial for chondrocytes, and joint activity maintains chondrocyte synthesis activity.

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273

Phenotypic changes

Phenotypic changes are a central feature of chondrocytes. This has been shown by several studies of chondrocyte differentiation in vivo in the cartilage of the foetal growth plate, potentially in the process of osteoarthritis, and from the analysis of chondrocyte behaviour in vitro. Some osteoarthritic chondrocytes synthesize type IIA collagen, characteristic of chondroprogenitor cells (Aigner et al., 1999), indicating that osteoarthritic chondrocytes are reverting to the foetal phenotype. In the deepest osteoarthritic cartilage area, the cells begin to express collagen type X, characteristic of hypertrophic chondrocytes (Girkontaite et al., 1996), become apoptotic and induce calcification of the cartilage matrix. Several studies carried out in chondrocytes of normal cartilage and osteoarthritis have detected activated chondrocytes expressing collagen type III, mainly in the middle cartilage of osteoarthritis (Aigner et al., 1992, 1993, 1997). Collagens type I and III are characteristic of the fibrocartilage that occurs at various locations in the mammalian organism (intervetebral discs and menisci). In fibrocartilage, chondrocytes have a typically spheroid form and are surrounded by a pericellular matrix compartment. However, the large interterritorial region contains an abundance of collagen fibrils and thick fibres which have poor shock-absorbing capacity. This lack of stability in osteoarthritic chondrocytes has been studied particularly in vitro, in a monolayer culture, where the cells adopt a more elongated, fibroblastic morphology and switch from synthesizing cartilage-specific extracellular matrix macromolecules (such as aggrecan and collagen type II) to type I collagen. This phenotype is known as dedifferentiated chondrocyte and appears after several passages of cells or after treatment with various factors, such as retinoic acid or interleukin-1 (Goldring et al., 1988).

11.5

Cell therapy for articular cartilage repair: chondrocytes and mesenchymal stem cells (MSCs)

The variety of cells available for use in cartilage tissue engineering ranges from undifferentiated pluripotent stem cells to well-differentiated chondrocytes (Mackay et al., 1998; Brittberg, 1999; Barry et al., 2001).

11.5.1 The use of chondrocytes in cartilage cell therapy Chondrocytes are a natural and logical choice for articular repair applications. However, the limited capacity of donor sites to provide a large quantity of chondrocytes is a major impediment to autologous chondrocyte transplantation. Indeed, for clinical applications, cartilage slices can only be harvested from the

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non-articulating area of the knee joint of the patient. A biopsy provides a small number of cells, on average 4:5  105 cells/100 mg of cartilage from patients aged 40. Autologous chondrocyte implantation requires 5 to 30 million chondrocytes, depending on the size of defect to be filled and the procedure used. Thus, in vitro monolayer expansion is required to generate sufficient cells for implantation, but this leads to chondrocytes dedifferentiating to fibroblast-like cells, inducing the risk of generating fibrocartilage after implantation. In order to be used for clinical applications, dedifferentiated cells must be redifferentiated and phenotypical stability, with synthesis of aggrecan and type II collagen, has to be restored. Two main approaches have been attempted to make chondrocytes regain their cartilaginous features: a mechanical approach, mimicking the matrix±cell interaction environment, and a biochemical approach, treating the culture with various differentiating agents. The mechanical approach includes the regulation of oxygen tension, the use of mechanical forces and the creation of an environment supporting a spherical morphology, such as the use of various three-dimensional substrata, including alginate beads and plating on coated substrates. The biochemical approach involves modified culture conditions, including adding constituents such as growth factors and chemical compounds. Human articular cartilage can be obtained from femoral heads of patients undergoing joint arthroplasty or post-mortem. We generally isolate cells from human cartilage on the day of the operation, but when this is not possible, tissue can be refrigerated overnight and still gives satisfactory yields of chondrocytes. This may be particularly useful with human tissue, where the supply can be erratic. Cartilage is cut into small shavings and placed in phosphate-buffered saline (PBS) serum 1X. The extract is digested at 37 ëC with pronase E (4 mg/ml in PBS, 45 min, Sigma-Aldrich) and Clostridium hystoliticum collagenase (1 mg/ml, overnight, Sigma-Aldrich) in Dulbecco's Modified Eagle's Medium (DMEM) containing 4 mM L-glutamine and 10% fetal calf serum (FCS, Life Technologies), penicillin (100 IU/ml, Roger Bellon), erythromycin (100 g/ml, Qualimed) and fungizone (0.25 g/ml, Serva). The cell suspension is filtered through a 70 m mesh nylon, and then centrifuged (600g for 5 min). The pellet is washed twice in DMEM containing 10% FCS and the cells are counted. The chondrocytes can then be seeded and cultured in DMEM + 10% FCS until the desired confluency is obtained. For standard monolayer cultures, cells are plated at 3  104 cells/cm2 in plastic vessels. From the moment the chondrocytes begin to attach to the tissue culture plastic and spread out, they change their phenotype. When possible, higher densities should be used to minimize cell dedifferentiation.

11.5.2 The use of MSC in cartilage cell therapy Much interest has recently been aroused regarding the use of adult MSCs for cartilage repair. These cells have the potential to differentiate into different

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lineages, including chondrocytes with aggrecan and collagen type II expression (reviewed in Hwang and Elisseeff, 2009), in response to changes in external stimuli. The use of in vitro models of chondrogenesis could also lead to a better understanding of the mechanisms involved in each step and allow the development of new strategies to induce or maintain chondrocyte phenotype. Choosing the appropriate MSC MSCs are derived from various tissues, such as bone marrow, adipose tissue, synovial membrane and other connective tissues (Mackay et al., 1998; Barry et al., 2001; Sakaguchi et al., 2005). Human bone marrow MSCs are the best characterized and have clinical advantages, including the routine harvest of cells under local anaesthesia instead of general anaesthesia, the ability to avoid significant damage to articular donor sites, the possibility of expanding cell numbers in vitro prior to re-implantation, and a lack of immune reaction to the cell membrane. Bone marrow aspirates can be obtained from the iliac crests of adult donors. Marrow is aspirated into 20 ml of PBS containing 100 U/ml sodium heparin. Cells are fractionated on a Hypaque-Ficoll density gradient by centrifuging at 600g for 10 min. The interface mononuclear cells are isolated, the cells are washed twice by centrifugation in PBS and then seeded at a density of 5  104 nucleated cells/cm2. Cells are cultured in alpha-modified eaglels medium (MEM) supplemented with 10% FCS (Invitrogen), 2 mM L-glutamine, 100 U/ml penicillin, 0.025 mg/ml fungizone, 0.1 mg/ml streptomycine and 1 ng/ ml FGF-2 (Sigma Chemical Co.), at 37 ëC in a humidified atmosphere with 5% CO2. After 48 hours, cultures are washed with PBS to remove non-adherent materials. During the expansion, the medium is changed twice a week. At nearly 80% confluence, cells are harvested by trypsinization (0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA), Invitrogen), counted and seeded again at 103 cells/cm2. When the desired number of cells is obtained (passage 3±4), they are used for the differentiation procedure. Although less invasive than chondrocyte harvesting, procedures for obtaining MSCs from bone marrow or other tissues are more invasive than muscle biopsies. In light of its availability and the relative ease of muscle isolation, skeletal muscle has become an attractive source of cells for use in cartilage tissue engineering applications. Muscle biopsy can be conducted in outpatient clinics without special instruments. Skeletal muscle represents up to 40±50% of total human body weight, and we can harvest adequate amounts of muscle tissue because the donor's tissue can regenerate after the injury created by the biopsy. Although the amount of skeletal muscle tissue necessary for such an application is difficult to determine at this point, it has been reported that an open biopsy of 200 mg of the quadriceps femoris is enough to produce myogenic cells. This open biopsy procedure is not harmful to the donor.

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Muscle tissue has been extensively investigated as a potential source for isolation of pluripotent stem cells. Several studies have provided evidence of the existence of pluripotent stem cells in skeletal muscle that can differentiate into various lineages, including myogenic, hematopoietic, osteogenic, endothelial, and neuronal. Concerning cartilage repair, Adachi et al. (2002) reported comparable healing of cartilage defects treated with collagen gel containing either muscle-derived cells (MDCs) or chondrocytes. However, the number of studies that have examined the use of these cells in the treatment of articular cartilage is limited. Andriamanalijaona et al. (2008) demonstrated the potential of two subpopulations of muscle-derived cells (MDCs: CD56ÿ and CD56‡) to differentiate into chondrogenic lineage and therefore to serve as a source of progenitor cells for cartilage repair. The behaviour of the MDCs in the system resembled the differentiation pathway of the chondro-osseous rudiment already observed in pellet cultures of human bone marrow MSCs (Muraglia et al., 2003), suggesting that MDCs, like bone marrow stromal cells (BMSCs), are capable of undergoing a process reminiscent of endochondral ossification, and could be considered as pluripotent cells. Culture methodology for MDCs has been described previously (Vilquin et al., 2005). Briefly, the fresh biopsy is washed and finely minced using scissors. The muscle fragments are digested using collagenase (Roche), followed by trypsin±EDTA (Hyclone). The cell suspension is filtered through 100 then 40 m cell strainers (Falcon), pelleted and seeded (density of 2000±5000 cells per cm2). The proliferation medium contains 80% modified custom-made MCDB medium (Hyclone), 20% defined FBS (Hyclone), Gentamicine (50 g/ml, Panpharma), 10 ng/ml human recombinant bFGF, 10ÿ6 M dexamethasone (Merck). The medium is changed on the following day to remove tissular and cellular debris and to harvest the nonadherent fraction. The cultures are settled for 6±8 days, then the cells are harvested by trypsinization and expanded before reaching 80% confluency. The cells are grown in this medium for two to three passages, then shifted towards standard MSC proliferation medium for one or two passages (alpha-MEM supplemented with 20% FBS, 10-6 M dexamethasone, 50 g/ml gentamicine). The cells are used for chondrogenic differentiation assays on passages three to four, by which time they had undergone 8 to 12 divisions. The big challenge in using MSC for cartilage repair While collagen type X is supposed to be a marker for hypertrophic chondrocytes, its expression has been detected during in vitro chondrogenesis of MSC from bone marrow and from the entire fetal circulating blood without the addition of growth factors (Naruse et al., 2004; Mwale et al., 2006). Another report, using MSCs cultured in pellets under standard chondrogenic medium supplemented with TGF 1 and dexamethasone, demonstrated that type X

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collagen messages were detected throughout the pellet culture, including in the undifferentiated MSCs (Barry et al., 2001). Pelttari et al. (2006) revealed that up-regulation of collagen type X and ALP enzyme activity during in vitro chondrogenesis leads to strong matrix calcification accompanied by vascular invasion and even micro-ossicle formation after ectopic transplantation into subcutaneous pouches of severe combined immunodeficient (SCID) mice. The predisposition of bone marrow-derived MSCs towards osteogenesis and matrix calcification is not due to their originating from bone, since MSCs from adipose tissue mineralized their surrounding matrix in vivo to a similar extent when transplanted subcutaneously (Hennig et al., 2007). Type X collagens have also been detected in undifferentiated MDCs (Andriamanalijaona et al., 2008). In conclusion, standard protocols for chondrogenesis produce chondrocytes that undergo premature hyperthrophy and develop into transient, endochondral cartilage instead of articular cartilage-like tissue. The development of new culture methods should focus on how to stop chondrogenesis before hypertrophy, which is not desirable in articular cartilage repair tissue. Chondrogenic phenotype assessment Evidence for MSC differentiation into chondrocytes can be obtained by analysing ECM components such as collagen types I, IIA, IIB and X, and aggrecan core protein. Using these markers, the differentiation stage ± prechondrogenic, mature chondrocyte or hypertrophic chondrocyte ± can be determined It is easy to measure RNA expression using real-time reverse transcriptase polymerase chain reaction (RT-PCR), which only requires a small amount of material. However, RNA analysis is not sufficient and protein analysis is essential to further characterize the tissue, mainly for collagen because RNA detection does not always reflect protein expression. Proteins can be detected by immunohistochemical methods or by western blot analysis. The latter can provide more quantitative data, but has the disadvantage of requiring a large amount of the protein (10±15 g). The production of a PG-rich ECM can also be detected using histochemical staining of sections with safranin'O, toluidine blue or alcian blue. Determining transcription factor expression (Nkx3, Sox family, Runx2, HIF1, etc.) can help to identify the precise differentiation stage. Transcription factor binding activity can be analysed using electrophoretic mobility shift assays. Alkaline phosphatase activity can also be measured to confirm the hypertrophic phase.

11.6

The use of chemical compounds to enhance matrix production

Several chemical compounds have been demonstrated to promote chondrogenic differentiation in vitro, including dexamethasone, thyroid hormone, 1,25-

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dihydroxyvitamin D3, prostaglandin E2 (PGE2), ascorbic acid, ethanol and staurosporine (reviewed in Heng et al., 2004). These compounds can be used in defining culture milieu that promote ECM synthesis in chondrocytes or that direct the differentiation of stem cells. Sodium ascorbate and dexamethasone have been used in several studies in chondrogenic medium culture. The medium used for chondrogenesis corresponds to DMEM with antibiotics, 100 nM dexamethasone, 50 g/ml ascorbic acid-2-phosphate, 40 g/ml proline, 1 mM sodium pyruvate and a 1 : 100 dilution of Insulin Transferin Selenium supplement (Sigma Chemical Co.). The use of this last compound prevents interference of growth factor in tests for other factors. It was shown that this medium induced chondrogenesis in human bone marrow-derived MSCs (Fig. 11.1) and MDCs (Andriamnalijaona et al., 2008).

11.1 Chondrogenic medium and alginate beads culture induce human bone marrow MSCs differentiation into chondrocyte. Human bone marrow MSCs were cultured in alginate beads and in a chondrogenic medium during 7 days. Quantitative PCR were performed to measure aggrecan (a) and Type II collagen (b) mRNA levels at the beginning of the culure (C0), at days 1, 3 and 7. Bars represent mean  SEM of three different samples from three donors.

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Dexamethasone is a potent inducer of chondrogenic differentiation in humanderived MSCs (reviewed in Heng et al. 2004), possibly through dose-dependent increases of Sox9 (Sekiya et al., 2001). For CD56 MDCs, a proliferation media was used that contained dexamethasone from the onset of culture, which may have promoted the differentiation pathway. Sodium ascorbate can modulate chondrocyte growth and differentiation in monolayer cultures, increasing type II collagen expression (Sandell and Daniel, 1988; Venezian et al., 1998; de Haart et al., 1999). However, it was also found that alginate culture of MDCs with a chondrogenic medium induced increased levels of type I and type X collagen proteins after 21 days of culture, which could be attributed to the sodium ascorbate. Indeed, previous reports have demonstrated that high concentrations of ascorbate can lead to terminal differentiation and hypertrophy in chondrocytes (Shapiro et al., 1991; Farquharson et al., 1998).

11.7

Strategies to maintain the chondrogenic phenotype: the use of three-dimensional systems

Several studies have shown that three-dimensional culture systems are essential for maintaining articular chondrocytes in a fully differentiated state. Dedifferentiated human chondrocytes can redifferentiate in a three-dimensional high density culture following one to five passages, but more extensive expansion limits this capacity (Benya and Shaffer, 1982; Schulze-Tanzil et al., 2002). However, Benya and Shaffer (1982) also observed that a small fraction of low passage dedifferentiated chondrocytes do not differentiate, which could indirectly affect integration through compromising normal chondrocyte phenotype and function (Yan and Yu, 2007). Many natural substances are suitable for three-dimensional systems, including fibrin, agarose, alginate, collagen, chitosan and hyaluronic acid (reviewed in Chiang and Jiang, 2009). Many of these are hydrogels and can be injectable in their liquid form, which blends well with chondrogenic cells (Chiang et al., 2005). Natural substances have also been used to design and produce scaffolds in a rich variety of configurations including woven and nonwoven meshes, sponges, foams, hydrogels, glues, composite bilayer and trilayer hybrid solutions and, more recently, electrospun nanofibres (Geutjes et al., 2006; Barnes et al., 2007). Synthetic polymers, such as poly-glycolic acid (PGA), poly-L-lactic acid (PLA) and copolymer poly-lactic-co-glycolic acid (PLGA), allow a better control of shape, surface morphology, mechanical and physico-chemical properties than natural polymers. However, most synthetic polymers, especially the widely used polyesters such as PLA, PGA or PLGA, induce some inflammation in vivo. The feasibility of recombinant human type II collagen gel as a threedimensional culture system for bovine chondrocytes was recently evaluated in

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vitro and the results showed that, during the 4-week cultivation period, the chondrocytes were evenly distributed inside the gels, maintained their round phenotype, and the amount of the extracellular matrix (GAGs) increased (Pulkkinen et al., 2008). Shakibaei et al. (2008) obtained a stable chondrogenic phenotype with human articular chondrocytes plated on collagen type II-treated surfaces, suggesting that type II collagen has potential for cell therapy in cartilage. Three-dimensional alginate bead culture provides a useful model for maintaining chondrocyte phenotypes and studying their response to cytokines (HaÈuselmann et al., 1992, 1994, 1996; Petit et al., 1996; ReÂdini et al., 1997; Demoor-Fossard et al., 1999; Barry et al., 2001). But, despite wide success in chondrogenesis in vitro, alginates could inhibit spontaneous repair if implanted alone, and could sometimes induce severe immunological responses if implanted as pre-engineered cell-alginate constructs. Matrices made from alginates also have limitations such as fast degradation rates and lack of the necessary mechanical properties required for cartilage tissue engineering. Thus the use of alginate as a biomaterial for in vivo transplantation needs to be improved. A recent study demonstrated that alginate can be used in vivo with human chondrocytes (Dobratz et al., 2009). However, the use of alginates in vitro to investigate the mechanisms involved in chondrogenesis and chondrocyte redifferentiation is still relevant in identifying new targets for therapeutic use. Alginate bead systems were used to differentiate MSCs in chondrogenic cells. The advantages of this system are, firstly, that cultures can be processed over several weeks, allowing the changes that occur over time to be studied, and secondly, that at harvest the beads can be entirely dissolved, leaving the cells as single cell suspensions for various analyses. The methodology used is described below. After expansion, human bone marrow MSCs and muscle-derived stem cells were re-suspended in sodium alginate (5  106 cells/ml). At passage five, cells were aliquoted, centrifuged, and re-suspended in sodium alginate. A density of 5  106 cells/ml was observed to lead to a more homogeneous cartilaginous matrix. The beads were formed by slowly dispensing droplets of alginate cell suspension from a 22-gauge needle into a 100 mM CaCl2 solution. After removal of the CaCl2 and washes with 0.15 M NaCl, the alginate beads were washed twice with chondrogenic medium and then cultured with the same medium, which was changed every 2 days. At harvest, some beads were rinsed with PBS and fixed for 2.5 h in 2.5% glutaraldehyde. They were then frozen directly in optimal cutting temperature (OCT) medium in liquid nitrogen for immune-histochemical analysis. The other beads were dissolved at 37 ëC in 55 mM sodium citrate, 150 mM NaCl, and gently centrifuged to prepare the RNA, nuclear and cystoplasmic proteins. For RNA, the pellet can be stored at ÿ20 ëC until extraction. Using this model of chondrogenesis, it was shown that the synthesis of cartilage markers increased significantly, associated with an increase in the

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number of isogenous groups, and was characteristic of cartilaginous tissue formation. Three stages in the process of differentiation were defined as the cells accumulate different elements of the new ECM. In stage I, in early differentiated cells (pre-chondrocytes) there was expression of types IIA, I and X collagens and aggrecan, GAG accumulation and Sox9 binding activity enhancement. During stage II (chondrocyte maturation), type IIB collagen appeared, aggrecan was enhanced, while type I collagen decreased. Finally, newly synthesized type I and X collagens represented stage III, where the cells become pre-hypertrophic (Andriamanalijaona et al., 2008). From a biological perspective, beneficial material features would include not only a three-dimensional environment for differentiation and biocompatibility, but also the promotion of release of factors supporting one or more of the biological aspects of repair. For example, chitosan microspheres have served as delivery vehicles for controlled release of TGF 1 (Cai et al., 2007). The use of PGLA microspheres was tested as a new delivery system for BMP-7 (GaveÂnis et al., 2007).

11.8

Use of exogenous growth factors to promote the chondrogenic phenotype

The selection of growth factors is a step forward in the development of a defined culture medium for directing chondrogenic differentiation or redifferentiation of chondrocytes. Several reported sets of data are still controversial, but differences could be related to differences in type of culture (monolayer, or threedimensional), species and age of the tissue.

11.8.1 Fibroblast growth factors Fibroblast growth factors (FGF) play an important role in chondrogenesis, as shown in limb bud cartilage formation (Ornitz and Marie, 2002). FGF-2 is the most potent mitogen for chondrocytes. Its use during articular chondrocytes expansion enhances the ability of the cells to redifferentiate upon transfer into a three-dimensional environment (reviewed in Steinert et al., 2007). Its use in the medium for expanding MSC could have a significant effect on the ability of cells to undergo chondrogenesis. By contrast, this factor may have the opposite effect on matrix deposition and accumulation, since it promotes both anabolic and catabolic activities. Recent studies have shown that FGF-2 stored in the adult cartilage matrix is released on mechanical injury or with loading, and that FGF-2 can inhibit the anabolic activities of insulin-like growth factor (IGF-1) and BMP-7 in primary chondrocytes (Loeser et al., 2005). FGF-9 and FGF-18 may be better candidates for promoting cartilage repair, since they increase matrix synthesis by mature chondrocytes, and FGF-18 has been shown to promote cartilage repair in a rat meniscal tear model of OA (Moore et al., 2005).

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11.8.2 Transforming growth factors TGF are already known to play critical roles in chondrogenesis during embryonic skeletal development and bone fracture repair. TGF 1 has emerged as one of the key factors promoting synthesis of type II collagen and aggrecan core protein and down-regulating metalloproteinases in chondrocytes. TGF 1 has also been shown to regulate chondrogenic events during development and to stimulate chondrogenesis of MSC. It was also shown to increase type II collagen synthesis in MSC in monolayer, in pellets and in chondrocyte-intervertebral disc cell-seeded tissue engineered constructs. Kuroda et al. (2006) have also observed that addition of TGF 1 to MDC cultures increased the number of type II collagen-positive colonies. The importance of TGF 1 in enhancing chondrogenesis was shown by demonstrating that it enhances Sox9 expression, GAGs deposition, aggrecan and type II collagen synthesis over 7 days following TGF 1 (10 ng/ml) addition to MDCs cultured in alginated beads (Andriamanalijaona et al., 2008). The influence of TGF 1 on MSCs and hypertrophy is still being debated. It has been demonstrated that in vitro terminal differentiation is repressed by TGF , which keeps chondrocytes in the prehypertrophic state (Zhang et al., 2004; Kawamura et al., 2005; Andriamanalijaona et al., 2008). However, Ma et al. (2005) demonstrated that only fibrous connective tissue, with type X collagen mRNA expression, rather than hyaline cartilage, was obtained when an MSCalginate construct was exposed to TGF 1 for one day before transplantation under the dorsal skin of nude mice. When these types of constructs were treated in vitro with TGF 1 for more than 1 week and transplanted, a neo-cartilage was generated at 8 weeks after transplantation (Ma et al., 2005). Lieb et al. (2004) demonstrated that TGF 1 is a potent stimulator of osteoblastic differentiation in rat marrow stromal cells. These two studies underline the fact that the effect of TGF 1 on MSC differentiation towards osteoblastic or chondrogenic types depends on culture conditions, cell type and doses used. A recent study using another member of the TGF family, TGF 3, demonstrated that short-term treatment with the growth factor from bone marrow MSC cultured in alginate beads could prevent alkaline phosphatase expression and osteoblastic differentiation (Mehlhorn et al., 2006), which reinforces the hypothesis that the TGF family may be more effective in suppressing chondrocyte terminal differentiation in vitro.

11.8.3 Bone morphogenetic proteins BMPs are important signalling molecules that induce limb bud development during embryogenesis, ectopical bone formation, and differentiation of mesenchymal progenitor cells into osteoblasts and chondrocytes. BMP-2, -4, -6, -7, -9 and -13, can enhance the synthesis of type II collagen and aggrecan by

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articular chondrocytes in vitro. BMP-2, -7 and -14 can also promote cartilage repair in various models of focal cartilage defects (reviewed in Goldring, 2006). Interestingly, a recent study has shown that BMP2 promotes redifferentiation of human chondrocytes from nasal cartilage after their expansion on plastic, (one, two or three passages), without inducing osteogenic expression. However, this stimulatory effect decreases with the number of cell passages (Salentey et al., 2008). BMP-7 has demonstrated great potential as a cartilage anabolic factor because of its ability to induce matrix synthesis and promote repair in cartilage (reviewed in Chubinskaya et al., 2007b). Addition of BMP-7 to chondrocyte cultures also caused an activation of the IGF-1 signalling pathway, namely expression of IGF-1, IGF-1 receptor, IGF-1 binding proteins and other downstream molecules, which may in part lead to the restoration of the responses to IGF-1 lost by human chondrocytes with ageing (Chubinskaya et al., 2007a; Loeser et al., 2003). Chondrogenic differentiation has also been observed in MSC cultured in alginate beads using BMPs. However, only BMP-2 and BMP-7 are currently approved for multiple indications in bone fracture repair and spinal fusion. Consistent with their roles in vivo in promoting endochondral ossification, they may promote chondrocyte hypertrophy in repair models and may serve as more effective anabolic factors for cartilage repair in juveniles, where chondroprogenitors are available, than in adults. Implantation of chondrocytes genetically modified to express BMP-7 has been shown to generate good hyaline cartilage repair tissue after 6 weeks in vivo, but only negative results have been reported after 1 year, with loss of 72% to 100% of the transplanted cells (Hidaka et al., 2003). This might be attributed to mechanisms of hypertrophic differentiation and subsequent apoptosis.

11.8.4 Insulin-like growth factor-1 IGF-1 has been shown to have potent stimulatory effects on chondrogenic differentiation and cartilage matrix synthesis. IGF-1 maintains chondrocyte metabolism in cartilage homeostasis, stimulates chondrocyte synthetic and mitotic activities, and inhibits chondrocyte-mediated matrix catabolism. Furthermore, IGF-1 is capable of redifferentiating chondrocytes that have lost their differentiated phenotype after several passages (P1 up to P7) (Shakibaei et al., 2006).

11.9

Use of gene therapy to deliver chondrogenic factors

In recent years, several factors have been identified that might be functional in augmenting different aspects of cartilage tissue repair. Of particular interest are transcription factors (Sox9, L-Sox5, Sox6), the TGF superfamily, including

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TGF s 1, 2 and 3, several BMPs and IGF-1, inhibitors of hypertrophic differentiation (IHH). Monolayer-expanded chondrocytes are readily transduced by viral vectors, such as Moloney Murine Leukemia Virus (MLV), lentivirus, adenovirus and adenovirus associated viruses (AAV). Adenoviral-mediated delivery of various transgenes, such as TGF 1, BMP-2, IGF-1 or BMP-7, has been shown to stimulate the production of a cartilage-specific matrix rich in collagen type II and proteoglycans, and shows a decreased tendency towards dedifferentiation (reviewed in Steinert et al., 2008). Transduction with the transcription factor Sox9 increased collagen type II expression of chondrocytes in three-dimensional culture in vitro and could restore the chondrogenic phenotype (reviewed in Hardingham et al., 2006). Further studies are necessary to generate higher levels of transgene expression with greater persistence in chondrocytes, as these cells are very difficult to transfect via plasmid DNA. Certain lipid-based formulations have been found to enhance the efficiency of DNA uptake (reviewed in Steinert et al., 2008). In vitro, the use of nucleofection methods improves the number of cells transfected and has given successful results for chondrocytes or MSCs. In order to avoid cartilage hypertrophy using MSCs, a better strategy may be the use of inhibitors of hypertrophic differentiation. Transduction of Sox9 alone or together with L-Sox5 and Sox6 into MSCs ex vivo may also be a good strategy for improving cartilage tissue engineering with Runx2 inhibition (Zhou et al., 2006). The disadvantage of directing chondrogenic differentiation through genetic modulation is the potential risk associated with using recombinant DNA technology in human clinical therapy. For example, the constitutive overexpression of any one particular protein or growth factor within transfected stem cells would certainly have unpredictable physiological effects on transplantation in vivo. This problem may be overcome by placing the recombinant expression of the particular protein under the control of switchable promoters, several of which have been developed for expression in eukaryotic systems (reviewed in Heng et al., 2004). Genetically modified stem cells may also run the risk of becoming malignant within the transplanted recipient. The potentially detrimental effects of transplanting genetically modified stem cells in vivo need to be investigated in animal models to address the safety aspects of such an approach.

11.10 Control of chondrocyte phenotype and chondrogenesis by hydrostatic pressure Since articular cartilage is subjected to varying loads in vivo and undergoes cyclic hydrostatic pressure during periods of loading, it is hypothesized that mimicking these conditions might enhance synthesis of important matrix components during cultivation in vitro. When applied with specific magnitudes

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and frequencies, physical stimuli can up-regulate the production of extracellular matrix components. While static compression of cartilage causes a decrease in the synthesis of PGs and collagens, periodic compression (such as in walking) increases the synthesis of these components (Buschmann et al., 1999). Chondrocytes thus respond to ECM deformation by changing shape and volume, thereby regulating their metabolic activity (reviewed in Kerin et al., 2002). Many researchers have attempted to capitalize on this phenomenon and develop bioreactors to apply mechanical stimuli to cell-seeded scaffolds or hydrogels in an effort to enhance cell differentiation and/or tissue development. The clear advantage of these methods is that cell seeding, cartilage growth and matrix deposition can be controlled in bioreactors to produce hyaline cartilage under optimal conditions. A number of studies have reported stimulated metabolism in response to dynamic compression, although these responses were greatly dependent upon the specific magnitude and/or frequency applied (Lee and Bader, 1995; Buschmann et al., 1995; Davisson et al., 2002; Lee et al., 2003, Hung et al., 2004). The influence of intermittent loading during redifferentiation of chondrocytes in alginate beads was also investigated. A statistically significant increased synthesis of GAG and collagen type II during redifferentiation of chondrocytes embedded in alginate beads, as well as an increase in GAG content of tissueengineered cartilage, was found compared to control without load (Heyland et al., 2006). It has also been suggested that in vitro hydrodynamic and biomechanical conditioning of grafts prior to their implantation may benefit lateral integration. Obradovic et al. studied cartilage disc composites cultured in bioreactors for up to 8 weeks and reported that tissue remodelling and adhesive strength of the integrating interface was generally higher for immature than for mature tissue. The highest integrative repair was attributed to active tissue remodelling by proliferative cells and required several weeks in culture in order to produce a hyaline-like repair tissue (Obradovic et al., 2001). Although mechanical conditioning has been proven to stimulate tissue development, further experiments are still needed to identify exactly the specific mechanical forces or regimes that should be applied.

11.11 Use of low oxygen tension in cartilage repair 11.11.1 Use of hypoxia to favour differentiated chondrocytes Being avascular, chondrocytes in articular cartilage exist in an environment of low oxygen tension in vivo. Most in vitro studies on isolated chondrocytes have so far been performed under atmospheric oxygen levels, thus exposing cells to oxygen tensions that are much higher than pathophysiological levels. In order to mimic the physiological environment of chondrocytes, a culture system was

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developed in which the oxygen tension was 5% O2 (Martin et al., 2004). The system consisted of a sealed chamber equipped with gloves, with a small culture incubator placed inside. Vessels were introduced via a special airlock. The chamber was equilibrated to 5% O2 by flushing it with a gas mixture (N2 ‡ CO2) without oxygen, and the O2 tension was monitored with a probe. The medium within the chamber had to be changed in order to avoid reoxygenation stress for the cells. Under these conditions, the effects of hypoxia on both the metabolism of bovine articular chondrocytes and their response to IL-1 in monolayer culture were investigated. As indicated in other studies (Grimshaw and Mason, 2000, 2001), it was confirmed that the type II collagen and aggrecan core protein mRNA levels were reduced in hypoxia compared with normoxia. In contrast, cells expressed more TGFû1 and TGFû receptor I mRNA than in normal oxygen conditions. It was also noticed that, under low oxygen tension, chondrocyte proliferation and elongation were reduced, limiting the dedifferentiation state. Moreover, it was demonstrated that the intensity of the chondrocyte response to IL-1 was generally greater in a low oxygen tension environment, with a marked down-regulation of type II collagen compared with normoxia conditions. This study demonstrated that low oxygen levels may increase the chondrocyte response to IL-1 and TGF and that some results previously obtained in normoxic cultures may need to be revisited to better understand the mechanisms of chondrocyte regulation. Chondrocyte culture in physiological conditions, i.e. under low oxygen tension, appears to be a necessary prerequisite for maintaining or restoring the chondrogenic phenotype, and more and more studies now focus on this model. For example, it has been demonstrated that dedifferentiated articular chondrocytes can recover their phenotype when cultured in alginate beads and transferred to hypoxia (Murphy and Sambanis, 2001; Domm et al., 2002). Scherer et al. (2004) described how 5% oxygen in combination with intermittent hydrostatic pressure promoted chondrogenic gene expression in articular chondrocytes and bone marrow cells. In murine and bovine chondrocytes, exposure to low oxygen levels (1% oxygen) leads to significantly increased amounts of type II collagen (Hansen et al., 2001; Domm et al., 2002; Pfander et al., 2003). It has also been reported that hypoxia-induced COL2A1 gene expression in three-dimensional culture systems involves increases in endogenous Sox9 expression (Domm et al., 2002; Murphy and Polak, 2004). Sox9 transduced articular chondrocytes in the presence of IGF1 and TGF 3 formed even larger cell aggregates when cultured under lowered oxygen (5%) conditions, with an increase in deposition of type II collagen (reviewed in Hardingham et al., 2006). HIF-1 is a heterodimeric protein consisting of HIF-1 and HIF-1 subunits (Semenza and Wang, 1992) and represents the most important transcription factor regulating gene expression under hypoxic conditions. Its availability is mainly determined by HIF-1, which is regulated in an oxygen-sensitive

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manner. The induction of hypoxia-sensitive genes takes place at functionally essential HIF-1 binding sites via HIF-1 binding to hypoxia-responsive elements (HRE). It has been clearly established that HIF-1 is of fundamental importance in growth plate development (Schipani et al., 2001). It has been further described to be expressed by articular chondrocytes in vivo (Aigner et al., 2001; Stokes et al., 2002) and HIF-1 activity was also shown to be necessary for collagen type II synthesis (Pfander et al., 2003).

11.11.2 Hypoxia-induced chondrogenic differentiation of stem cells Several findings strongly suggest that commitment of bone marrow stromal cells to chondrocytes is favoured by low oxygen environments. It must be remembered that formation of endochondral bone normally occurs in an avascular environment, and that the chondrocytes of the developing growth plate develop under low oxygen tension. As bone is vascularized and the tissue becomes oxygenated, osteoblasts arise from the bone marrow and begin to deposit and mineralize osteoid tissue along the cartilage matrix. Interestingly, the cellular events that occur following fracture largely recapitulate those that occur during endochondral bone formation. Disruption of vascular supply to the fracture site, and subsequent hypoxia, result in the appearance of chondrocytes and formation of a cartilaginous callus. This is followed by vascular in-growth and the appearance of osteoclasts and osteoblasts, which remodel the cartilaginous callus to form mineralized bone. Since chondrocytes and osteoblasts are believed to originate from a common stromal stem cell (Bradham et al., 1995; Mackay et al., 1998), it has been suggested that the local tissue level of O2 might influence the developmental fate of bone progenitor cells (HaÈuselmann et al., 1992; Petit et al., 1996). Some studies have described the effect of hypoxia on primary human bone marrow stromal cells. This pathway was investigated using the author's culture system and alginate beads, and demonstrated that hypoxia-induced human bone marrow-derived MSC showed chondrogenic differentiation with an increase in type II collagen, aggrecan mRNA synthesis and Sox9 binding activity on the col2a1 enhancer sequence used as a probe (Fig. 11.2). Scherer et al. (2004) described how 5% oxygen in combination with intermittent hydrostatic pressure promoted chondrogenic gene expression in articular chondrocytes and bone marrow cells. Robins et al. (2005) described how 1% oxygen up-regulated Sox9 gene expression in monolayer human MSC lines (ST2 stromal cells). Hirao et al. (2006) demonstrated that a lower oxygen tension promoted BMP-2-induced chondrogenesis in the murine C3H10T1/2 mesenchymal cell line. Using other cell types, Wang et al. (2005) reported that human adiposederived MSCs cultured in a 5% O2 environment suspended in alginate beads demonstrated an increase in chondrogenic differentiation. In contrast, Malladi et

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11.2 Expression of chondrogenic markers in hBM cells under hypoxia treatment. Human bone marrow stromal cells were cultured for differentiation in alginate beads in 5% O2. Quantitative PCR were performed to measure Aggrecan (a) and Type II collagen (b) mRNA levels. Bars represent mean  SEM of three different of three different samples from three donors. Statistical significance was assessed between 21% and 5% O2 using the Student t-test (n ˆ 3): * p < 0:05; ** p < 0:01; *** p < 0:001. Electrophoretic mobility shift assay (EMSA) were performed, using nuclear proteins from undifferentiated cells (C0), normoxia (N) and hypoxia-treated (H) cells. The treatment was made during 30 minutes, 3, 6 or 24 hours. A 48-bp COL2A1 enhancer sequences was used as a probe (c).

al. (2006) demonstrated that 2% O2 treatment markedly reduced TGF 1induced chondrogenesis of murine adipose-derived MSCs in micromass culture (Malladi et al., 2006). However, the contradictory data can be explained by the variety of species involved and by the ambient oxygen level chosen. A 2% O2 environment may cause severe stress for stem cells, as they observed a decrease in DNA content, suggesting possible cell apoptosis, whereas 5% O2 might be more viable for primary stem cells. Evidence for the involvement of HIF-1 in cartilage development is supported by observations in conditional HIF-1 knockout mice. In these animals, deletion of HIF-1 in chondrocytes of the developing growth plate resulted in premature programmed cell death. In murine embryos lacking HIF1, strongly reduced type II collagen signals were detected in the central, hypoxic areas of the growth plate (Schipani et al., 2001). Furthermore, HIF-1

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null chondrocytes exposed to hypoxia had markedly decreased expression of the HIF-1 target genes col2a and aggrecan (Pfander et al., 2003), suggesting the importance of this pathway in chondrocyte lineage progression. Transient transfections were performed of human bone marrow MSCs with a HIF-1 expression vector (pcDNA3-HIF1) and pcDNA3-HIF1DN construct, which encodes a dominant negative form of HIF1 lacking the N-terminal and Cterminal transactivation domains. The HIF1DN heterodimerizes with HIF-1 , leading to the formation of a non-functional HIF-1 dimer. Finally, transient transfections of cells with a Sox9 expression vector (pcDNA3-UT-Flag-Sox9) were also performed in order to verify the involvement of Sox9 in the commitment of cells to the chondrocyte phenotype. Electrophoretic mobility shift assay (EMSA) analysis was performed using HIF-1 consensus sequences and COL2A1 enhancer sequences as probes. Data showed enhanced protein DNA binding to HIF-1 consensus (Fig. 11.3a(i)) induced by hypoxia and HIF1 or HIF-1-DN over-expression. Using the 48 bp col2a1 enhancer sequence as probe, an increased binding activity of Sox proteins under hypoxia or HIF-1 or Sox9 over-expression was shown (Fig. 11.3a(ii)). In contrast, over-expression of the HIF-1-DN protein strongly reduced the hypoxia-induced binding activity of Sox proteins on the COL2A1 enhancer sequences. It was also shown that HIF-1 over-expression induced Sox9, Sox5, Sox6, type II collagen and aggrecan mRNA expression (Fig. 11.3b). The HIF-1-DN protein enhanced HIF-1 mRNA expression, whereas it abolished the stimulatory effect of hypoxia on Sox factors, type II collagen and aggrecan mRNA levels (Fig. 11.3c). These preliminary data reinforce the hypothesis that HIF-1 is involved in the regulation of cartilage markers in MSC.

11.11.3 Hypertrophy inhibition by hypoxia When chondrogenic cells are cultured under hypoxic conditions, expression of Col10a1 is inhibited as a result of down-regulation of Runx2 expression (Hirao et al., 2006). Hypoxia effects in these cells could be mediated by activation of HDAC4. Indeed, silencing Hdac4 expression counteracts hypoxia-induced suppression of both Runx2 and Col10a1 expression, indicating that it plays a role in mediating the effects of hypoxia on chondrocyte hypertrophy. Hypoxia effects on type X collagen could also be due to the Sox9 upregulation in hypoxia-treated MSCs, as studies have shown that Sox9 inhibits maturation to hypertrophic chondrocytes by mediating PTHrP signalling (de Crombrugghe et al., 2000).

11.12 Conclusions The observations presented above clearly demonstrate the complexity and the challenges in using cell therapy for cartilage repair. Strategies developed in the

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11.3 Effects of HIF-1, HIF-1-DN proteins overexpression on binding activity and chondrogenic markers expression. hBM cells were transfected by HIF-1 (pcDNA3-HIF1), HIF-1-DN (pcDNA3-DN-HIF1) or Sox9 (pcDNA3-UT-Flag-Sox9) expression vectors or corresponding empty vectors. The medium was changed 12 h after transfection. After transfection with HIF1 negative dominant, cells were cultured in hypoxia. Cultures were harvested 24 h after the medium replacement. (a) Nuclear extracts were used for EMSA using HIF-1 consensus (i) and the 48 bp COL2A1 enhancer (ii) sequences as probes. RNA was used in RT-PCR to determine HIF-1, Sox9, L-Sox5, Sox6, type IIB collagen (COL2) and aggrecan (AGGR) mRNAs expression. Bars represent mean  SEM of three separate experiments. (b) HIF-1 overexpression. Statistical significance between empty vector and expression vectors was evaluated by the Student's t-test (*p < 0:05, ** p < 0:01, *** p < 0:001). (c) HIF-1 negative dominant overexpression. Statistical significance was evaluated by the Student's t-test (* p < 0:05, ** p < 0:01, *** p < 0:001).

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laboratory need to consider and anticipate the consequences and constraints in relation to clinical application. For cartilage repair, a tissue with specific biomechanical features is needed. As the majority of in vivo studies show fibrocartilage or calcified tissue formation, standard in vitro conditions will have to be improved. Beneficial material features would include not only a three-dimensional environment for differentiation and biocompatibility, but also the release of factors supporting one or more of the biological aspects of repair. Chemical, biomaterial and biological factors may be used to promote ECM production in culture. However, for human safety, it is crucial to better understand the molecular mechanisms involved in each pathway and their long-term consequences in vivo. Cell culture has to be performed using a serum-free medium in order to avoid interference from other factors and problems of pathogen transmission. As the use of growth factors is still controversial, oxygen tension and dynamic mechanical stimulation could be factors of interest in preparing cells. The capacity of hypoxia to restore the chondrogenic phenotype, to suppress chondrogenic hypertrophy and to induce chondrocyte maturation in vitro suggests that hypoxia represents a promising method for conditioning cells before transplantation. The use of hypoxia systems in vitro is also relevant for testing other promising agents on dedifferentiated chondrocytes or MSCs, as the molecular mechanisms involved in these phenomena could be made more realistic. The experiments also allow us to better understand the mechanism by which muscle-derived cells differentiate towards a chondrogenic lineage, and further support the notion that muscle tissue may become a valuable resource for chondro-progenitor cells, which can be used in the clinical setting to improve cartilage healing. The standardization of cell expansion has already been established for clinical use. Further in vivo study is needed to confirm the hypothesis. Thus the different factors described above can be combined to improve the maintenance of the chondrocyte phenotype. However, the cost of developing grafts has also to be considered, and a clinically applicable model for cartilage regeneration should be kept as simple as possible.

11.13 Acknowledgements I would like to thank the Laboratory of Normal and Pathological Extracellular Matrix (University of Caen, France) where all my research studies were performed. I was also supported by a grant from the European Community, as part of the GENOSTEM Integrated Project (6th Framework Program). I thank Mr Delorme and Mr Chambord (University of Tours, Tours, France) for their teaching on human bone marrow cell culture. I thank Mr Vilquin, Mr Marolleau

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(Pierre et Marie Curie University, Paris, France), the company Myosix SA and Mr. FreÂdeÂric Chereau for giving access to myoblast culture methodology. I wish to thank Brigitte Ternaux, Marie-Noelle Lacassagne, Isabelle Robert, JeÂroÃme Larghero (HoÃpital Saint Louis, Paris, France) and Dr Bruno Dalle (Myosix SA, Paris, France) for their assistance and teaching. I thank Dr Geffard and Dr Benateau (University Hospital Center, Caen, France) and Dr Leclerc (Clinique St Martin, Caen, France) for providing human samples. I am grateful to Professor Pouyssegur (Nice, France) for providing pcDNA3-HIF-1 and pcDNA3-HIF-1DN expression vectors and Professor de Crombrugghe (Houston, Texas, USA) for providing the pcDNA3-UT-Flag-Sox9 vector.

11.14 References and further reading Adachi N, Sato K, Usas A, Fu FH, Ochi M, Han CW, Niyibizi C and Huard J (2002), Muscle derived, cell based ex vivo gene therapy for treatment of full thickness articular cartilage defects, J Rheumatol, 29, 1920±1930. Aigner T and Dudhia J (1997), Phenotypic modulation of chondrocytes as a potentiel therapeutic target in osteoarthritis: a hypothesis, Ann Rheum Dis, 56, 287. Aigner T, StoÈû H, Weseloh G, Zeiler G and von der Mark (1992), Activation of collagen type II expression in osteoarthritic and rheumatoid cartilage, Virchows Arch B, 62, 337±345. Aigner T, Bertling W, StoÈû H, Weseloh G, Zeiler G and von der Mark (1993), Independent expression of fibril-forming collagens I, II and III in human osteoarthritic cartilage, J Clin Invest, 91, 829±837. Aigner T, Vornhem SI, Zeiler G, Dudhia J, von de Mark K and Bayliss MT (1997), Suppression of cartilage matrix gene expression in upper zone chondrocytes of osteoarthritic cartilage, Arthritis Rheum, 40, 562±569. Aigner T, Zhu Y, Chansky HH, Matsen FA, Maloney WJ and Sandell LJ (1999), Reexpression of type IIA procollagen by adulte articular chondrocytes in osteoarthritic cartilage, Arthritis Rheum, 42, 1443±1450. Aigner T, Zien A, Gehrsitz A, Gebhard PM and McKenna L (2001), Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA-array technology, Arthritis Rheum, 44, 2777±2789. Akiyama H, Chaboissier MC, Martin JF, Schedl A and de Crombrugghe B (2002), The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6, Genes Dev, 16, 2813±2828. Andriamanalijaona R, Duval E, Raoudi M, Lecourt S, Vilquin JT, Marolleau JP, Pujol JP, Galera P and Boumediene K (2008), Differentiation potential of human musclederived cells towards chondrogenic phenotype in alginate beads culture, Osteoarthritis Cartilage, 16, 1509±1518. Archer CW and Francis-West P (2002), The chondrocyte, Int J Biochem Cell Biol, 32, 401±404. Arnold MA, Kim Y, Czubryt MP, Phan D, McAnally J, Qi X, Shelton JM, Richardson JA, Bassel-Duby R and Olson EN (2007), MEF2C transcription factor controls chondrocyte hypertrophy and bone development, Dev Cell, 12, 377±329. Bae JS, Gutierrez S, Narla R, Pratap J, Devados R, van Wijnen AJ, Stein JL, Stein GS,

Cell therapies for articular cartilage repair

293

Lian JB and Javed A (2007), Reconstitution of Runx2/Cbfa1-null cells identifies a requirement for BMP2 signaling through a Runx2 functional domain during osteoblast differentiation, J Cell Biochem, 100, 434±449. Barnes CP, Sell SA, Boland ED, Simpson DG and Bowlin GL (2007), Nanofiber technology: designing the next generation of tissue engineering scaffolds, Adv Drug Deliv Rev, 59, 1413±1433. Barry F, Boynton RE, Liu B and Murphy JM (2001), Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components, Exp Cell Res, 268, 189±200. Benya PD and Shaffer JD (1982), Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels, Cell, 30, 215±224. Bidanset DJ, Guidry C, Rosenberg LC, Choi HU, Timpl R and Hook M (1992), Binding of the proteoglycan decorin to collagen type VI, J Biol Chem, 267, 5250±5256. Bradham DM, Passaniti A and Horton WE Jr (1995), Mesenchymal cell chondrogenesis is stimulated by basement membrane matrix and inhibited by age-associated factors, Matrix Biol, 14, 561±571. Brighton CT and Heppenstall RB (1971), Oxygen tension in zones of the epiphyseal plate, the metaphysis and diaphysis. An in vitro and in vivo study in rats and rabbits, J Bone Joint Surg Am, 53, 719±728. Brittberg M (1999), Autologous chondrocyte transplantation, Clin Orthop Relat Res, 147±155. Buckwalter JA and Mankin HJ (1998), Articular cartilage: tissue design and chondrocyte±matrix interactions, AAOS Instruction Course Lectures, 47, 477±486. Buschmann MD, Gluzband YA, Grodzinsky AJ and Hunziker EB (1995), Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture, Cell Sci, 108, 1497±1508. Buschmann MD, Kim YJ, Wong M, Frank E, Hunziker EB and Grodzinsky AJ (1999), Stimulation of aggrecan synthesis in cartilage explants by cyclic loading is localized to regions of high interstitial fluid flow, Arch Biochem Biophys, 366, 1±7. Cai DZ, Zeng C, Quan DP, Bu LS, Wang K, Lu HD and Li XF (2007), Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-beta1 delivery for cartilage tissue engineering, Chin Med J, 120, 197± 203. Erratum in: Chin Med J, 120, 293. Cancedda R, Descalzi-Cancedda F and Castagnola P (1995), Chondrocyte differentiation, Int Rev Cytol, 159, 265±358. Capasso O, Gionti E, Pontarelli G, Ambesi-Impiombato FS, Nitsch L, Tajana G and Cancedda R (1982), The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. 1. Characterization of the chondrocyte specific phenotypes, Exp Cell Res, 142, 197±206. Chang J, Nakajima H and Poole A (1997), Structural colocalisation of type VI collagen and fibronectin in agarose cultured chondrocytes and isolated chondrons extracted from adult canine tibial cartilage, J Anat, 190, 523±532. Chiang H and Jiang CC (2009), Repair of articular cartilage defects: review and perspectives, J Formos Med Assoc, 108, 87±101. Chiang H, Kuo TF, Tsai CC, Lin MC, She BR, Huang YY, Lee HS, Shieh CS, Chen MH, Ramshaw JA, Werkmeister JA, Tuan RS and Jiang CC (2005), Repair of porcine articular cartilage defect with autologous chondrocyte transplantation, J Orthop Res, 23, 584±593. Chubinskaya S, Hakimiyan A, Pacione C, Yanke A, Rappoport L, Aigner T, Rueger DC and Loeser RF (2007a), Synergistic effect of IGF-1 and OP-1 on matrix formation

294

Regenerative medicine and biomaterials in connective tissue repair

by normal and OA chondrocytes cultured in alginate beads, Osteoarthritis Cartilage, 15, 421±430. Chubinskaya S, Hurtig M and Rueger DC (2007b), OP-1/BMP-7 in cartilage repair, Int Orthop, 31, 773±781. Cohn MJ, IzpisuÂa-Belmonte JC, Abud H, Heath JK and Tickle C (1995), Fibroblast growth factors induce additional limb development from the flank of chick embryos, Cell, 80, 739±746. Davisson T, Sah RL and Ratcliffe A (2002), Perfusion increases cell content and matrix synthesis in chondrocyte three-dimensional cultures, Tissue Eng, 8, 807±816. de Crombrugghe B, Lefebvre V, Behringer RR, Bi W, Murakami S and Huang W (2000),Transcriptional mechanisms of chondrocyte differentiation, Matrix Biol, 19, 389±394. de Crombrugghe B, Lefebvre V and Nakashima K (2001), Regulatory mechanisms in the pathways of cartilage and bone formation, Curr Opin Cell Biol, 13, 721±727. de Haart M, Marijnissen WJ, van Osch GJ and Verhaar JA (1999), Optimization of chondrocyte expansion in culture. Effect of TGF beta-2, bFGF and L-ascorbic acid on bovine articular chondrocytes, Acta Orthop Scand, 70, 55±61. DeLise AM, Fischer L and Tuan RS (2000), Cellular interactions and signaling in cartilage development, Osteoarthritis Cartilage, 8, 309±334. Demoor-Fossard M, Boittin M, Redini F and Pujol JP (1999), Differential effects of interleukin-1 and transforming growth factor beta on the synthesis of small proteoglycans by rabbit articular chondrocytes cultured in alginate beads as compared to monolayers, Mol Cell Biochem, 199, 69±80. Dobratz EJ, Kim SW, Voglewede A and Park SS (2009), Injectable cartilage: using alginate and human chondrocytes, Arch Facial Plast Surg, 11, 40±47. Domm C, SchuÈnke M, Christesen K and Kurz B (2002), Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension, Osteoarthritis Cartilage, 10, 13±22. Duprez DM, Coltey M, Amthor H, Brickley PM and Tickle C (1996), Bone morphogenetic-2 (BMP-2) inhibits muscle development and promotes cartilage formation in chick limb bud cultures, Dev Biol, 174, 448±452. Farquharson C, Berry JL, Mawer EB, Seawright E and Whitehead CC (1998), Ascorbic acid-induced chondrocyte terminal differentiation: the role of the extracellular matrix and 1,25-dihydroxyvitamin D, Eur J Cell Biol, 76, 110±118. GaveÂnis K, Klee D, Pereira-Paz RM, von Walter M, Mollenhauer J, Schneider U and Schmidt-Rohlfing B (2007), BMP-7 loaded microspheres as a new delivery system for the cultivation of human chondrocytes in a collagen type-I gel, J Biomed Mater Res B Appl Biomater, 82, 275±283. Geutjes PJ, Daamen WF, Buma P, Feitz WF, Faraj KA and van Kuppevelt TH (2006), From molecules to matrix: construction and evaluation of molecularly defined bioscaffolds, Adv Exp Med Biol, 585, 279±295. Girkontaite I, Frischolz S, Lamni P, Wagner K, Swoboda B, Aigner T and von der Mark K (1996), Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies, Matrix Biol, 15, 231±238. Goldring MB (2006), Update on the biology of the chondrocyte and new approaches to treating cartilage diseases, Best Pract Res Clin Rheumatol, 20, 1003±1025. Goldring MB, Birkead J, Kimura T and Krane SM (1988), Interleukin-1 suppresses expression of cartilage specific types II and IX collagens and increases types I and III collagens in human chondrocytes, J Clin Invest, 82, 2026±2037. Grimshaw MJ and Mason RM (2000), Bovine articular chondrocyte function in vitro

Cell therapies for articular cartilage repair

295

depends upon oxygen tension, Osteoarthritis Cartilage, 8, 386±392. Grimshaw MJ and Mason RM (2001), Modulation of bovine articular chondrocyte gene expression in vitro by oxygen tension, Osteoarthritis Cartilage, 9, 357±364. Grover J and Roughley PJ (1995), Expression of cell surface proteoglycan mRNA by human artricular chondrocytes, Biochem J, 309, 963±968. Guo J, Chung UI, Yang D, Karsenty G, Bringhurst FR and Kronenberg HM (2006), PTH/ PTHrP receptor delays chondrocyte hypertrophy via both Runx2-dependent and independent pathways, Dev Biol, 292, 116±128. Hall B and Miyake T (2000), All for one and one for all: condensations and the initiation of skeletal development, Bioassays, 22, 138±147. Hangody L, Kish G, Karpati Z, Udvarhelyi I, Szigeti I and Bely M (1998), Mosaicplasty for the treatment of articular cartilage defects: application in clinical practice, Orthopedics, 21, 751±756. Hansen U, SchuÈnke M, Domm C, Ioannidis N, Hassenpflug J, Gehrke T and Kurz B (2001), Combination of reduced oxygen tension and intermittent hydrostatic pressure: a useful tool in articular cartilage tissue engineering, J Biomech, 34, 941±949. Hardingham TE, Fosang AJ and Dudhia J (1994), The strucutre, function and turnover of aggrecan, the large aggregating proteoglycan from cartilage, Eur J Chem Clin Biochem, 32, 249±257. Hardingham TE, Oldershaw RA and Tew SR (2006), Cartilage, SOX9 and Notch signals in chondrogenesis, Anatomy, 209, 469±480. HaÈuselmann HJ, Aydelotte MB, Schumacher BL, Kuettner KE, Gitelis SH and Thonar EJ (1992), Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads, Matrix, 12, 116±129. HaÈuselmann HJ, Fernandes RJ, Mok SS, Schmid TM, Block JA, Aydelotte MB, Kuettner KE and Thonar EJ (1994), Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads, J Cell Sci, 107, 17±27. HaÈuselmann HJ, Masuda K, Hunziker EB, Neidhart M, Mok SS, Michel BA and Thonar EJ (1996), Adult human chondrocytes cultured in alginate form a matrix similar to native human articular cartilage, Am J Physiol, 271, C742±C752. Heng BC, Cao T and Lee EH (2004), Directing stem cell differentiation into the chondrogenic lineage in vitro, Stem Cells, 22, 1152±1167. Hennig T, Lorenz H, Thiel A, Goetzke K, Dickhut A, Geiger F and Richter W (2007), Reduced chondrogenic potential of adipose tissue derived stromal cells correlates with an altered TGFbeta receptor and BMP profile and is overcome by BMP-6, J Cell Physiol, 211, 682±691. Heyland J, Wiegandt K, Goepfert C, Nagel-Heyer S, Ilinich E, Schumacher U and PoÈrtner R (2006), Redifferentiation of chondrocytes and cartilage formation under intermittent hydrostatic pressure, Biotechnol Lett, 28, 1641±1648. Hidaka C, Goodrich LR, Chen CT, Warren RF, Crystal RG and Nixon AJ (2003), Acceleration of cartilage repair by genetically modified chondrocytes over expressing bone morphogenetic protein-7, J Orthop Res, 21, 573±583. Higashikawa A, Saito T, Ikeda T, Kamekura S, Kawamura N, Kan A, Oshima Y, Ohba S, Ogata N, Takeshita K, Nakamura K, Chung UI and Kawaguchi H (2009), Identification of the core element responsive to runt-related transcription factor 2 in the promoter of human type X collagen gene, Arthritis Rheum, 60, 166±178. Hildebrand A, Romaris M, Rasmussen LM, Heinegard D, Twarzik DR, Border WA and Rouslahti E (1994), Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor , Biochem J, 302, 527± 534.

296

Regenerative medicine and biomaterials in connective tissue repair

Hirao M, Tamai N, Tsumaki N, Yoshikawa H and Myoui A (2006), Oxygen tension regulates chondrocyte differentiation and function during endochondral ossification, J Biol Chem, 281, 31079±31092. Hung CT, Mauck RL, Wang CC, Lima EG and Ateshian GA (2004), A paradigm for functional tissue engineering of articular cartilage via applied physiologic deformational loading, Ann Biomed Eng, 32, 35±49. Erratum in: Ann Biomed Eng, 32, 510. Hwang NS and Elisseeff J (2009), Application of stem cells for articular cartilage regeneration, J Knee Surg, 22, 60±71. Javed A, Afzal F, Bae JS, Gutierrez S, Zaidi K, Pratap J, van Wijnen AJ, Stein JL, Stein GS and Lian JB (2009), Specific residues of RUNX2 are obligatory for formation of BMP2-induced RUNX2-SMAD complex to promote osteoblast differentiation, Cells Tissues Organs, 189, 133±137. Katagiri T and Takahashi N (2002), Regulatory mechanisms of osteoblast and osteoclast differentiation, Oral Dis, 8, 147±159. Kawaguchi H (2008), Endochondral ossification signals in cartilage degradation during osteoarthritis progression in experimental mouse models, Mol Cells, 25, 1±6. Kawamura K, Chu CR, Sobajima S, Robbins PD, Fu FH, Izzo NJ and Niyibizi C (2005), Adenoviral-mediated transfer of TGF-beta1 but not IGF-1 induces chondrogenic differentiation of human mesenchymal stem cells in pellet cultures, Exp Hematol, 33, 865±872. Kerin A, Patwari P, Kuettner K, Cole A and Grodzinsky A (2002), Molecular basis of osteoarthritis: biomechanical aspects, Cell Mol Life Sci, 59, 27±35. Kulyk WM, Upholt WB and Kosher RA (1989), Fibronectin gene expression during limb cartilage differentiation, Development, 106, 449±455. Kuroda R, Usas A, Kubo S, Corsi K, Peng H, Rose T, Cummins J, Fu FH and Huard J (2006), Cartilage repair using bone morphogenetic protein 4 and muscle-derived stem cells, Arthritis Rheum, 54, 433±442. Lee CR, Grodzinsky AJ, Hsu HP and Spector M (2003), Effects of cultured autologous chondrocyte-seeded type II collagen scaffold on the haling of a chondral defect in canine model, J Orthop Res, 21, 272±281. Lee DA and Bader DL (1995), The development and characterization of an in vitro system to study strain-induced cell deformation in isolated chondrocytes, In Vitro Cell Dev Biol Anim, 31, 828±835. Lefebvre V and de Crombrugghe B (1998), Topward understanding SOX9 function in chondrocyte differentiation, Matrix Biol, 16, 529±540. Lefebvre V and Smits P (2005), Transcriptional control of chondrocyte fate and differentiation, Birth Defects Res C Embryo Today, 75, 200±212. Lefebvre V, Behringer RR and de Crombrugghe B (2001), L-Sox5, Sox6 and Sox9 control essential steps of the chondrocyte differentiation pathway, Osteoarthritis Cartilage, 9 Suppl A, S69±75. Leitinger B and Hohenester E (2007), Mammalian collagen receptors, Matrix Biol, 26, 146±155. Lieb E, Vogel T, Milz S, Dauner M and Schulz MB (2004), Effects of transforming growth factor beta1 on bonelike tissue formation in three-dimensional cell culture. II: Osteoblastic differentiation, Tissue Eng, 10, 1414±1425. Loeser RF, Pacione CA and Chubinskaya S (2003), The combination of insulin-like growth factor 1 and osteogenic protein 1 promotes increased survival of and matrix synthesis by normal and osteoarthritic human articular chondrocytes, Arthritis Rheum, 48, 2188±2196.

Cell therapies for articular cartilage repair

297

Loeser RF, Chubinskaya S, Pacione C and Im HJ (2005), Basic fibroblast growth factor inhibits the anabolic activity of insulin-like growth factor 1 and osteogenic protein 1 in adult human articular chondrocytes, Arthritis Rheum, 52, 3910±3917. Long F, Sonenshein GE and Linsenmayer TF (1998), Multiple transcriptional elements in the avian type X collagen gene. Identification of Sp1 family proteins as regulators for high level expression in hypertrophic chondrocytes, J Biol Chem, 273, 6542± 6549. Ma HL, Chen TH, Low-Tone Ho L and Hung SC (2005), Neocartilage from human mesenchymal stem cells in alginate: implied timing of transplantation, J Biomed Mater Res A, 74, 439±446. Mackay AM, Beck SC, Murphy JM, Barry FP, Chichester CO and Pittenger MF (1998), Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow, Tissue Eng, 4, 415±428. Magee C, Nurminskaya M, Faverman L, Galera P and Linsenmayer TF (2005), SP3/SP1 transcription activity regulates specific expression of collagen type X in hypertrophic chondrocytes, J Biol Chem, 280, 25331±25338. Malladi P, Xu Y, Chiou M, Giaccia AJ and Longaker MT (2006), Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells, Am J Physiol Cell Physiol, 290, C1139±1146. Martin G, Andriamanalijaona R, GraÈssel S, Dreier R, Mathy-Hartert M, Bogdanowicz P, BoumeÂdiene K, Henrotin Y, Bruckner P and Pujol JP (2004), Effect of hypoxia and reoxygenation on gene expression and response to interleukin-1 in cultured articular chondrocytes, Arthritis Rheum, 50, 3549±3560. Mehlhorn AT, Schmal H, Kaiser S, Lepski G, Finkenzeller G, Stark GB and SuÈdkamp NP (2006), Mesenchymal stem cells maintain TGF-beta-mediated chondrogenic phenotype in alginate bead culture, Tissue Eng, 12, 1393±1403. Moore EE, Bendele AM, Thompson DL, Littau A, Waggie KS, Reardon B and Ellsworth JL (2005), Fibroblast growth factor-18 stimulates chondrogenesis and cartilage repair in a rat model of injury-induced osteoarthritis, Osteoarthritis Cartilage, 13, 623±631. Muraglia A, Corsi A, Riminucci M, Mastrogiacomo M, Cancedda R, Bianco P and Quarto R (2003), Formation of a chondro-osseous rudiment in micromass cultures of human bone-marrow stromal cells, J Cell Sci, 116, 2949±2955. Murphy CL and Polak JM (2004), Control of human articular chondrocyte differentiation by reduced oxygen tension, J Cell Physiol, 199, 451±459. Murphy CL and Sambanis A (2001), Effect of oxygen tension and alginate encapsulation on restoration of the differentiated phenotype of passaged chondrocytes, Tissue Eng, 7, 791±803. Mwale F, Stachura D, Roughley P and Antoniou J (2006), Limitations of using aggrecan and type X collagen as markers of chondrogenesis in mesenchymal stem cell differentiation, J Orthop Res, 24, 1791±1798. Naruse K, Urabe K, Mukaida T, Ueno T, Migishima F, Oikawa A, Mikuni-Takagaki Y and Itoman M (2004), Spontaneous differentiation of mesenchymal stem cells obtained from fetal rat circulation, Bone, 35, 850±858. O'Driscoll SW (1999), Articular cartilage regeneration using periosteum, Clin Orthop Relat Res, 186±203. Oberlender SA and Tuan RS (1994), Expression and functional involvement of Ncadherin in embryonic limb chondrogenesis, Development, 120, 177±187. Obradovic B, Martin I, Padera RF, Treppo S, Freed LE and Vunjak-Novakovic G (2001), Integration of engineered cartilage, J Orthop Res, 19, 1089±1097.

298

Regenerative medicine and biomaterials in connective tissue repair

Oegema TR and Thompson RC (1990). Cartilage bone interface (tidemark), in Brand KD (ed), Cartilage Changes in Osteoarthritis, Indianpolis, 43±52. Ornitz DM and Marie PJ (2002), FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease, Genes Dev, 16, 1446±1465. Pelttari K, Winter A, Steck E, Goetzke K, Hennig T, Ochs BG, Aigner T and Richter W (2006), Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice, Arthritis Rheum, 54, 3254±3266. Petit B, Masuda K, D'Souza AL, Otten L, Pietryla D, Hartmann DJ, Morris NP, Uebelhart D, Schmid TM and Thonar EJ (1996), Characterization of crosslinked collagens synthesized by mature articular chondrocytes cultured in alginate beads: comparison of two distinct matrix compartments, Exp Cell Res, 225, 151±161. Pfander D, Cramer T, Schipani E and Johnson RS (2003), HIF-1alpha controls extracellular matrix synthesis by epiphyseal chondrocytes, J Cell Sci, 116, 1819± 1826. Pizette S and Niswander L (2000), BMPs are required at two steps of limb chondrogenesis: formation of prechondrogenic condensations and their differentiation into chondrocytes, Dev Biol, 219, 237±249. Provot S, Kempf H, Murtaugh LC, Chung UI, Kim DW, Chyung J, Kronenberg HM and Lassar AB (2006), Nkx3.2/Bapx1 acts as a negative regulator of chondrocyte maturation, Development, 133, 651±662. Pulkkinen HJ, Tiitu V, Valonen P, Hamalainen ER, Lammi MJ and Kiviranta I (2008), Recombinant human type II collagen as a material for cartilage tissue engineering, Int J Artif Organs, 31, 960±969. Rajpurohit R, Koch CJ, Tao Z, Teixeira CM and Shapiro IM (1996), Adaptation of chondrocytes to low oxygen tension: relationship between hypoxia and cellular metabolism, J Cell Physiol, 168, 424±432. ReÂdini F (2001), Structure et reÂgulation de l'expression des proteÂoglycanes du cartilage articulaire, Pathol Biol, 49, 364±375. ReÂdini F, Min W, Demoor-Fossard M, Boittin M and Pujol JP (1997), Differential expression of membrane-anchored proteoglycans in rabbit articular chondrocytes cultured in monolayers and in alginate beads. Effect of transforming growth factorbeta 1, Biochim Biophys Acta, 1355, 20±32. Robins JC, Akeno N, Mukherjee A, Dalal RR, Aronow BJ, Koopman P and Clemens TL (2005), Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9, Bone, 37, 313±322. Roughley PJ and Lee ER (1994), Cartilage proteoglycans: structure and potential functions, Microsc Res Tech, 28, 385±397. Sakaguchi Y, Sekiya I, Yagishita K and Muneta T (2005), Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source, Arthritis Rheum, 52, 2521±2529. Salentey V, Claus S, Bougault C, Paumier A, Aubert-Foucher E, Perrier-Groult E, RonzieÁre MC, Freyria AM, GaleÂra P, Beauchef G, Duterque-Coquillaud M, Piperno M, Damour O, Herbage B and Mallein-Gerin F (2008), Human chondrocyte responsiveness to bone morphogenetic protein-2 after their in vitro dedifferentiation: potential use of bone morphogenetic protein-2 for cartilage cell therapy, Pathol Biol, 57, 282±289. Sandell LJ and Daniel JC (1988), Effects of ascorbic acid on collagen mRNA levels in short term chondrocyte cultures, Connect Tissue Res, 17, 11±22.

Cell therapies for articular cartilage repair

299

Sandell LJ, Nalin AM and Reife RA (1994), Alternative splice form of type II procollagen mRNA (IIA) is predominant in skeletal precursors and noncartilaginous tissues during early mouse development, Dev Dyn, 199, 129±140. Scherer K, SchuÈnke M, Sellckau R, Hassenpflug J and Kurz B (2004), The influence of oxygen and hydrostatic pressure on articular chondrocytes and adherent bone marrow cells in vitro, Biorheology, 41, 323±333. Schipani E, Ryan HE, Didrickson S, Kobayashi T, Knight M and Johnson RS (2001), Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival, Genes Dev, 15, 2865±2876. Schmid TM and Conrad HE (1982), A unique low molecular weight collagen secreted by cultured chick embryo chondrocytes, J Biol Chem, 257, 12444±12450. Schulze-Tanzil G, de Souza P, Villegas Castrejon H, John T, Merker HJ, Scheid A and Shakibaei M (2002), Redifferentiation of dedifferentiated human chondrocytes in high-density cultures, Cell Tissue Res, 308, 371±379. Sekiya I, Koopman P, Tsuji K, Mertin S, Harley V, Yamada Y, Shinomiya K, Nifuji A and Noda M (2001), Dexamethasone enhances SOX9 expression in chondrocytes, J Endocrinol, 169, 573±579. Semenza GL and Wang GL (1992), A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation, Mol Cell Biol, 12, 5447±5454. Shakibaei M, Seifarth C, John T, Rahmanzadeh M and Mobasheri A (2006), Igf-I extends the chondrogenic potential of human articular chondrocytes in vitro: molecular association between Sox9 and Erk1/2, Biochem Pharmacol, 72, 1382±1395. Shakibaei M, Csaki C, Rahmanzadeh M and Putz R (2008), Interaction between human chondrocytes and extracellular matrix in vitro: a contribution to autologous chondrocyte transplantation, Orthopade, 37, 440±447. Shapiro IM, Leboy PS, Tokuoka T, Forbes E, DeBolt K, Adams SL and Pacifici M (1991), Ascorbic acid regulates multiple metabolic activities of cartilage cells, Am J Clin Nutr, 54, 1209S±1213S. Silver IA (1975), Measurement of pH and ionic composition of pericellular sites, Philos Trans R Soc Lond B Biol Sci, 271, 261±272. Smits P, Dy P, Mitra S and Lefebvre V (2004), Sox5 and Sox6 are needed to develop and maintain source, columnar, and hypertrophic chondrocytes in the cartilage growth plate, J Cell Biol, 164, 747±758. Steinert AF, Ghivizzani SC, Rethwilm A, Tuan RS, Evans CH and NoÈth U (2007), Major biological obstacles for persistent cell-based regeneration of articular cartilage, Arthritis Res Ther, 9, 213. Steinert AF, NoÈth U and Tuan RS (2008), Concepts in gene therapy for cartilage repair, Injury, 39 Suppl 1, S97±113. Stokes DG, Liu G, Coimbra IB, Piera-Velazquez S, Crowl RM and JimeÂnez SA (2002), Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis, Arthritis Rheum, 46, 404±419. Treppo S, Koepp H, Quan EC, Cole AA, Kuettner KE and Grodzinsky AJ (2000), Comparison of biomechanical an d biochemical properties of cartilage from human knee and ankle pairs, J Orthop Res, 18, 739±748. Valcourt U, Gouttenoire J, Aubert-Foucher E, Herbage D and Mallein-Gerin F (2003), Alternative splicing of type II procollagen pre-mRNA in chondrocytes is oppositely regulated by BMP-2 and TGF-beta1, FEBS Lett, 545, 115±119. van der Rest M and Mayne R (1988), Type IX collagen proteoglycan from cartilage is covently cross-linked to type II collagen, J Biol Chem, 263, 1615±1618.

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Regenerative medicine and biomaterials in connective tissue repair

Vega RB, Matsuda K, Oh J, Barbosa AC, Yang X, Meadows E, McAnally J, Pomajzl C, Shelton JM, Richardson JA, Karsenty G and Olson EN (2004), Histone deacetylase 4 controls chondrocyte hypertrophy during skeletogenesis, Cell, 119, 555±566. Venezian R, Shenker BJ, Datar S and Leboy PS (1998), Modulation of chondrocyte proliferation by ascorbic acid and BMP-2, J Cell Physiol, 174, 331±341. Vilquin JT, Marolleau JP, Sacconi S, Garcin I, Lacassagne MN, Robert I, et al. (2005), Normal growth and regenerating ability of myoblasts from unaffected muscles of facioscapulohumeral muscular dystrophy patients. Gene Ther, 12, 1651±1662. Wang DW, Fermor B, Gimble JM, Awad HA and Guilak F (2005), Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells, Cell Physiol, 204, 184±191. Yan H and Yu C (2007), Repair of full-thickness cartilage defects with cells of different origin in a rabbit model, Arthroscopy, 23, 178±187. Yang C, Li SW, Helminen HJ, Khillan JS, Bao Y and Prockop DJ (1997), Apoptosis of chondrocytes in transgenic mice lacking collagen II, Exp Cell Res, 235, 370±373. Yoshida CA, Yamamoto H, Fujita T, Furuichi T, Ito K, Inoue K, Yamana K, Zanma A, Takada K, Ito Y and Komori T (2004), Runx2 and Runx3 are essential for chondrocyte maturation, and Runx2 regulates limb growth through induction of Indian hedgehog, Genes Dev, 18, 952±963. Zhang X, Ziran N, Goater JJ, Schwarz EM, Puzas JE, Rosier RN, Zuscik M, Drissi H and O'Keefe RJ (2004), Primary murine limb bud mesenchymal cells in long-term culture complete chondrocyte differentiation: TGF-beta delays hypertrophy and PGE2 inhibits terminal differentiation, Bone, 34, 809±817. Zhou G, Zheng Q, Engin F, Munivez E, Chen Y, Sebald E, Krakow D and Lee B (2006), Dominance of SOX9 function over RUNX2 during skeletogenesis, Proc Natl Acad Sci USA, 103, 19004±19009.

12

Scaffolds for musculoskeletal tissue engineering

H . L I and J . H . E L I S S E E F F , Johns Hopkins University, USA

Abstract: Tissue engineering, with the use of cells and scaffolds, has important clinical application in regenerative medicine. Additionally, these technologies can also be applied to the study of cellular biology, specifically, the cellular interactions within organized scaffolds, and the effects of paracrine communication between different cell types. The main focus of this chapter will be on the cell±matrix interactions and the communications between normal and diseased cells within organized scaffolds. In addition, this chapter will review the different cell types utilized in musculoskeletal tissue engineering with a focus on the application of mesenchymal stem cells in therapeutic scenarios, summarize the use of natural and synthetic biomaterials in tissue engineering and their combination into organized scaffolds, and discuss how cytokines affect tissue growth and disease development through the stimulation of matrix metalloproteinases. Key words: osteoarthritis, matrix metalloproteinases, scaffolds, mesenchymal stem cells, paracrine communication.

12.1

Introduction

The promise of tissue engineering is to develop new strategies to address the challenge of tissue loss. The field can be divided into three components: the cells that synthesize the extracellular matrix (ECM) and produce functional tissue, the scaffolds that provide a three-dimensional support for new tissue growth, and the signaling molecules that stimulate cells to proliferate, differentiate, and synthesize the ECM (Chapekar 2000; Griffith and Naughton 2002). These components have been applied either alone or combined in an attempt to repair tissues in the musculoskeletal system. An example of musculoskeletal tissue is hyaline cartilage, which can suffer traumatic injuries or progressively degenerate due to osteoarthritis (OA). OA affects millions of people within the United States, targeting the elderly population and younger adults who have suffered joint trauma (Brittberg et al. 1994; Johnson-Nurse and Dandy 1985; Lawrence et al. 2008). It is characterized by degradation of the articular cartilage, along with synovial inflammation and osteophyte formation, as well as changes in the underlying subchondral bone. Tissue engineering therapies are ideal for regenerating cartilage since hyaline cartilage has a naturally low capacity for repair due to its avascular and aneural phenotype. Current treatments, such as

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surgical microfracture or cell therapies, have shown inconsistent results and poor long-term repair, while nutritional supplements that are orally administered, such as glucosamine and chondroitin sulfate, have also failed to result in significant improvement in recent clinical studies (Gregory et al. 2008). As the field of tissue engineering has matured, new scaffold systems have been designed that support the ability to organize different cell types into a hierarchical structure and mimic the biological niches for cell growth and function. This organization can aid in recapitulating native tissue structure and recreating the interfacial zones between different tissues (Phillips et al. 2008; Sharma and Elisseeff 2004; Sharma et al. 2007). This architecture is important not only for the structural aspects of the tissue, but also in guiding the paracrine communication between different cells, which plays a crucial role in new tissue formation and maintenance. Remodeling is another important aspect of tissue regeneration as it contributes to the imbalance of tissue metabolism associated with diseases. Therefore, a better understanding of the proteases and cytokines that control this process is required for regenerating tissues in both a normal and a diseased environment. In addition to addressing the three main components of tissue engineering in the context of musculoskeletal tissues, this chapter will focus on the importance of co-culturing cells in hierarchical scaffolds, and the resulting effects of cell±cell communication on the homeostasis of both normal and osteoarthritic cartilage.

12.2

Cell types utilized for tissue regeneration

12.2.1 Autologous primary cells Mature articular cartilage comprises the superficial, transitional, and deep zones (Ulrich-Vinther et al. 2003). While chondrocytes are the primary cells that reside within all the zones, each layer varies in cellular morphology and density as well as ECM fibril formation. Chondrocytes in the superficial zone are flattened and the collagen fibrils are parallel to the cartilage surface, while the transitional zone is composed of obliquely oriented fibrils that are randomly organized. In the deep zone, the cells and collagen fibrils are vertical and columnar in formation. In each zone, the chondrocytes are surrounded by an ECM that is composed of type II collagen, aggrecan, smaller proteoglycans, and other collagenous and noncollagenous proteins (Ulrich-Vinther et al. 2003). When trauma or defect occur, the tissue's initial response is signaled from the chondrocytes, which proliferate and increase synthesis of the ECM components in an attempt to repair the defect. However, this intrinsic effort is very limited due to the lack of blood supply and innervation. Instead of a new hyaline cartilage, a fibrocartilage is formed, which lacks the same mechanical structure as hyaline cartilage. Surgical techniques are the dominant therapies implemented today in cartilage repair. Microfracture is a commonly performed surgical process

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designed to induce repair in a defect site by drilling or creating holes into the subchondral bone. This creates an influx of bone marrow mesenchymal stem cells (MSCs) into the blood clot that forms at the site, which results in the formation of fibrocartilage (Mithoefer et al. 2005). However, this treatment is effective primarily in small defects, and may cause damage to the subchondral bone. Autologous chondrocyte transplantation (ACT) is another surgical method that is applied to patients with larger defects. This technique requires cell isolation and monolayer expansion from enzymatic digestion of a tissue biopsy that is usually harvested from all three zones of the cartilage, after which the cells are introduced to the defect site (Brittberg 2008; Roberts et al. 2008). Patients are required to limit their mobility of that operated joint, while going through rehabilitation to gradually increase the defect's weight-bearing load and range of motion. While chondrocytes are the obvious choice of cells to use in generating new and functional tissue, they have low metabolism, and primarily serve to maintain cartilage homeostasis (Goldring and Goldring 2007). In addition, the cells begin dedifferentiating once they are outside their native environment and cultured in monolayer, thus decreasing their ability to express and produce ECM components. Therefore, three-dimensional culture systems are currently in use today to aid the chondrocytes in regaining as much of their original phenotype as possible. In vitro and preclinical studies have investigated the application of chondrocytes to a variety of gel-based and solid polymer scaffolds, such as gelatin, chitosan, agarose, poly(ethylene glycol) (PEG), poly(lactic-co-glycolic acid) (PLGA), and collagen (Getgood et al. 2009). The cells are either seeded on or encapsulated within the scaffolds, and are able to secrete a matrix of proteoglycans and collagens typical of hyaline cartilage. Translation to large animal preclinical models and clinical application is in progress for some of the technologies (Brehm et al. 2006; Cancedda et al. 2003; Dell'Accio et al. 2003; Frisbie et al. 2006; Gotterbarm et al. 2008). The ability of osteoarthritic chondrocytes to secrete a functional matrix or repair tissue is also being investigated. Patients with no history of OA, but who have had a traumatic cartilage injury, are at significant risk for developing OA. Therefore, a better understanding of OA chondrocytes is critical as surgeons attempt the application of ACT in patients who have some degree of OA, and thus potentially isolate biopsies with diseased cells. Researchers are investigating if these diseased cells can be rescued with the correct stimulus or culture environment. One study has demonstrated that culturing OA chondrocytes in collagen matrices resulted in higher cell death over a 2-week period as indicated by DNA quantity when compared with healthy chondrocytes isolated from both an elderly patient and an adolescent undergoing surgeries unrelated to OA (Dorotka et al. 2005). There was also diminished biological activity of the OA chondrocytes, as demonstrated by the glycosaminoglycan (GAG) production.

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In another study, isolating OA chondrocytes and forming three-dimensional pellets resulted in good proliferation capabilities and comparable proteoglycan accumulation as compared with ACT donors; however, there was significantly less total collagen when the OA chondrocytes underwent redifferentiation (Tallheden et al. 2005). A third study profiled the gene expressions of osteoarthritic chondrocytes, and compared it with those isolated from healthy cartilage and also from patients undergoing ACT (Stoop et al. 2007). Both the OA population and healthy chondrocytes exhibited high aggrecan gene expressions, and type II collagen expression was higher in OA chondrocytes after expansion when compared with those isolated from ACT. Expanded OA chondrocytes were also seeded onto collagen scaffolds and implanted into severe combined immunodeficient (SCID) mice. When seeded at high enough densities, OA chondrocytes from all the donors in the experiment were able to secrete cartilage matrix in levels comparable to those from healthy chondrocytes. These studies indicate that more experiments need to be conducted to reveal the full potential of osteoarthritic chondrocytes in cell-based therapies, and perhaps to optimize the conditions of culturing osteoarthritic chondrocytes in order to restore their original phenotype and to regenerate functional cartilage tissue.

12.2.2 Adult stem cells The number of chondrocytes that can be isolated from a small biopsy is limited, and changes during cell expansion decrease their efficiency in building tissues. Furthermore, cells isolated from a diseased environment may be less efficient in producing extracellular matrix. Consequently, much attention has been given to adult stem cells as an alternative cell source for musculoskeletal tissue engineering. These stem cells reside in numerous tissues, such as bone marrow, synovium, muscles, and adipose (Barry and Murphy 2004; Caplan 2007; De Bari et al. 2001; Haynesworth et al. 1992; Lee et al. 2000; Pittenger et al. 1999; Shi and Gronthos 2003; Zuk et al. 2001). The two main properties of adult stem cells are the ability to self-renew, and the ability to differentiate into several lineages, such as cartilage, bone, and fat (Caplan 2007). Bone marrow-derived MSCs are a class of adult stem cells that have been extensively characterized in tissue formation, and translated to clinical applications. Much progress has been made with the application of MSC in musculoskeletal tissue regeneration. They have been demonstrated to enhance repair in osteochondral defects, femoral defects, muscular injuries, and tendon and ligament healing (Caplan 2007). As mentioned before, microfracture surgery not only increases the blood flow from the subchondral bone to the site of cartilage defect, but it also stimulates migration of progenitor stem cells, thus promoting repair and the formation of fibrocartilage (Endres et al. 2007; Shapiro et al. 1993). Studies have also indicated that donor MSCs have the ability to home specifically into tissue defect sites before instigating repair (Barry and Murphy

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2004; Rombouts and Ploemacher 2003). Numerous experiments have demonstrated the ability of MSCs to undergo chondrogenesis and osteogenesis when provided the appropriate stimuli (Caplan 2005; Jaiswal et al. 1997; Johnstone et al. 1998; Marolt et al. 2006; Williams et al. 2003). As with chondrocytes, MSCs are also able to secrete ECM when cultured with various biomaterials (Dawson et al. 2008; Elisseeff et al. 2005). In vivo animal models have additionally shown that MSCs can be employed to generate new bone and cartilage tissues (Lennon et al. 2001; Pelttari et al. 2006; Wang et al. 2007). While MSCs prove to be a promising cell source, studies so far have been unable to demonstrate the ability of MSCs to generate robust cartilage with ECM production and mechanical properties that match that of native cartilage. The primary advantage of utilizing MSCs may rest in the trophic factors they secrete (Caplan and Dennis 2006). Animal studies have suggested that MSCs are capable of repairing tissue even without undergoing much differentiation themselves. This implies that their regenerative effects may be a result of the trophic factors that they secrete more than the actual tissue they become. MSCs have been used in various animal models that resulted in the repair of brain tissue, enhancement of heart function, increased vascular endothelial growth factor (VEGF) production in ischemic myocardium, and regeneration of the meniscus in surgically induced joint defects (Caplan and Dennis 2006; Chen et al. 2003; Li et al. 2005; Min et al. 2002; Murphy et al. 2003; Shake et al. 2002; Tang et al. 2004). Since these preclinical studies did not always demonstrate significant MSC differentiation in the new tissue formation, trophic secretions were hypothesized as the mode of action. MSCs have also exhibited immuno-suppressive effects, providing more evidence for their role as secretory cells of trophic factors (Aggarwal and Pittenger 2005; Chen and Tuan 2008). The fact that MSCs secrete bioactive factors that can suppress the immune system offers an explanation for why allogeneic cells are not rejected by host patients, and also provides a unique donor opportunity for patients who can not use autologous cells. A study by Aggarwal et al. examined the interactions between MSCs and different immune cell lines, such as dendritic cells, T cells, and natural killer cells, in co-culture systems (Aggarwal and Pittenger 2005). The results showed that MSCs inhibited important proinflammatory cytokines and increased suppressive cytokines. This could potentially explain the safety of transplanting allogeneic MSCs in treating patients with severe graft-versus-host-disease (GVHD) (Le Blanc et al. 2008). The mechanism, however, by which MSCs improve disease states is still unknown. A systematic characterization of paracrine interactions between MSCs and other cell types in controlled environments can help explain the mechanism of repair.

12.2.3 Embryonic stem cells Embryonic stem cells (ESCs) are another potential cell source for tissue regeneration. An advantage of using ESCs relies in their unlimited proliferative

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capacity and their ability to differentiate into virtually all tissue types (Elisseeff 2004; Hwang et al. 2008; Shamblott et al. 1998; Thomson et al. 1998). ESCs are derived from the inner cell mass of a blastocyst, and can proliferate, differentiate, and secrete ECM when placed into three-dimensional biomaterial structures such as PEG and PLGA (Hwang et al. 2008; Levenberg et al. 2003). However, determining the appropriate biological signals to induce efficient and safe differentiation and tissue production remains a challenge. In the case of cartilage, a number of methodologies have recently been defined to guide ESCs towards chondrogenesis. For example, supplementation of culture medium with TGF- and BMP can induce ESCs to differentiate into chondrocytes, while exposing ESCs to morphogenetic factors secreted by fully differentiated cells also controls the differentiation of ESCs (Hwang et al. 2006a,b; Lee et al. 2008). Human ESCs that were co-cultured in a Transwell system with bovine chondrocytes for three weeks demonstrated an up-regulation of cartilagespecific gene markers, such as aggrecan and type II collagen, while ESCs not exposed to the co-culture system expressed multiple lineage markers (Hwang et al. 2008). ESCs are also capable of undergoing osteogenic differentiation through endochondral ossification (Jukes et al. 2008). Mineralized ECM was observed in the nodules that were formed from ESCs cultured in osteogenic supplements, along with a uniform distribution of type I collagen. Levenberg et al. also observed that culturing ESCs in a three-dimensional structure was important in supporting structure and matrix formation, and that the mechanical stiffness of the three-dimensional environment affects cell growth and tissue organization (Levenberg et al. 2003). While interest and knowledge in ESCs have accelerated over the last decade, their use has generated a lot of controversy because of the source of the cells (Elisseeff 2004). Human ESCs might have the proliferation and differentiation properties that tissue engineers want, but the method of isolating these cells from blastocysts and fetal tissue has prevented them from being fully studied in tissue engineering. However, ESCs have the advantage over MSCs in that they can proliferate indefinitely and maintain their ability to differentiate into multiple lineages. Much work still needs to be done to determine the optimal factors and environment to culture ESCs to generate fully functional tissue.

12.3

Scaffolds for engineering musculoskeletal tissue

Scaffolds provide a three-dimensional structural framework in which cells grow and secrete ECM. Biomaterial scaffolds can be implanted into the body with or without cells, and stimulate local cell activity to induce cellular infiltration (Leach and Mooney 2008; Silva and Mooney 2004). Requirements for biomaterial scaffolds include biocompatibility and diffusivity to various growth factors and nutrients. Ideally, a scaffold should be biodegradable at a

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rate that matches with the anabolic activity of the cells to create space for new tissue.

12.3.1 Natural and synthetic materials The choice of materials for scaffolds depends on the application, and can range from biologically derived or natural polymers to synthetic, artificial materials. The most commonly studied natural material in tissue engineering is collagen because it is a major constituent of all connective tissue; thus, extensive research has been conducted that focuses on either using collagen derived from different sources or developing biomaterials that mimic the fibrillar composition of native collagen-based ECM (Grande et al. 1997; Khew and Tong 2008; Nesic et al. 2006; Wakitani et al. 1994). Hyaluronic acid (HA) is another natural material that is extensively studied especially in cartilage tissue engineering. It is a glycosaminoglycan that gives cartilage its viscoelastic property owing to its ability to absorb and retain water (Bastow et al. 2008). Within the synovial fluid, it also provides lubrication for the joint and shock absorption. HYAFF-11 is an example of an HA scaffold used to retain seeded chondrocytes and MSCs for cartilage tissue regeneration, and studies with this biomaterial show that it enhances chondrogenesis and cartilage ECM production (Lisignoli et al. 2005, 2006). Other natural materials that have been used in cartilage tissue engineering include chitosan and alginate, while hydroxyapatite and demineralized bone are often incorporated into scaffolds for bone regeneration (Li and Zhang 2005; Yoshikawa et al. 2009). Synthetic materials are frequently applied in tissue engineering due to their highly controlled physical properties, consistency, and ease in manufacturing (Ratner and Bryant 2004). In addition, utilizing synthetic materials can reduce the risk of inducing an immune response or disease transmission from nonautologous materials. Examples include PEG, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), PLGA, polycaprolactone (PCL), poly(vinyl alcohol) (PVA) and poly(methyl methacrylate) (PMMA). Modification of synthetic materials with biological segments are often applied to direct adhesion, cell growth, or cell migration to mimic natural tissue behavior (Lutolf et al. 2003a,b). Hydrogels are one class of biomaterials that are either synthetic or natural, which fulfill many of the necessary scaffold requirements, such as biocompatibility, tissue-like elasticity, high water content and efficacy in allowing nutrient diffusion to aid tissue growth (Van Tomme et al. 2008). In addition, hydrogels are among the class of polymers that can be injected into tissue defect sites, thus presenting a minimally invasive alternative to the current surgical procedures. Numerous cell types related to the musculoskeletal system have been evaluated with hydrogels, including chondrocytes, osteoblasts, fibroblasts and MSCs (Bryant and Anseth 2002; Burdick and Anseth 2002; Nuttelman et al.

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2005; Zheng Shu et al. 2004). Specifically, results from applying PEG hydrogels to cartilage tissue engineering are very promising in supporting chondrogenic growth and ECM production within the matrix (Williams et al. 2003). Biomimetic scaffolds are designed to elicit cellular responses that lead to new tissue formation by attempting to mimic the natural environment that cells reside within (Ma 2008; Moutos et al. 2007). Self-assembling peptide-based hydrogels are an example of biomimetic scaffolds that form beta-sheet structures. This creates a nanofibrous structure which can induce specific cell±matrix interactions to promote tissue formation. The nanofibrillar structure also provides a matrix that is more similar to that experienced by cells in their native environment. Synthesizing hydrogels that have been crosslinked with protease substrates to mimic the enzymatic biodegradability of a tissue's ECM to induce remodeling is another form of creating biomimetic scaffolds (Ma 2008). An example of this is a matrix metalloproteinase (MMP)-sensitive hydrogel designed by Jeffrey Hubbell's group that incorporates substrates cleavable by MMPs (Fig. 12.1). Their in vivo studies demonstrated that by incorporating bone morphogenetic protein 2 (BMP-2) with MMP-sensitive hydrogels, there was distinctly more cellular invasion into the rat cranial defects from the surrounding host tissue as well as better bone healing (Lutolf et al. 2003b). This controlled release of a growth factor is a third technique in which biomimetic scaffolds attempt to recreate the native cellular milieu. The use of these materials allows cells to receive stimulatory signals at a more natural rate. Such subtle changes in growth factor concentrations have been shown to affect gene expression and, ultimately, tissue production.

12.3.2 Composite scaffolds to repair tissue With a greater understanding of cellular and developmental biology and novel biomaterials, tissue engineers are now better equipped to fabricate more specialized composite scaffolds. Two important goals in choosing scaffolds for cartilage tissue engineering are recreating the stratified layers that naturally occur in cartilage, and mimicking the mechanical properties of healthy articular cartilage. While polymers such as hydrogels meet the requirement for biocompatibility and diffusivity of nutrients, their mechanical properties do not entirely match that of native cartilage. Composite scaffolds are composed of multiple biomaterials, sometimes combining synthetic and natural materials. Taking advantage of the greater mechanical strength often provided by a synthetic material while still maintaining the biomimetic properties of natural materials, these composite materials have shown to both promote tissue production and provide structural integrity. Composite scaffolds can be fabricated in several ways. One method includes reinforcing the scaffolds with embedded materials to improve mechanical properties or cell seeding, such as using a polyglycolic±polylactic copolymer

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12.1 Histological samples with Goldner Trichrome demonstrating how the presence of MMP-sensitive sequences in the PEG hydrogel with incorporated recombinant human BMP-2 resulted in better bone healing than the controls (a, b) as well as collagen positive control gel (d). Scale bar ˆ 1 mm. Reprinted with permission from Macmillan Publishers Ltd: Nature Biotechnology, Lutolf, M.P., Weber, F.E., Schmoekel, H.G., Schense, J.C., Kohler, T., Muller, R. & Hubbell, J.A., `Repair of bone defects using synthetic mimetics of collagenous extracellular matrices', vol. 21, no. 5, pp. 513±518. Copyright 2003.

scaffold with alginate carriers for even cell distribution (Marijnissen et al. 2002). Another study involved scaffolds composed of PCL and poly-L-lactide acid (PLLA) fibers to address biocompatibility with lower degradability and stronger mechanical properties, while providing guidance for cellular migration (Guarino et al. 2008). Multilayered composite scaffolds are also created to address the zonal structures of tissues. These layers can be composed of either the same

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material but with different cell types (i.e. zonal chondrocytes layered within PEG diacrylate), or different materials to mimic the stratified layers. Gao et al. provide an example in which a composite scaffold composed of injectable calcium phosphate and degradable HA material was implanted into osteochondral defects surgically created in New Zealand white rabbits (Gao et al. 2002). Within 4 weeks, they observed integration of the calcium phosphate and the HA sponge, as well as clusters of chondrocytes in the chondral layer of the composite scaffolds. The zonal layers of healthy cartilage were also detected after 12 weeks of implantation. Arranging fibers into three-dimensional structures is a third technique to create woven architecture with porous structures. Moutos and Guilak (2008) developed a three-dimensional weaving method that provides the anisotrophy and viscoelasticity that are similar to those found in healthy articular cartilage. An example is creating multiple layers of perpendicularly oriented PGA fibers that were reinforced with the third set of fibers to mimic the mechanical properties of native cartilage (Moutos et al. 2007). Moutos et al. were able to dictate fiber specifications, such as the spacing and volume fraction of the fibers, and also specific selection of each fiber in the construct. Using these fibers to reinforce agarose hydrogels resulted in higher aggregate moduli and Young's moduli when compared with unreinforced agarose gels. Electrospinning is another fibrous technique that fabricates continuous fibers in both the micro- and nanoscales. By applying voltage to a solution that usually consists of polymer through the tip of a spinneret, a stream of liquid is generated, and collected on a grounded collector to form fibers (Pham et al. 2006; Teo et al. 2006). Advantages of electrospinning include a high surface to volume ratio for improved adhesion of cells to the material, control of fiber dimensions as well as porosity of the scaffold, and the ability to create aligned structures to aid in cell proliferation and elongation. Catledge et al. (2007) created a nanostructured scaffold with three different fibrous phases comprising PCL, collagen and hydroxyapatite. This composite formed a scaffold with interconnected pores, and allowed a slower degradation rate while matching mechanical properties and using biomaterials commonly found in bone. In another study, Erisken et al. (2008) applied a hybrid twin-screw method to create PCL electrospun scaffolds with incorporated -tricalcium phosphate nanoparticles for potential use in generating bone±cartilage interface.

12.4

Tissue remodeling

12.4.1 Metalloproteinases and their inhibitors The remodeling of the extracellular matrix is a constant process that occurs in healthy tissues, thus making it another focus in the field of tissue engineering. Endogenous proteases and their natural inhibitors are actively involved in the

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remodeling of healthy tissue to maintain the tissue's homeostasis (Bramono et al. 2004; Clark and Parker 2003; Gepstein et al. 2002; Zhang et al. 2003). They act by degrading old matrix components and stimulating resident cells to synthesize new matrix proteins. Matrix metalloproteinases (MMPs) are zincand calcium-dependent enzymes that play a prominent role in the remodeling process, and they exhibit a low basal activity in normal articular cartilage that is balanced with their tissue inhibitors of metalloproteinase (TIMPs) in a stoichiometric ratio. MMPs are also present in numerous other tissues and they degrade the ECM by cleaving various matrix fibrils at specific sites. MMPs are grouped into several families based on their main catalytic activity. Most of the MMP research for cartilage repair has focused on the collagenases (MMP-1, -8, -13), which specifically cleave collagen fibrils, and gelatinases (MMP-2, -9), which cleave collagen fibrils that are partially hydrolyzed. In addition to the remodeling of the ECM, MMPs aid in the migration of cells through protease-mediated breakdown of tissues (Vu and Werb 2000). As previously mentioned, Lutolf et al. engineered synthetic MMP-sensitive hydrogels, and observed infiltration of host cells as well as substantial bone formation within rat cranial defects with the addition of BMP-2 (Lutolf et al. 2003a,b). Additionally, several studies have shown that inhibiting an MMP activity during an arterial injury or creating animal models with an MMP knockout resulted in a significant decrease in the migration of smooth muscle cells, suggesting a connection between MMPs and the formation of new arterial wall tissue at sites of injury (Bendeck et al. 1996; Galis et al. 2002). Collagen breakdown plays a crucial role in cancer, where tumor cells infiltrate the basement membrane as part of the development of metastases. The presence of denatured collagen has been shown to result in higher MMP-2, TIMP-1, and TIMP-2 expressions, indicating a potential feedback connection between cleaved matrix components and MMP/TIMP transcription and activity that affects cellular migration (Abraham et al. 2007). These studies delineate the importance of manipulating MMP proteolytic activity during tissue regeneration. The ADAMs (a disintegrin and metalloproteinase) constitute another family of proteinases that are prominent in various physiological activities. This family is also zinc-dependent, and it is divided into two groups: the ADAMS which are membrane-anchored, and the ADAMTS which are secreted although some can also bind to ECM (Clark and Parker 2003; Porter et al. 2005). While MMPs and ADAMS have several similar structural domains, the major difference is the additional disintegrin-like region in ADAMS that potentially aid in cellular adhesion to other cells or matrices. ADAMTSs have additional thrombospondin motifs, and two of the ADAMTSs (ADAMTS-4 and ADAMTS-5) are commonly studied aggrecanases in cartilage. ADAMTSs -4 and -5 have also been identified in other tissues, thus suggesting alternative substrates such as other members of the proteoglycan family (Matthews et al. 2000; Sandy et al. 2001). Because the aggrecanases cleave at a different site fromn MMPs during cartilage

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metabolism, it has been suggested that the ADAMTSs are the more prominent proteinases in the degradation of aggrecan in arthritis (Porter et al. 2005; Sandy and Verscharen 2001). In addition, animal studies have demonstrated that knocking out another aggrecanase, ADAMTS-1, has resulted in growth retardation, malformations in the adipose tissue, as well as changes in the female reproductive system of mice (Porter et al. 2005; Shindo et al. 2000). These data demonstrate the importance of studying different families of proteinases to elucidate a clearer picture of different tissue development as well as pathological processes. TIMPs are also important in affecting tissue development. Four TIMPs have been identified so far. Three are secreted molecules that diffuse freely and one is attached to the ECM (Burrage and Brinckerhoff 2007). Their mechanism of inhibition involves non-covalent binding to the MMP and chelating the zinc ion within the active site to prevent the MMP from further cleavage of surrounding fibrils. TIMPs have so far been used therapeutically in rheumatoid arthritis cartilage models, aneurysm repair models, and to control metastases (Peterson 2004). In addition, scientists have observed that within TIMP-3-null mice, there are abnormal enlargements in the pulmonary airspaces and increased degradation of the ECM (Leco et al. 2001). However, other studies have demonstrated increased apoptosis when TIMP-3 was overexpressed, while some TIMP gene delivery models have increased tumorigenesis instead of decreasing it (Baker et al. 1998; Guedez et al. 2001; Jiang et al. 2001, 2002). The effects of MMPS and TIMPs in tissue growth are more clearly defined when comparing their roles in the development of healthy tissue and in the pathophysiology of diseased tissue. MMP transcriptional expression is regulated through hormones, growth factors, and cytokines, particularly through the process of paracrine interactions (Nelson et al. 2000). In healthy tissues, TIMPs bind themselves to MMPs, and prevent them from causing degenerative processes by decreasing their catalytic activity. However, in diseased states, such as OA and cancer, MMP expression is enhanced, and its activity is substantially up-regulated. TIMP secretion is down-regulated, which adds to the upset in the balance between MMPs and TIMPs, thus resulting in faster catabolism and slower anabolism. Hence, MMPs have become therapeutic targets in the study of various diseases. Numerous studies have focused on the use of MMP inhibitors, both native and synthetic, to inhibit MMP activity in hopes of attenuating their degradative effects (Fingleton 2007). Initial studies utilized natural inhibitors, such as the TIMPs; however, their low oral bioavailability was an obstacle. Examples of synthetic inhibitors that went through clinical trials include Batimastat and Marimastat, which are two peptide-mimetic inhibitors (Hoekstra et al. 2001; Parsons et al. 1997; Rosemurgy et al. 1999). Incidentally, many of the current inhibitors are non-specific, thus resulting in adverse side effects such as musculoskeletal pain observed in cancer clinical trials. Therefore, specific inhibitors have been developed to target particular MMPs in different diseases;

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however, other MMPs may compensate for the inhibited MMP due to the ability of these proteases to cleave more than one type of substrate.

12.4.2 Cytokines in osteoarthritis In the pathogenesis of OA, the environment stimulates chondrocytes to increase the production of proteases, thus promoting a catabolic phenotype, and resulting in the irreversible degradative process of OA (Goldring and Goldring 2007). During the early stages of OA, there is an increase in synthetic activity as cells try to compensate for the increase in degradation. Cytokines are molecules that affect both the degradative and synthetic activities through various mechanisms, such as paracrine, autocrine, and/or juxtacrine communications. The connection between cytokines and OA progression is well established in the literature. There are increased levels of IL-1 and tumor necrosis factor alpha (TNF-) in the joint tissue and the surrounding synovial fluid in patients with OA (Cortial et al. 2006; Fernandes et al. 2002; Lawrence et al. 2008). These proinflammatory cytokines also increase the expression and activity of other cytokines such as IL-6, IL-17, IL-18, and leukemia inhibitor factor (LIF) (Goldring and Goldring 2004). Tetlow et al. compared the expression of six MMPs and two proinflammatory cytokines in patients who had either healthy cartilage or an OA diagnosis (Tetlow et al. 2001). The presence of MMPs was significantly higher in superficial zones than in deep zones, with stronger expression and labeling in samples taken from patients with severe OA. The degree of MMP expression corresponded to the expression of pro-inflammatory cytokines, IL-1 and TNF-, while healthy cartilage and deep zones of OA samples had undetectable levels of these cytokines. Although the presence of these pro-inflammatory factors is well documented, the exact mechanism is not understood. The use of a co-culture system can help elucidate the downstream mechanism of action employed by these proteases, thus identifying potential therapeutic targets to halt the degradative process and even restore cartilage to its native phenotype. Currently, there are therapies that block cytokines to treat patients with rheumatoid arthritis. TNF blockers and IL-1 antagonists are two such treatments. Clinical trials have shown some improvements in patients receiving the blocker or antagonist compared to those receiving placebo treatments (Furst et al. 2005). Additionally, animal models that have been tested with IL-1 antagonists have also demonstrated a decrease in osteophyte formation, a common symptom in OA (Caron et al. 1996). While there is evidence that these treatments can slow the progression of arthritis, they are not without their own adverse side effects.

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Matrix stimulation and cell±cell communications in tissue regeneration

12.5.1 Cell±scaffold interactions Interactions between scaffolds and cells affect cellular phenotype and the secretion of soluble factors, providing cues that could potentially lead to tissue regeneration (Lutolf and Hubbell 2005). Addressing the complexity of these interactions has been a challenge because communication between matrices and cells is bi-directional, and achieving the delicate balance remains an obstacle. An example of this complexity can be observed in the studies of HA in tissue engineering. When MSCs were seeded on HYAFF-11, it resulted in a downregulation in MMP-13 and TIMP-1 expressions, but an increase in MMP-3 expression (Lisignoli et al. 2006). Additionally, CXCL13, the chemokine responsible for modulating B cell recruitment and homing, was also increased. This study is an example of how scaffolds can be used to manipulate inflammatory mediators and protease activity during chondrogenesis. However, another example demonstrates how the presence of HA oligosaccharides resulted in a decrease of both proteoglycan and type II collagen in human articular cartilage ECM (Ohno et al. 2006). In addition, HA hexassacharides were able to induce MMP-13 expression by eliciting the same promoter activity for MMP-13 as IL-1 does. These studies serve to illustrate how scaffold composition can directly affect cellular behavior. Small changes in the biomaterial components can have a large impact on tissue regeneration. The challenge, however, is identifying which materials have these effects and defining the range of their effects, so as to utilize them in ways that promote maximal tissue regeneration. The presence of matrix fragments has also been demonstrated to affect cellular functions. Whole collagen matrix that was digested from bovine articular cartilage into collagen fragments changed MMP expression from bovine chondrocytes and explants (Fichter et al. 2006). The presence of whole collagen fragments (Colf), type II collagen fragments (Col2f), and a synthetic Ntelopeptide (Ntelo) of type II collagen resulted in a dose-dependent increase in the latent form of one of the gelatinases of the MMP family. This study leads to the hypothesis that the increased level of ECM fragments seen in a disease state can potentially play a feedback role by increasing the gene and protein expressions of various MMPs involved in cartilage remodeling. Therefore, targeting the ECM fragments could be another therapeutic approach for OA patients. A second study involving matrix fragments investigated the effects of loading cartilage fragments into a scaffold, and utilizing the fragments as the source of cells for generating cartilage tissue (Lu et al. 2006). A comparison of the fragment-loaded scaffolds to scaffolds seeded with cells by Lu et al. revealed that although the cellular morphologies were comparable, there was more intense matrix production from fragment-loaded scaffolds, and also complete

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integration of the fragment-loaded scaffolds with the neighboring cartilage and subchondral bone in a caprine trochlear defect model (Fig. 12.2). This study demonstrates an alternative approach to the ACT methodology that can decrease the number of surgeries and avoid the monolayer expansion to attain enough cells for implantation. Fabricating a concentration gradient of biomaterials into a composite scaffold can aid in determining the movement, proliferation, and differentiation of cells. Mimura et al. observed that a gradient of type I collagen induced significantly more MSCs to migrate from the periphery to the center of the cartilage defect (Mimura et al. 2008). By embedding two different concentrations of collagen into a scaffold and then transplanting them into full-thickness defects in rabbits, they observed a faster distribution of MSCs from the periphery to the center as compared with the control non-composite scaffolds. Immunolabeling also revealed stronger type II collagen presence in both regions of the composite scaffolds, thus demonstrating the effects that a slight change in the surrounding matrix can have in controlling cellular phenotypes and functions.

12.5.2 Paracrine communication through organized scaffolds Cellular signals and interactions influence tissue regeneration; thus, recreating these signals and interactions is of great interest in tissue engineering. There are several different pathways through which cells can communicate: via the bloodstream, the nervous system, through released signals and membrane receptors, or by gap junctions (Hendriks et al. 2007). These cellular communications can be mimicked and studied with co-culture systems, helping to identify key cellular regulatory components in tissue formation. Cartilage is an example of stratified tissue, whose organization relies heavily on the communication and interaction that takes place at the cellular level. Although each of the three cartilage zones is composed of chondrocytes, the morphology and physiology of the chondrocytes vary greatly from layer to layer, thus creating a stratified tissue (Sharma and Elisseeff 2004; Sharma et al. 2007; Ulrich-Vinther et al. 2003). The signaling that occurs among the different chondrocytes helps regulate cartilage growth and maintain stable mechanical properties. Therefore, recreating the natural stratification present in cartilage could be crucial for the successful engineering of replacement cartilage tissue. In one study, PEG hydrogels were utilized to encapsulate chondrocytes from the superficial and deep zones to study paracrine interactions between chondrocytes of different layers (Sharma and Elisseeff 2004; Sharma et al. 2007). In single-layered controls, deep zone cells produced more collagen and GAG than the superficial zone cells. When cocultured together in a bilayer, a much greater difference was observed between the two chondrocyte types. Safranin-O staining for GAG production confirmed that placing the cells in a heterogeneous system resulted in significantly enhanced GAG production in both the superficial and deep layers when compared with

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12.2 Safranin-O histological samples demonstrating how loading a PGA/PLA or PGA/PCL scaffold with cartilage fragments (a1) resulted in more hyalinelike cartilage tissue within the trochlear groove defects than only plain scaffold (b1), or empty defects (c1). Triangles indicate the margins of the defects, * indicates staple intrusion site. Reprinted with permission from John Wiley & Sons, Inc., `Minced cartilage without cell culture serves as an effective intraoperative cell source for cartilage repair', Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society, vol. 24, no. 6, 2006, pp. 1261±1270. Copyright 2006, Lu, Y., Dhanaraj, S., Wang, Z., Bradley, D.M., Bowman, S.M., Cole, B.J. & Binette, F.

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their respective control single layers. Mechanical testing also demonstrated that building a zonal organization of the construct resulted in significantly higher average shear modulus when compared with individual encapsulations of the different cells. Because the cells were encapsulated in a way that did not promote the mixing of two cell types, the effects that the chondrocytes had on each other was most likely through soluble paracrine factors. Organized scaffolds can also be utilized to layer different cell types in order to study the crosstalk between the cells and across tissue interfaces. An example involves reproducing the fibrocartilage interface within anterior cruciate ligament (ACL) tears. Similar to cartilage, ligaments have limited repair capacity and lack of vascularization (Bray et al. 2002). Surgical options are commonly used to apply autografts to the site of defects. However, there is a high morbidity rate at donor sites, and significant scar formation. Wang and Lu (2006) applied the concept of layering in tissue engineering and created a tri-culture system consisting of osteoblasts, chondrocytes, and fibroblasts, and studied the interactions among these cells and the effects they had in the regeneration of the interface. The format of the layering was created to mimic the graft, the interface, and the bone regions. Tri-culturing with osteoblasts resulted in an increase in mineralization and expressions of both type I and type II collagen from the fibroblasts. Cellular signaling seemed to modulate the phenotypes and expressions of various cells used in this study, thus creating a model that better mimics the interface. Skin is another tissue that is composed of several layers of different cell types; thus, creating a system with layered scaffolds may therefore be advantageous to aid in wound healing. Collagen±PCL composite membranes were fabricated to support the culture of keratinocytes and fibroblasts that were seeded on respective sides of the membrane, and resulted in good proliferation and attachment of both cell types (Dai et al. 2005). Organized scaffolds can be implemented to recreate an entire diseased environment between two tissue types to better understand what role neighboring cells play in the disease. Although seen in greater amounts in patients with rheumatoid arthritis, inflammation is also present in joints of patients with OA. A co-culture study of monocytes/macrophages with human articular chondrocytes was conducted to better understand the role that chondrocytes play during inflammation, and the cells were cultured in serum-free media to exclude effects of exogenous soluble factors and mediators (Dreier et al. 2001). Both the monocytes and macrophages were observed to survive longer in the co-culture system than in monolayer cultures. This suggests that chondrocytes secreted mediators that enhanced the survival of neighboring cells that could explain the presence of inflammation in OA. Other models have focused on the interaction between osteoarthritic chondrocytes and the subchondral osteoblasts that underlie the diseased cartilage. While osteoarthritis is an erosion of the articular cartilage in joints, the subchondral bone also plays a role in affecting chondrocytic phenotype. Sanchez et al. (2005) observed that monolayer sclerotic

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subchondral osteoblasts isolated from osteoarthritic joints produced higher amounts of osteocalcin and alkaline phosphatase. Culturing these osteoblasts with OA chondrocytes in alginate beads resulted in a decrease in aggrecan content, and increases in both MMP-3 and MMP-13 proteins. Exposing normal osteoblasts to IL-1 and then co-culturing them with OA chondrocytes also resulted in a decrease in aggrecan production and significant up-regulations of MMP-3 and MMP-13 mRNA levels, an effect not seen in unexposed normal osteoblasts. Greater stimulation of MMP gene expression was also the result when the sclerotic osteoblasts were stimulated with Il-1 and co-cultured with chondrocytes. These studies demonstrate the utility of organized, layered in vitro scaffolds for studying paracrine communication in diseases. Recent studies in regenerative medicine are focusing on the transitional interfaces between tissues to create continuous integrations of engineered tissues composed of different cell types (Phillips et al. 2008). Phillips et al. focused on the interface between bone and soft tissue in an attempt to monitor the communication between different cell types. A biomaterial-mediated gene transfer approach was implemented, and results showed that a spatial distribution of Runx2 fixed to the biomaterial created a zonal organization of osteoblastic and fibroblastic phenotypes. This study attempted to form a continuous interface between bone and soft tissue, and demonstrated that seeding just one cell type can lead to a transitional interface of different tissue types.

12.6

Future trends and perspectives

Tissue engineering has progressed tremendously over the years, and the tools and knowledge to improve the field have expanded exponentially. The current trends of tissue engineering reveal that future work will require the combination of multiple cell types along with different material composites to create hierarchical structures that mimic native tissue. Current knowledge about the pathophysiology of OA raises the concern of balancing catabolic processes with anabolic processes. Enzymatic activity has been observed to be crucial in normal tissue development because it stimulates cells to secrete extracellular components and continuously remodel the ECM. Therefore, attenuating the up-regulated protease activity in OA is only part of the potential solution in treating patients with this disease. The act of controlling proteolysis needs to be coupled with factors that encourage tissue regeneration. Various challenges still stand in the way of progress. The use of human embryonic stem cells generates controversial debates, and that has prevented the complete incorporation of those cells in tissue engineering. Instead, murine ESCs are used, and studies directing them through chondrogenesis and osteogenesis have been accomplished with relative success. The use of MSCs and autologous primary cells bypass the cell source controversy; however, MSCs do not have the ability to proliferate indefinitely, and as monolayer expansion

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continues, they lose their efficiency in differentiating into different tissue types. Similarly, primary cells start dedifferentiating when removed from their native environment, and the number of cells obtained from biopsies is very limited. In addition, the use of MSCs or autologous cells from the patients themselves could prove to be a disadvantage since old age and damage to cells could restrict the cells' original synthetic ability. The scaffolds available today also hold potential issues. Various biodegradable materials that are used have acidic by-products, thus generating unwanted side effects (Agrawal and Athanasiou 1997). Specifically within cartilage tissue engineering, the mechanical stability of most engineered matrices does not match the native tissue's values. This poses a significant problem as patients would not have a stable and strong enough joint tissue to withstand the daily wear and pressure, especially during recovery. Lack of mechanical stability also does not alleviate the pain associated with osteoarthritis. Other materials do not have good cell adhesive properties, which can alter the cells' ability to synthesize new tissue. While these challenges have yet to be met, existing research has shown that the ultimate solution in tissue regeneration will involve a better understanding of how the cytokines are involved in the pathological stage, the specificities of the paracrine communication between different cell types and how the cellular secretions affect surrounding neighbor cells, and the application of composite scaffolds to address various structural issues of different tissues and potentially manipulate cellular crosstalk in a co-culture system. Accomplishing these steps can also help determine the combination of soluble factors that will lead osteoarthritic chondrocytes, MSCs and/or ESCS to create robust cartilage that will be more hyaline and less fibrocartilage. Excellent references on this topic include website sources in addressing the musculoskeletal diseases include the National Institute of Arthritis and Musculoskeletal and Skin Diseases, the Pittsburgh Tissue Engineering Initiative, and Cell Medicine. Journal literature such as Biomaterials, Tissue Engineering, Cell, and the Annals of Biomedical Engineering address many of the advances and issues related to tissue engineering.

12.7

References

Abraham, L.C., Dice, J.F., Lee, K. and Kaplan, D.L. 2007, `Phagocytosis and remodeling of collagen matrices', Experimental Cell Research, vol. 313, no. 5, pp. 1045±1055. DOI: 10.1016/j.yexcr.2006.12.019 Aggarwal, S. and Pittenger, M.F. 2005, `Human mesenchymal stem cells modulate allogeneic immune cell responses', Blood, vol. 105, no. 4, pp. 1815±1822. DOI: 10.1182/blood-2004-04-1559 Agrawal, C.M. and Athanasiou, K.A. 1997, `Technique to control pH in vicinity of biodegrading PLA-PGA implants', Journal of Biomedical Materials Research, vol. 38, no. 2, pp. 105±114.

320

Regenerative medicine and biomaterials in connective tissue repair

Baker, A.H., Zaltsman, A.B., George, S.J. and Newby, A.C. 1998, `Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis', The Journal of Clinical Investigation, vol. 101, no. 6, pp. 1478±1487. DOI: 10.1172/JCI1584 Barry, F.P. and Murphy, J.M. 2004, `Mesenchymal stem cells: clinical applications and biological characterization', The International Journal of Biochemistry and Cell Biology, vol. 36, no. 4, pp. 568±584. DOI: 10.1016/j.biocel.2003.11.001 Bastow, E.R., Byers, S., Golub, S.B., Clarkin, C.E., Pitsillides, A.A. and Fosang, A.J. 2008, `Hyaluronan synthesis and degradation in cartilage and bone', Cellular and Molecular Life Sciences: CMLS, vol. 65, no. 3, pp. 395±413. DOI: 10.1007/s00018007-7360-z Bendeck, M.P., Irvin, C. and Reidy, M.A. 1996, `Inhibition of matrix metalloproteinase activity inhibits smooth muscle cell migration but not neointimal thickening after arterial injury', Circulation Research, vol. 78, no. 1, pp. 38±43. Bramono, D.S., Richmond, J.C., Weitzel, P.P., Kaplan, D.L. and Altman, G.H. 2004, `Matrix metalloproteinases and their clinical applications in orthopaedics', Clinical Orthopaedics and Related Research, vol. 428, no. 428, pp. 272±285. Bray, R.C., Leonard, C.A. and Salo, P.T. 2002, `Vascular physiology and long-term healing of partial ligament tears', Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society, vol. 20, no. 5, pp. 984±989. Brehm, W., Aklin, B., Yamashita, T., Rieser, F., Trub, T., Jakob, R.P. and Mainil-Varlet, P. 2006, `Repair of superficial osteochondral defects with an autologous scaffoldfree cartilage construct in a caprine model: implantation method and short-term results', Osteoarthritis and Cartilage/OARS, Osteoarthritis Research Society, vol. 14, no. 12, pp. 1214±1226. DOI: 10.1016/j.joca.2006.05.002 Brittberg, M. 2008, `Autologous chondrocyte implantation±technique and long-term follow-up', Injury, vol. 39 Suppl 1, pp. S40±49. DOI: 10.1016/j.injury.2008.01.040 Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O. and Peterson, L. 1994, `Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation', The New England Journal of Medicine, vol. 331, no. 14, pp. 889± 895. Bryant, S.J. and Anseth, K.S. 2002, `Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels', Journal of Biomedical Materials Research, vol. 59, no. 1, pp. 63±72. Burdick, J.A. and Anseth, K.S. 2002, `Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering', Biomaterials, vol. 23, no. 22, pp. 4315±4323. Burrage, P.S. and Brinckerhoff, C.E. 2007, `Molecular targets in osteoarthritis: metalloproteinases and their inhibitors', Current Drug Targets, vol. 8, no. 2, pp. 293±303. Cancedda, R., Dozin, B., Giannoni, P. and Quarto, R. 2003, `Tissue engineering and cell therapy of cartilage and bone', Matrix Biology: Journal of the International Society for Matrix Biology, vol. 22, no. 1, pp. 81±91. Caplan, A.I. 2005, `Review: mesenchymal stem cells: cell-based reconstructive therapy in orthopedics', Tissue Engineering, vol. 11, no. 7±8, pp. 1198±1211. DOI: 10.1089/ ten.2005.11.1198 Caplan, A.I. 2007, `Adult mesenchymal stem cells for tissue engineering versus regenerative medicine', Journal of Cellular Physiology, vol. 213, no. 2, pp. 341± 347. DOI: 10.1002/jcp.21200

Scaffolds for musculoskeletal tissue engineering

321

Caplan, A.I. and Dennis, J.E. 2006, `Mesenchymal stem cells as trophic mediators', Journal of Cellular Biochemistry, vol. 98, no. 5, pp. 1076±1084. DOI: 10.1002/ jcb.20886 Caron, J.P., Fernandes, J.C., Martel-Pelletier, J., Tardif, G., Mineau, F., Geng, C. and Pelletier, J.P. 1996, `Chondroprotective effect of intraarticular injections of interleukin-1 receptor antagonist in experimental osteoarthritis. Suppression of collagenase-1 expression', Arthritis and Rheumatism, vol. 39, no. 9, pp. 1535±1544. Catledge, S.A., Clem, W.C., Shrikishen, N., Chowdhury, S., Stanishevsky, A.V., Koopman, M. and Vohra, Y.K. 2007, `An electrospun triphasic nanofibrous scaffold for bone tissue engineering', Biomedical Materials, vol. 2, no. 2, pp. 142± 150. DOI: 10.1088/1748-6041/2/2/013 Chapekar, M.S. 2000, `Tissue engineering: challenges and opportunities', Journal of Biomedical Materials Research, vol. 53, no. 6, pp. 617±620. Chen, F.H. and Tuan, R.S. 2008, `Mesenchymal stem cells in arthritic diseases', Arthritis Research and Therapy, vol. 10, no. 5, p. 223. DOI: 10.1186/ar2514 Chen, J., Li, Y., Katakowski, M., Chen, X., Wang, L., Lu, D., Lu, M., Gautam, S.C. and Chopp, M. 2003, `Intravenous bone marrow stromal cell therapy reduces apoptosis and promotes endogenous cell proliferation after stroke in female rat', Journal of Neuroscience Research, vol. 73, no. 6, pp. 778±786. DOI: 10.1002/jnr.10691 Clark, I.M. and Parker, A.E. 2003, `Metalloproteinases: their role in arthritis and potential as therapeutic targets', Expert Opinion on Therapeutic Targets, vol. 7, no. 1, pp. 19±34. DOI: 10.1517/14728222.7.1.19 Cortial, D., Gouttenoire, J., Rousseau, C.F., Ronziere, M.C., Piccardi, N., Msika, P., Herbage, D., Mallein-Gerin, F. and Freyria, A.M. 2006, `Activation by IL-1 of bovine articular chondrocytes in culture within a 3D collagen-based scaffold. An in vitro model to address the effect of compounds with therapeutic potential in osteoarthritis', Osteoarthritis Cartilage, vol. 14, no. 7, pp. 631±40. DOI: S10634584(06)00009-4 [pii] 10.1016/j.joca.2006.01.008 Dai, N.T., Yeh, M.K., Liu, D.D., Adams, E.F., Chiang, C.H., Yen, C.Y., Shih, C.M., Sytwu, H.K., Chen, T.M., Wang, H.J., Williamson, M.R. and Coombes, A.G. 2005, `A co-cultured skin model based on cell support membranes', Biochemical and Biophysical Research Communications, vol. 329, no. 3, pp. 905±908. DOI: 10.1016/j.bbrc.2005.02.059 Dawson, E., Mapili, G., Erickson, K., Taqvi, S. and Roy, K. 2008, `Biomaterials for stem cell differentiation', Advanced Drug Delivery Reviews, vol. 60, no. 2, pp. 215±228. DOI: 10.1016/j.addr.2007.08.037 De Bari, C., Dell'Accio, F., Tylzanowski, P. and Luyten, F.P. 2001, `Multipotent mesenchymal stem cells from adult human synovial membrane', Arthritis and Rheumatism, vol. 44, no. 8, pp. 1928±1942. DOI: 2-P Dell'Accio, F., Vanlauwe, J., Bellemans, J., Neys, J., De Bari, C. and Luyten, F.P. 2003, `Expanded phenotypically stable chondrocytes persist in the repair tissue and contribute to cartilage matrix formation and structural integration in a goat model of autologous chondrocyte implantation', Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society, vol. 21, no. 1, pp. 123±131. DOI: 10.1016/S0736-0266(02)00090-6 Dorotka, R., Bindreiter, U., Vavken, P. and Nehrer, S. 2005, `Behavior of human articular chondrocytes derived from nonarthritic and osteoarthritic cartilage in a collagen matrix', Tissue Engineering, vol. 11, no. 5±6, pp. 877±886. DOI: 10.1089/ ten.2005.11.877 Dreier, R., Wallace, S., Fuchs, S., Bruckner, P. and Grassel, S. 2001, `Paracrine

322

Regenerative medicine and biomaterials in connective tissue repair

interactions of chondrocytes and macrophages in cartilage degradation: articular chondrocytes provide factors that activate macrophage-derived pro-gelatinase B (pro-MMP-9)', Journal of Cell Science, vol. 114, no. Pt 21, pp. 3813±3822. Elisseeff, J.H. 2004, `Embryonic stem cells: potential for more impact', Trends in Biotechnology, vol. 22, no. 4, pp. 155±156. Elisseeff, J., Puleo, C., Yang, F. and Sharma, B. 2005, `Advances in skeletal tissue engineering with hydrogels', Orthodontics and Craniofacial Research, vol. 8, no. 3, pp. 150±161. DOI: 10.1111/j.1601-6343.2005.00335.x Endres, M., Neumann, K., Haupl, T., Erggelet, C., Ringe, J., Sittinger, M. and Kaps, C. 2007, `Synovial fluid recruits human mesenchymal progenitors from subchondral spongious bone marrow', Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society, vol. 25, no. 10, pp. 1299±1307. DOI: 10.1002/ jor.20394 Erisken, C., Kalyon, D.M. and Wang, H. 2008, `Functionally graded electrospun polycaprolactone and beta-tricalcium phosphate nanocomposites for tissue engineering applications', Biomaterials, vol. 29, no. 30, pp. 4065±4073. DOI: 10.1016/j.biomaterials.2008.06.022 Fernandes, J.C., Martel-Pelletier, J. and Pelletier, J.P. 2002, `The role of cytokines in osteoarthritis pathophysiology', Biorheology, vol. 39, no. 1±2, pp. 237±246. Fichter, M., Korner, U., Schomburg, J., Jennings, L., Cole, A.A. and Mollenhauer, J. 2006, `Collagen degradation products modulate matrix metalloproteinase expression in cultured articular chondrocytes', Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society, vol. 24, no. 1, pp. 63±70. DOI: 10.1002/jor.20001 Fingleton, B. 2007, `Matrix metalloproteinases as valid clinical targets', Current Pharmaceutical Design, vol. 13, no. 3, pp. 333±346. Frisbie, D.D., Morisset, S., Ho, C.P., Rodkey, W.G., Steadman, J.R. and McIlwraith, C.W. 2006, `Effects of calcified cartilage on healing of chondral defects treated with microfracture in horses', The American Journal of Sports Medicine, vol. 34, no. 11, pp. 1824±1831. DOI: 10.1177/0363546506289882 Furst, D.E., Breedveld, F.C., Kalden, J.R., Smolen, J.S., Burmester, G.R., Bijlsma, J.W., Dougados, M., Emery, P., Keystone, E.C., Klareskog, L. and Mease, P.J. 2005, `Updated consensus statement on biological agents, specifically tumour necrosis factor  (TNF) blocking agents and interleukin-1 receptor antagonist (IL-1ra), for the treatment of rheumatic diseases, 2005', Annals of the Rheumatic Diseases, vol. 64 Suppl 4, pp. iv2±14. DOI: 10.1136/ard.2005.044941 Galis, Z.S., Johnson, C., Godin, D., Magid, R., Shipley, J.M., Senior, R.M. and Ivan, E. 2002, `Targeted disruption of the matrix metalloproteinase-9 gene impairs smooth muscle cell migration and geometrical arterial remodeling', Circulation Research, vol. 91, no. 9, pp. 852±859. Gao, J., Dennis, J.E., Solchaga, L.A., Goldberg, V.M. and Caplan, A.I. 2002, `Repair of osteochondral defect with tissue-engineered two-phase composite material of injectable calcium phosphate and hyaluronan sponge', Tissue Engineering, vol. 8, no. 5, pp. 827±837. DOI: 10.1089/10763270260424187 Gepstein, A., Shapiro, S., Arbel, G., Lahat, N. and Livne, E. 2002, `Expression of matrix metalloproteinases in articular cartilage of temporomandibular and knee joints of mice during growth, maturation, and aging', Arthritis and Rheumatism, vol. 46, no. 12, pp. 3240±3250. DOI: 10.1002/art.10690 Getgood, A., Brooks, R., Fortier, L. and Rushton, N. 2009, `Articular cartilage tissue engineering: today's research, tomorrow's practice?', The Journal of Bone and

Scaffolds for musculoskeletal tissue engineering

323

Joint Surgery. British volume, vol. 91, no. 5, pp. 565±576. DOI: 10.1302/0301620X.91B5.21832 Goldring, S.R. and Goldring, M.B. 2004, `The role of cytokines in cartilage matrix degeneration in osteoarthritis', Clinical Orthopaedics and Related Research, vol. (427 Suppl), no. 427 Suppl, pp. S27±36. Goldring, M.B. and Goldring, S.R. 2007, `Osteoarthritis', Journal of Cellular Physiology, vol. 213, no. 3, pp. 626±634. DOI: 10.1002/jcp.21258 Gotterbarm, T., Breusch, S.J., Schneider, U. and Jung, M. 2008, `The minipig model for experimental chondral and osteochondral defect repair in tissue engineering: retrospective analysis of 180 defects', Laboratory Animals, vol. 42, no. 1, pp. 71± 82. DOI: 10.1258/la.2007.06029e Grande, D.A., Halberstadt, C., Naughton, G., Schwartz, R. and Manji, R. 1997, `Evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts', Journal of Biomedical Materials Research, vol. 34, no. 2, pp. 211±220. Gregory, P.J., Sperry, M. and Wilson, A.F. 2008, `Dietary supplements for osteoarthritis', American Family Physician, vol. 77, no. 2, pp. 177±184. Griffith, L.G. and Naughton, G. 2002, `Tissue engineering ± current challenges and expanding opportunities', Science (New York.), vol. 295, no. 5557, pp. 1009±1014. DOI: 10.1126/science.1069210 Guarino, V., Causa, F., Taddei, P., di Foggia, M., Ciapetti, G., Martini, D., Fagnano, C., Baldini, N. and Ambrosio, L. 2008, `Polylactic acid fibre-reinforced polycaprolactone scaffolds for bone tissue engineering', Biomaterials, vol. 29, no. 27, pp. 3662±3670. DOI: 10.1016/j.biomaterials.2008.05.024 Guedez, L., McMarlin, A.J., Kingma, D.W., Bennett, T.A., Stetler-Stevenson, M. and Stetler-Stevenson, W.G. 2001, `Tissue inhibitor of metalloproteinase-1 alters the tumorigenicity of Burkitt's lymphoma via divergent effects on tumor growth and angiogenesis', The American Journal of Pathology, vol. 158, no. 4, pp. 1207±1215. Haynesworth, S.E., Goshima, J., Goldberg, V.M. and Caplan, A.I. 1992, `Characterization of cells with osteogenic potential from human marrow', Bone, vol. 13, no. 1, pp. 81±88. Hendriks, J., Riesle, J. and van Blitterswijk, C.A. 2007, `Co-culture in cartilage tissue engineering', Journal of Tissue Engineering and Regenerative Medicine, vol. 1, no. 3, pp. 170±178. DOI: 10.1002/term.19 Hoekstra, R., Eskens, F.A. and Verweij, J. 2001, `Matrix metalloproteinase inhibitors: current developments and future perspectives', The Oncologist, vol. 6, no. 5, pp. 415±427. Hwang, N.S., Kim, M.S., Sampattavanich, S., Baek, J.H., Zhang, Z. and Elisseeff, J. 2006a, `Effects of three-dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells', Stem Cells (Dayton, OH), vol. 24, no. 2, pp. 284±291. DOI: 10.1634/stemcells.2005-0024 Hwang, N.S., Varghese, S., Theprungsirikul, P., Canver, A. and Elisseeff, J. 2006b, `Enhanced chondrogenic differentiation of murine embryonic stem cells in hydrogels with glucosamine', Biomaterials, vol. 27, no. 36, pp. 6015±6023. DOI: 10.1016/j.biomaterials.2006.06.033 Hwang, N.S., Varghese, S. and Elisseeff, J. 2008, `Derivation of chondrogenicallycommitted cells from human embryonic cells for cartilage tissue regeneration', PLoS ONE, vol. 3, no. 6, pp. e2498. DOI: 10.1371/journal.pone.0002498 Jaiswal, N., Haynesworth, S.E., Caplan, A.I. and Bruder, S.P. 1997, `Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro', Journal of Cellular Biochemistry, vol. 64, no. 2, pp. 295±312.

324

Regenerative medicine and biomaterials in connective tissue repair

Jiang, Y., Wang, M., Celiker, M.Y., Liu, Y.E., Sang, Q.X., Goldberg, I.D. and Shi, Y.E. 2001, `Stimulation of mammary tumorigenesis by systemic tissue inhibitor of matrix metalloproteinase 4 gene delivery', Cancer Research, vol. 61, no. 6, pp. 2365±2370. Jiang, Y., Goldberg, I.D. and Shi, Y.E. 2002, `Complex roles of tissue inhibitors of metalloproteinases in cancer', Oncogene, vol. 21, no. 14, pp. 2245±2252. DOI: 10.1038/sj.onc.1205291 Johnson-Nurse, C. and Dandy, D.J. 1985, `Fracture-separation of articular cartilage in the adult knee', The Journal of Bone and Joint Surgery. British volume, vol. 67, no. 1, pp. 42±43. Johnstone, B., Hering, T.M., Caplan, A.I., Goldberg, V.M. and Yoo, J.U. 1998, `In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells', Experimental Cell Research, vol. 238, no. 1, pp. 265±272. DOI: 10.1006/ excr.1997.3858 Jukes, J.M., Both, S.K., Leusink, A., Sterk, L.M., van Blitterswijk, C.A. and de Boer, J. 2008, `Endochondral bone tissue engineering using embryonic stem cells', Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 19, pp. 6840±6845. DOI: 10.1073/pnas.0711662105 Khew, S.T. and Tong, Y.W. 2008, `Template-assembled triple-helical peptide molecules: mimicry of collagen by molecular architecture and integrin-specific cell adhesion', Biochemistry, vol. 47, no. 2, pp. 585±596. DOI: 10.1021/bi702018v Lawrence, R.C., Felson, D.T., Helmick, C.G., Arnold, L.M., Choi, H., Deyo, R.A., Gabriel, S., Hirsch, R., Hochberg, M.C., Hunder, G.G., Jordan, J.M., Katz, J.N., Kremers, H.M. and Wolfe, F. 2008, `Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II', Arthritis and Rheumatism, vol. 58, no. 1, pp. 26±35. DOI: 10.1002/art.23176 Le Blanc, K., Frassoni, F., Ball, L., Locatelli, F., Roelofs, H., Lewis, I., Lanino, E., Sundberg, B., Bernardo, M.E., Remberger, M., Dini, G., Egeler, R.M., Bacigalupo, A., Fibbe, W., Ringden, O. and Developmental Committee of the European Group for Blood and Marrow Transplantation 2008, `Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study', Lancet, vol. 371, no. 9624, pp. 1579±1586. DOI: 10.1016/S01406736(08)60690-X Leach, J.K. and Mooney, D.J. 2008, `Synthetic extracellular matrices for tissue engineering', Pharmaceutical Research, vol. 25, no. 5, pp. 1209±1211. DOI: 10.1007/s11095-008-9541-3 Leco, K.J., Waterhouse, P., Sanchez, O.H., Gowing, K.L., Poole, A.R., Wakeham, A., Mak, T.W. and Khokha, R. 2001, `Spontaneous air space enlargement in the lungs of mice lacking tissue inhibitor of metalloproteinases-3 (TIMP-3)', The Journal of Clinical Investigation, vol. 108, no. 6, pp. 817±829. DOI: 10.1172/JCI12067 Lee, H.J., Yu, C., Chansakul, T., Varghese, S., Hwang, N.S. and Elisseeff, J.H. 2008, `Enhanced chondrogenic differentiation of embryonic stem cells by coculture with hepatic cells', Stem Cells and Development, vol. 17, no. 3, pp. 555±563. DOI: 10.1089/scd.2007.0177 Lee, J.Y., Qu-Petersen, Z., Cao, B., Kimura, S., Jankowski, R., Cummins, J., Usas, A., Gates, C., Robbins, P., Wernig, A. and Huard, J. 2000, `Clonal isolation of musclederived cells capable of enhancing muscle regeneration and bone healing', The Journal of Cell Biology, vol. 150, no. 5, pp. 1085±1100. Lennon, D.P., Edmison, J.M. and Caplan, A.I. 2001, `Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo

Scaffolds for musculoskeletal tissue engineering

325

osteochondrogenesis', Journal of Cellular Physiology, vol. 187, no. 3, pp. 345±355. DOI: 10.1002/jcp.1081 Levenberg, S., Huang, N.F., Lavik, E., Rogers, A.B., Itskovitz-Eldor, J. and Langer, R. 2003, `Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds', Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 22, pp. 12741±12746. DOI: 10.1073/ pnas.1735463100 Li, Y., Chen, J., Zhang, C.L., Wang, L., Lu, D., Katakowski, M., Gao, Q., Shen, L.H., Zhang, J., Lu, M. and Chopp, M. 2005, `Gliosis and brain remodeling after treatment of stroke in rats with marrow stromal cells', Glia, vol. 49, no. 3, pp. 407± 417. DOI: 10.1002/glia.20126 Li, Z. and Zhang, M. 2005, `Chitosan-alginate as scaffolding material for cartilage tissue engineering', Journal of Biomedical Materials Research. Part A, vol. 75, no. 2, pp. 485±493. DOI: 10.1002/jbm.a.30449 Lisignoli, G., Cristino, S., Piacentini, A., Toneguzzi, S., Grassi, F., Cavallo, C., Zini, N., Solimando, L., Mario Maraldi, N. and Facchini, A. 2005, `Cellular and molecular events during chondrogenesis of human mesenchymal stromal cells grown in a three-dimensional hyaluronan based scaffold', Biomaterials, vol. 26, no. 28, pp. 5677±5686. DOI: 10.1016/j.biomaterials.2005.02.031 Lisignoli, G., Cristino, S., Piacentini, A., Cavallo, C., Caplan, A.I. and Facchini, A. 2006, `Hyaluronan-based polymer scaffold modulates the expression of inflammatory and degradative factors in mesenchymal stem cells: involvement of Cd44 and Cd54', Journal of Cellular Physiology, vol. 207, no. 2, pp. 364±373. DOI: 10.1002/ jcp.20572 Lu, Y., Dhanaraj, S., Wang, Z., Bradley, D.M., Bowman, S.M., Cole, B.J. and Binette, F. 2006, `Minced cartilage without cell culture serves as an effective intraoperative cell source for cartilage repair', Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society, vol. 24, no. 6, pp. 1261±1270. DOI: 10.1002/jor.20135 Lutolf, M.P. and Hubbell, J.A. 2005, `Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering', Nature Biotechnology, vol. 23, no. 1, pp. 47±55. DOI: 10.1038/nbt1055 Lutolf, M.P., Lauer-Fields, J.L., Schmoekel, H.G., Metters, A.T., Weber, F.E., Fields, G.B. and Hubbell, J.A. 2003a, `Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics', Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 9, pp. 5413±5418. DOI: 10.1073/pnas.0737381100 Lutolf, M.P., Weber, F.E., Schmoekel, H.G., Schense, J.C., Kohler, T., Muller, R. and Hubbell, J.A. 2003b, `Repair of bone defects using synthetic mimetics of collagenous extracellular matrices', Nature Biotechnology, vol. 21, no. 5, pp. 513± 518. DOI: 10.1038/nbt818 Ma, P.X. 2008, `Biomimetic materials for tissue engineering', Advanced Drug Delivery Reviews, vol. 60, no. 2, pp. 184±198. DOI: 10.1016/j.addr.2007.08.041 Marijnissen, W.J., van Osch, G.J., Aigner, J., van der Veen, S.W., Hollander, A.P., Verwoerd-Verhoef, H.L. and Verhaar, J.A. 2002, `Alginate as a chondrocytedelivery substance in combination with a non-woven scaffold for cartilage tissue engineering', Biomaterials, vol. 23, no. 6, pp. 1511±1517. Marolt, D., Augst, A., Freed, L.E., Vepari, C., Fajardo, R., Patel, N., Gray, M., Farley, M., Kaplan, D. and Vunjak-Novakovic, G. 2006, `Bone and cartilage tissue constructs grown using human bone marrow stromal cells, silk scaffolds and rotating

326

Regenerative medicine and biomaterials in connective tissue repair

bioreactors', Biomaterials, vol. 27, no. 36, pp. 6138±6149. DOI: 10.1016/ j.biomaterials.2006.07.015 Matthews, R.T., Gary, S.C., Zerillo, C., Pratta, M., Solomon, K., Arner, E.C. and Hockfield, S. 2000, `Brain-enriched hyaluronan binding (BEHAB)/brevican cleavage in a glioma cell line is mediated by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family member', The Journal of Biological Chemistry, vol. 275, no. 30, pp. 22695±22703. DOI: 10.1074/ jbc.M909764199 Mimura, T., Imai, S., Kubo, M., Isoya, E., Ando, K., Okumura, N. and Matsusue, Y. 2008, `A novel exogenous concentration-gradient collagen scaffold augments fullthickness articular cartilage repair', Osteoarthritis and Cartilage/OARS, Osteoarthritis Research Society, vol. 16, no. 9, pp. 1083±1091. DOI: 10.1016/ j.joca.2008.02.003 Min, J.Y., Sullivan, M.F., Yang, Y., Zhang, J.P., Converso, K.L., Morgan, J.P. and Xiao, Y.F. 2002, `Significant improvement of heart function by cotransplantation of human mesenchymal stem cells and fetal cardiomyocytes in postinfarcted pigs', The Annals of Thoracic Surgery, vol. 74, no. 5, pp. 1568±1575. DOI: Mithoefer, K., Williams, R.J.,3rd, Warren, R.F., Potter, H.G., Spock, C.R., Jones, E.C., Wickiewicz, T.L. and Marx, R.G. 2005, `The microfracture technique for the treatment of articular cartilage lesions in the knee. A prospective cohort study', The Journal of Bone and Joint Surgery. American volume, vol. 87, no. 9, pp. 1911± 1920. DOI: 10.2106/JBJS.D.02846 Moutos, F.T. and Guilak, F. 2008, `Composite scaffolds for cartilage tissue engineering', Biorheology, vol. 45, no. 3±4, pp. 501±512. Moutos, F.T., Freed, L.E. and Guilak, F. 2007, `A biomimetic three-dimensional woven composite scaffold for functional tissue engineering of cartilage', Nature Materials, vol. 6, no. 2, pp. 162±167. DOI: 10.1038/nmat1822 Murphy, J.M., Fink, D.J., Hunziker, E.B. and Barry, F.P. 2003, `Stem cell therapy in a caprine model of osteoarthritis', Arthritis and Rheumatism, vol. 48, no. 12, pp. 3464±3474. DOI: 10.1002/art.11365 Nelson, A.R., Fingleton, B., Rothenberg, M.L. and Matrisian, L.M. 2000, `Matrix metalloproteinases: biologic activity and clinical implications', Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, vol. 18, no. 5, pp. 1135±1149. Nesic, D., Whiteside, R., Brittberg, M., Wendt, D., Martin, I. and Mainil-Varlet, P. 2006, `Cartilage tissue engineering for degenerative joint disease', Advanced Drug Delivery Reviews, vol. 58, no. 2, pp. 300±322. DOI: 10.1016/j.addr.2006.01.012 Nuttelman, C.R., Tripodi, M.C. and Anseth, K.S. 2005, `Synthetic hydrogel niches that promote hMSC viability', Matrix Biology: Journal of the International Society for Matrix Biology, vol. 24, no. 3, pp. 208±218. DOI: 10.1016/j.matbio.2005.03.004 Ohno, S., Im, H.J., Knudson, C.B. and Knudson, W. 2006, `Hyaluronan oligosaccharides induce matrix metalloproteinase 13 via transcriptional activation of NFkappaB and p38 MAP kinase in articular chondrocytes', The Journal of Biological Chemistry, vol. 281, no. 26, pp. 17952±17960. DOI: 10.1074/jbc.M602750200 Parsons, S.L., Watson, S.A. and Steele, R.J. 1997, `Phase I/II trial of batimastat, a matrix metalloproteinase inhibitor, in patients with malignant ascites', European Journal of Surgical Oncology: the Journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology, vol. 23, no. 6, pp. 526±531. Pelttari, K., Winter, A., Steck, E., Goetzke, K., Hennig, T., Ochs, B.G., Aigner, T. and Richter, W. 2006, `Premature induction of hypertrophy during in vitro

Scaffolds for musculoskeletal tissue engineering

327

chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice', Arthritis and Rheumatism, vol. 54, no. 10, pp. 3254±3266. DOI: 10.1002/art.22136 Peterson, J.T. 2004, `Matrix metalloproteinase inhibitor development and the remodeling of drug discovery', Heart Failure Reviews, vol. 9, no. 1, pp. 63±79. DOI: 10.1023/ B:HREV.0000011395.11179.af Pham, Q.P., Sharma, U. and Mikos, A.G. 2006, `Electrospinning of polymeric nanofibers for tissue engineering applications: a review', Tissue Engineering, vol. 12, no. 5, pp. 1197±1211. DOI: 10.1089/ten.2006.12.1197 Phillips, J.E., Burns, K.L., Le Doux, J.M., Guldberg, R.E. and Garcia, A.J. 2008, `Engineering graded tissue interfaces', Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 34, pp. 12170±12175. DOI: 10.1073/pnas.0801988105 Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S. and Marshak, D.R. 1999, `Multilineage potential of adult human mesenchymal stem cells', Science (New York), vol. 284, no. 5411, pp. 143±147. Porter, S., Clark, I.M., Kevorkian, L. and Edwards, D.R. 2005, `The ADAMTS metalloproteinases', The Biochemical Journal, vol. 386, no. Pt 1, pp. 15±27. DOI: 10.1042/BJ20040424 Ratner, B.D. and Bryant, S.J. 2004, `Biomaterials: where we have been and where we are going', Annual Review of Biomedical Engineering, vol. 6, pp. 41±75. DOI: 10.1146/annurev.bioeng.6.040803.140027 Roberts, S.J., Howard, D., Buttery, L.D. and Shakesheff, K.M. 2008, `Clinical applications of musculoskeletal tissue engineering', British Medical Bulletin, vol. 86, pp. 7±22. DOI: 10.1093/bmb/ldn016 Rombouts, W.J. and Ploemacher, R.E. 2003, `Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture', Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, UK, vol. 17, no. 1, pp. 160±170. DOI: 10.1038/sj.leu.2402763 Rosemurgy, A., Harris, J., Langleben, A., Casper, E., Goode, S. and Rasmussen, H. 1999, `Marimastat in patients with advanced pancreatic cancer: a dose-finding study', American Journal of Clinical Oncology, vol. 22, no. 3, pp. 247±252. Sanchez, C., Deberg, M.A., Piccardi, N., Msika, P., Reginster, J.Y. and Henrotin, Y.E. 2005, `Osteoblasts from the sclerotic subchondral bone downregulate aggrecan but upregulate metalloproteinases expression by chondrocytes. This effect is mimicked by interleukin-6, -1beta and oncostatin M pre-treated non-sclerotic osteoblasts', Osteoarthritis and Cartilage/OARS, Osteoarthritis Research Society, vol. 13, no. 11, pp. 979±987. DOI: 10.1016/j.joca.2005.03.008 Sandy, J.D. and Verscharen, C. 2001, `Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo', The Biochemical Journal, vol. 358, no. Pt 3, pp. 615±626. Sandy, J.D., Westling, J., Kenagy, R.D., Iruela-Arispe, M.L., Verscharen, C., RodriguezMazaneque, J.C., Zimmermann, D.R., Lemire, J.M., Fischer, J.W., Wight, T.N. and Clowes, A.W. 2001, `Versican V1 proteolysis in human aorta in vivo occurs at the Glu441-Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4', The Journal of Biological Chemistry, vol. 276, no. 16, pp. 13372± 13378. DOI: 10.1074/jbc.M009737200

328

Regenerative medicine and biomaterials in connective tissue repair

Shake, J.G., Gruber, P.J., Baumgartner, W.A., Senechal, G., Meyers, J., Redmond, J.M., Pittenger, M.F. and Martin, B.J. 2002, `Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects', The Annals of Thoracic Surgery, vol. 73, no. 6, pp. 1919±25; discussion 1926. Shamblott, M.J., Axelman, J., Wang, S., Bugg, E.M., Littlefield, J.W., Donovan, P.J., Blumenthal, P.D., Huggins, G.R. and Gearhart, J.D. 1998, `Derivation of pluripotent stem cells from cultured human primordial germ cells', Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 23, pp. 13726±13731. Shapiro, F., Koide, S. and Glimcher, M.J. 1993, `Cell origin and differentiation in the repair of full-thickness defects of articular cartilage', The Journal of Bone and Joint Surgery. American volume, vol. 75, no. 4, pp. 532±553. Sharma, B. and Elisseeff, J.H. 2004, `Engineering structurally organized cartilage and bone tissues', Annals of Biomedical Engineering, vol. 32, no. 1, pp. 148±159. Sharma, B., Williams, C.G., Kim, T.K., Sun, D., Malik, A., Khan, M., Leong, K. and Elisseeff, J.H. 2007, `Designing zonal organization into tissue-engineered cartilage', Tissue Engineering, vol. 13, no. 2, pp. 405±414. DOI: 10.1089/ ten.2006.0068 Shi, S. and Gronthos, S. 2003, `Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp', Journal of Bone and Mineral Research: the Official Journal of the American Society for Bone and Mineral Research, vol. 18, no. 4, pp. 696±704. Shindo, T., Kurihara, H., Kuno, K., Yokoyama, H., Wada, T., Kurihara, Y., Imai, T., Wang, Y., Ogata, M., Nishimatsu, H., Moriyama, N., Oh-hashi, Y., Morita, H., Ishikawa, T., Nagai, R., Yazaki, Y. and Matsushima, K. 2000, `ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function', The Journal of Clinical Investigation, vol. 105, no. 10, pp. 1345±1352. DOI: 10.1172/JCI8635 Silva, E.A. and Mooney, D.J. 2004, `Synthetic extracellular matrices for tissue engineering and regeneration', Current Topics in Developmental Biology, vol. 64, pp. 181±205. DOI: 10.1016/S0070-2153(04)64008-7 Stoop, R., Albrecht, D., Gaissmaier, C., Fritz, J., Felka, T., Rudert, M. and Aicher, W.K. 2007, `Comparison of marker gene expression in chondrocytes from patients receiving autologous chondrocyte transplantation versus osteoarthritis patients', Arthritis Research and Therapy, vol. 9, no. 3, p. R60. DOI: 10.1186/ar2218 Tallheden, T., Bengtsson, C., Brantsing, C., Sjogren-Jansson, E., Carlsson, L., Peterson, L., Brittberg, M. and Lindahl, A. 2005, `Proliferation and differentiation potential of chondrocytes from osteoarthritic patients', Arthritis Research and Therapy, vol. 7, no. 3, pp. R560±568. DOI: 10.1186/ar1709 Tang, Y.L., Zhao, Q., Zhang, Y.C., Cheng, L., Liu, M., Shi, J., Yang, Y.Z., Pan, C., Ge, J. and Phillips, M.I. 2004, `Autologous mesenchymal stem cell transplantation induce VEGF and neovascularization in ischemic myocardium', Regulatory Peptides, vol. 117, no. 1, pp. 3±10. Teo, W.E., He, W. and Ramakrishna, S. 2006, `Electrospun scaffold tailored for tissuespecific extracellular matrix', Biotechnology Journal, vol. 1, no. 9, pp. 918±929. DOI: 10.1002/biot.200600044 Tetlow, L.C., Adlam, D.J. and Woolley, D.E. 2001, `Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes', Arthritis and Rheumatism, vol. 44, no. 3, pp. 585±594. DOI: 2-C

Scaffolds for musculoskeletal tissue engineering

329

Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S. and Jones, J.M. 1998, `Embryonic stem cell lines derived from human blastocysts', Science (New York), vol. 282, no. 5391, pp. 1145±1147. Ulrich-Vinther, M., Maloney, M.D., Schwarz, E.M., Rosier, R. and O'Keefe, R.J. 2003, `Articular cartilage biology', The Journal of the American Academy of Orthopaedic Surgeons, vol. 11, no. 6, pp. 421±430. Van Tomme, S.R., Storm, G. and Hennink, W.E. 2008, `In situ gelling hydrogels for pharmaceutical and biomedical applications', International Journal of Pharmaceutics, vol. 355, no. 1±2, pp. 1±18. DOI: 10.1016/j.ijpharm.2008.01.057 Vu, T.H. and Werb, Z. 2000, `Matrix metalloproteinases: effectors of development and normal physiology', Genes and Development, vol. 14, no. 17, pp. 2123±2133. Wakitani, S., Goto, T., Pineda, S.J., Young, R.G., Mansour, J.M., Caplan, A.I. and Goldberg, V.M. 1994, `Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage', The Journal of Bone and Joint Surgery. American volume, vol. 76, no. 4, pp. 579±592. Wang, H., Li, Y., Zuo, Y., Li, J., Ma, S. and Cheng, L. 2007, `Biocompatibility and osteogenesis of biomimetic nano-hydroxyapatite/polyamide composite scaffolds for bone tissue engineering', Biomaterials, vol. 28, no. 22, pp. 3338±3348. DOI: 10.1016/j.biomaterials.2007.04.014 Wang, I.N. and Lu, H.H. 2006, `Role of cell-cell interactions on the regeneration of soft tissue-to-bone interface', Conference Proceedings: Annual International Conference of the IEEE Engineering in Medicine and Biology Society.IEEE Engineering in Medicine and Biology Society. Conference, vol. 1, pp. 783±786. DOI: 10.1109/IEMBS.2006.259456 Williams, C.G., Kim, T.K., Taboas, A., Malik, A., Manson, P. and Elisseeff, J. 2003, `In vitro chondrogenesis of bone marrow-derived mesenchymal stem cells in a photopolymerizing hydrogel', Tissue Engineering, vol. 9, no. 4, pp. 679±688. DOI: 10.1089/107632703768247377 Yoshikawa, H., Tamai, N., Murase, T. and Myoui, A. 2009, `Interconnected porous hydroxyapatite ceramics for bone tissue engineering', Journal of the Royal Society, Interface/the Royal Society, vol. 6 Suppl 3, pp. S341±348. DOI: 10.1098/ rsif.2008.0425.focus Zhang, J., Bai, S., Zhang, X., Nagase, H. and Sarras, M.P.,Jr 2003, `The expression of novel membrane-type matrix metalloproteinase isoforms is required for normal development of zebrafish embryos', Matrix Biology: Journal of the International Society for Matrix Biology, vol. 22, no. 3, pp. 279±293. Zheng Shu, X., Liu, Y., Palumbo, F.S., Luo, Y. and Prestwich, G.D. 2004, `In situ crosslinkable hyaluronan hydrogels for tissue engineering', Biomaterials, vol. 25, no. 7±8, pp. 1339±1348. Zuk, P.A., Zhu, M., Mizuno, H., Huang, J., Futrell, J.W., Katz, A.J., Benhaim, P., Lorenz, H.P. and Hedrick, M.H. 2001, `Multilineage cells from human adipose tissue: implications for cell-based therapies', Tissue Engineering, vol. 7, no. 2, pp. 211± 228. DOI: 10.1089/107632701300062859

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Outcome measures of articular cartilage repair M . E . T R I C E , Johns Hopkins University School of Medicine, USA

Abstract: Outcome measurement of articular cartilage repair is an essential feature in the development of this important treatment modality. Subjective outcome measures are well established for other knee problems, but are less so for articular cartilage injuries. Objective outcome measures are of particular importance with articular cartilage repair since most repair alternatives have limited long-term follow-up. Although early studies of the outcomes of articular cartilage repair have been performed using tools designed for other surgical procedures, tools designed specifically for cartilage repair are becoming available. Validated tools for both subjective and objective outcome measurement of articular cartilage repair may prove essential to ensure that repairs approximate the functional and structural characteristic of hyaline articular cartilage. Key words: articular cartilage, outcome measures, validity, utility, methodology.

13.1

Introduction

Outcome measures have become increasingly important in orthopaedic surgery. There is perhaps no other area of this profession that outcomes are proving more vital than in that of cartilage repair. There are no universally accepted measures of outcome at present. In fact, there is a wide variation in the number of available measures, most of which are not designed for cartilage repair This chapter will provide an overview of the currently accepted approaches to measuring outcomes, with an emphasis on commonly used measures. Then a critical assessment of this should provide the reader with a thorough understanding of where we are with outcomes of cartilage repair at present and provide insights as to where we might go in the future. Johnson1 cited the Webster's definition of outcome as `. . . the way something turns out, a result, consequence or an effect' in his 1994 Presidential address to the American Orthopaedic Society for Sports Medicine. Keller2 drew the distinction between patient-based outcome measures and measures of `the process of care'. Patient-based (subjective) outcomes measure the patient's perception of the results of their care. Process-centered (objective) measures assess technical factors that clinicians employ to quantitate success (e.g. range of motion, radiographic findings). While there are a myriad of measures of both

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types, the major challenge with all outcomes research is that patient-based outcomes and process measures are often divergent. For example, while a negative posterior drawer after a posterior cruciate ligament (PCL) surgery may confirm a successful outcome from a process of care standpoint, it does not always correspond to an optimal patient-based outcome. Some of the more recent outcome measurement in articular cartilage repair is particularly dependent on a combination of patient-based and process measures because of the relative newness of biologic treatments for orthopaedic pathology. Accurate outcome assessment of articular cartilage repair is especially important for a number of reasons. First, articular cartilage lesions have long been considered irreparable. This is likely the basis for some skepticism that cartilage repair treatments actually work. Thus, the burden of proof of efficacy drives the necessity for a strong outcome database. Second, while other novel treatments have revolutionized orthopaedics over the past 50 years, even some of these very successful interventions have been spawned from dismal early results. For example, while Charnley's total hip arthroplasty has ultimately proven an irrefutable success, his early efforts were marked by dramatic failures. Attentiveness to early outcomes provided the foundation upon which developed one of the most highly regarded procedures orthopaedists perform today. Third, since outcome measure validation is best achieved by comparison with an established `standard' and there is no such standard for articular cartilage repair; those attempting to assess the utility of outcome measures are at quite a disadvantage. Finally, cartilage repair provides new challenges because of the unique manner in which it and other biologic interventions actually work. Many of our currently accepted treatments succeed because we complement the body's capacity to repair itself (e.g. fracture fixation) or replace compromised tissue (e.g. arthroplasty). Rapidly developing articular cartilage procedures actually use biologic tissue to replace defective tissue. This newly implanted tissue may necessitate an entirely new outcome paradigm because it may function very differently over time. For example, some implants may result in repairs that are actually improving over time because like other living tissue, this repair tissue may function with an ongoing healing process. The outcome of such treatments that have improved over time may pose different challenges. Much of the evolution in outcome measures in orthopaedics, in general, and cartilage repair, in specific, can be attributed to the development of and utilization of measurement instruments. The features of quality instruments have been well defined. Suk et al.3 contended there are three components of quality outcome instrument: content, methodology, and clinical utility. Content is the instrument's purpose, in essence what it attempts to measure, methodology is the means through which the instrument measures, and clinical utility is the degree to which the instrument is practical in clinical settings. Each of these components has subcategories that help us to better define the usefulness of an outcome measure.

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Content may be divided into three subcategories: type, scale, and interpretation. The type of an outcome measure is defined by whether it is a clinician-based or patient-reported measure. The scale is represented by the questions that make up the instrument and how the answers are scored. Interpretation is the meaning that can be attributed to a given score. For example, in some scales higher scores are interpreted as successful outcomes. Methodology may be divided into three subcategories: validity, reliability, and responsiveness. Validity is the extent the tool measures what it is attempting to measure. reliability is the extent to which the instrument can provide similar results when measured more than once, and responsiveness is the capacity that the instrument has to manifest a change when the patient's condition is altered. Since much of articular cartilage repair is relatively new technology, the importance of methodology may well be magnified. Validity, reliability, and responsiveness of newer interventions are confounded by the lack of an established reference criterion. For example, the most concrete method of establishing validity is called criterion validity. Assessment of criterion validity requires that the results of an intervention approach that of a gold standard. In articular cartilage repair there is no universally agreed gold standard, making criteria validity much more difficult to establish. There are alternative types of validation such as construct validity. Construct validity, which assesses whether an instrument relates to other tools as would be expected by a hypothesis of what the instrument measures, may be more suitable for articular cartilage repair. Another example of the unique challenges forced by cartilage researchers is that responsiveness is likely of extraordinary importance as a parameter for outcome measurement of cartilage repair, since any scale that had limitations with measure improvement would be particularly impotent for assessment of this new technology. The last pertinent component of an outcome instrument is its clinical utility. This component is divided into two subcategories; acceptability and feasibility. Acceptability is how patient-friendly an instrument is. Feasibility is determined by how easy it is for a clinician, or a clinician's staff, to use a measure. Suk suggests that the overall quality of a given outcome instrument is determined by its strength in the above categories (content, methodology, and clinical utility). Accordingly, these criteria provide useful guidelines for the assessment of outcome measures. This chapter will provide a general overview of commonly used outcome measures of articular cartilage repair. The first part of this chapter will focus on `patient-based' outcome measures. A review of process measures will follow with emphasis on Suk's criteria for quality measures. Each of these measures will be discussed individually. Special attention will be given to those instruments particularly suited for the measurement of outcomes in articular cartilage repair.

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13.2

333

Patient-based (subjective) outcome measures

Patient-based outcome measures have taken on great importance in the measure of a variety of orthopaedic outcomes. Many of these measures have been derived from information obtained with questionnaires. While both self-administered and observer-administered questionnaires have been used to measure orthopaedic outcomes, self-administered questionnaires may be preferred because of a discrepancy between observer and patient reporting. Most of the patient-based outcome measures were designed as observer-administered devices. More recently, there has been a trend towards self-administered outcome measures. This trend is supported by data suggesting patient-reported outcomes are more reflective of the actual level of patient satisfaction.4 Although patient-based outcome measures are very popular they are by no means universally accepted. Some critics argue that these measures are inherently subjective and consequently of less utility than process of care or objective measures.

13.2.1 Lysholm The Lysholm scale was one of the first widely recognized outcome measures for knee problems.5 Designed for knee ligament injury, it was originally validated against a scale developed previously by Larson.6 Lysholm sought to generate a scoring scale that was designed specifically for the assessment of knee ligament injury, contending that there was a need for more specific scales. Ironically, this scale has since been widely used in outcome measurement of a variety of knee problems including articular cartilage repair. From a content standpoint, this score was designed as a clinician-based measure. It attempts to measure symptoms of instability. It is scored by the clinician under the patient's direction. A score of 100 is excellent and the worst possible score is 0. It consists of eight items (limp, support, stairclimbing, squatting, instability, pain, swelling, and atrophy of thigh) with pain and instability weighted most heavily. The methodology of the Lysholm score has been extensively evaluated. Validation has been done for a number of knee conditions.7±9 It has also been confirmed to be reliable and responsive as well for anterior cruciate ligament (ACL) surgery.8 Its methodological efficacy has been demonstrated for numerous knee problems. Perhaps that is why it has been so frequently employed. The utility of the Lysholm score seems to be acceptable. The Lysholm score has been accepted as a clinically reliable instrument. It does have the limitations of being clinician administered, but the scale is patient and clinician friendly. It is relatively brief and easy to comprehend. Kocher and co-workers8 provided a detailed assessment of the reliability,

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validity, and responsiveness of this scale. While they thought it had some value in this regard, they were critical of its ability to differentiate functional status for chondral pathology. Also reliability was less than optimal. Irrgang et al.10 also looked at the Lysholm scale for a variety of knee problems. While the validity of this scale for variety of disorders has been established, its validity as a measure for chondral injuries must be considered unclear. It has been used in a number of studies of articular cartilage repair.11,12 However, there has been no validation of this tool for these purposes. Some criticisms of the Lysholm score is that it is biased by being clinician administered. Sgaglione et al.13 suggested that the Lysholm scale was inherently so subjective that it might best be supplemented with another score (e.g. Tegner). Wright14 suggests this score might `best be used in conjunction with more modern patient reported outcome measures'.

13.2.2 Tegner The Tegner scale15 is a brief clinician-administered scale which grades the ability to work or perform sports. It was initially referenced in comparison with the Lysholm score and subsequently recommended as a complement to it. The scoring is from 1 to 10, 10 being able to participate in competitive sports, 0 equating to disability. The validity of this scale is somewhat controversial. Some content has been validated, while others have challenged its validity.14 Its reliability is also in question, while a number of studies have verified its responsiveness. It is quite simple and brief. Its patient and clinician friendliness are unquestioned, which may well be why it is one of the most popular component of outcomes for a variety of knee disorders. It is not to my knowledge used as an isolated measure of outcomes for research purposes.

13.2.3 Cincinnati knee rating scale Noyes et al.16 developed this widely used scale specifically for ACL outcome measurement. It is a three-part scale designed for standardization of knee evaluation. One part was meant to be `subjective' and was to be used in either a patient-based or clinician-based manner. The other two parts, including a knee examination portion were meant for physician-based `objective' assessment. The scale has undergone a number of modifications and in its most current rendition it is a 100-point scale with six subscales that include symptoms, functional activities, knee examination, knee stability testing, radiographic finds, and functional testing. A higher score is associated with a better outcome, a lower score with a worse one. From a methodologic standpoint, parts of this tool have been validated for ACL surgeries and sports in general. Reliability has been tested as well as

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responsiveness, and the curves have been deemed valid, reliable and responsive.17 As for its clinical utility, this scale is quite comprehensive and perhaps somewhat involved. This may make it something of a burden for both patients and observers. Another potential shortcoming is that the scale is for the most part observer-administered and this has been challenged as a potential source of error. This measure has also been used to evaluate outcomes with disorders other than ACL surgery. The methodology has been considered adequate in some cases. As for its utility in articular cartilage repair, it has been employed in a number of studies,18,19 but has not been validated for these purposes.

13.2.4 Medical outcome study 36-item short form The medical outcome study 36-item short-form (SF-36) is a questionnaire that is a widely recognized tool. Its content is geared towards assessing general health. This, unlike some other tools, was actually designed for patient administration.20 It was developed to be `comprehensive, psychometrically sound, and brief' with the intent to serve the purpose of a valid and reproducible measure of the general health of the patient. The use of this measure is so widespread that its use for articular cartilage repair studies is self-evident. It was designed as a shortened version of 149 validated health-related questions originally reported as a part of a 22 000 patient medical outcome study.21 While this tool is used very frequently, it is rarely used alone in any orthopaedic outcome study. Since it is a validated measure of general health it is often used as a complementary measure to another tool that assesses more specific disease. Consequently, while it is used frequently in articular cartilage repair studies, it has not to my knowledge been validated specifically for this purpose. The SF-36 consists of 35 questions that are divided into eight subscales including physical functioning, bodily pain, and role limitations due to physical problems and even social functioning. Scores are weighed and calculated by a 100-point scale (0 is severe disability and 100 is no disability). A modification of this scale divided components into physical (PCS) and mental (MCS) summary scores. The scores can be analyzed with standard deviation assessment to quantitate statistically significant differences in patient groups. According to Patel et al.21 it can be utilized for four distinct goals: (1) to determine the effect of a condition on the patient's quality of health, (2) to determine the outcome of a treatment, (3) to compare the effect of treatment options for a condition, and (4) to compare the effect of a procedure against unrelated procedure and condition. The methodology of the SF-36 is one of the reasons it is so prevalent. It has been extensively validated, and validated translations are available in many

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languages.22,23 It has also been assessed for relativity and responsiveness. This outcome measure is also valued for its utility. Specifically it is patient friendly and a shorter version has even improved this. Also it provides the latitude of being designed for patient-driven outcomes but can be utilized in a providerderived format. In many ways, the SF-36 is an optimal outcome measure. It has many, if not all, of the features sought for these measures. Its shortcoming is that it may be too general and consequently is rarely used along and perhaps rightly so. The SF-36 has been used in the study of articular cartilage repair.24 The instrument is used with other measures to provide a general health assessment of surgical outcomes. While it serves that purpose well it may prove that a shorter modification of this (e.g. SF-12) might be preferable because of the extensive battery of measures that may be indicated in assessment of these early outcomes. If there are more measures, the combined length of these measures and the SF36 may prove less patient and clinical friendly.

13.2.5 International Knee Documentation Committee subjective knee form (IKDC) The International Knee Documentation Committee subjective knee form (IKDC) was initially published in 1993 and modified in 1997.25 It was designed as a knee-specific measure to detect change in symptoms, function, and sports activity for patients with various problems including ligament and meniscal injuries, and articular cartilage injuries as well. It is a patient-based outcome measure, with an 18-item scale including questions that assess pain, stiffness, instability, activity level, etc. The IKDC form is scored by scoring individual items adding them together and then transforming the scale to a score that ranges from 0 to 100. A score of 100 means no limitations of activities and 0 means extreme disability. This score has been validated beginning with the careful validation completed in its development. Both reliability and responsiveness have been confirmed for a variety of knee problems.26,27 The extensive verification of this methodology is complemented by other important useful studies that have provided data based on age and sex. The clinical utility of this measure is foretold by its format. It is a one-page 18-question instrument that is especially patient and clinician friendly. Tanner et al.28 compared knee-specific instruments including the IKDC form for their ability to detect problems important to the patient. Evaluation of patients with ACL injury, meniscal pathology, and osteoarthritis suggested that this measure was particularly relevant to what patients find important. This instrument has been utilized for a variety of knee problems including articular cartilage repair.29,30 While this instrument was not designed specifically for any knee problem, it is both knee specific and patient relevant,

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which makes it particularly attractive as measure of outcomes of articular cartilage repair.

13.2.6 Knee injury and osteoarthritis outcome score (KOOS) The knee injury and osteoarthritis outcome score (KOOS) score31 was designed as a patient-based measure with an objective of measuring knee surgery outcomes. Based on a WOMAC (Western Ontario and McMasters Universities) scale, a validated outcome measure for osteoarthritis, it is a 42-item questionnaire which goes from 0 to 100. Higher scores equate to better outcome while lower scores are associated with poor ones. Methodology of this measure has been extensively assessed. This scale has been validated repeatedly.32 It has been tested for reliability and demonstrated test±retest reliability. It has also demonstrated to be responsive and correlated well with post-operative improvement. In fact a change of about 10 points on the KOOS scale has been confirmed by KOOS to be a meaningful measure of incremental improvement.33 The questionnaire seems relatively patient and clinician friendly, taking about 10 minutes to complete, although it requires completion of 42 items. It has been used extensively in outcome assessment for a variety of procedures, including articular cartilage repair.34 Its strengths seem to be its content and methodology. The fact that it was partly derived from a measure designed for osteoarthritis (WOMAC) may make it particularly applicable to articular cartilage repair outcomes.

13.2.7 Marx activity rating scale Marx et al.35 proposed a rating scale to measure the activity level of patients in 2001. The Marx scale sought to provide scores that would facilitate stratification of activity level for the purposes of comparison of two treatment groups. They set goals of developing a patient-based, patient-friendly instrument that could allow for comparison of patients who participate in a variety of sports. The developers seemed to optimize content, methodology, and utility in the development of this instrument. This scale is a patient-based outcome measure that seeks to measure activity level for sports in general. There are four items in the scale: running, cutting, decelerating, and pivoting. (These items were chosen with a selection process to ensure clinical relevance.) The scale also accounts for frequency of activity from one to four times per week. Thus, with four items and four options for frequency of activity the maximum score or activity level is 16 and the minimum is 0. The scale is designed to determine the highest level of function over the past year. From a methodologic standpoint, Marx et al. confirmed both reliability and validity in development. They confirmed test±retest reliability with 40 subjects.

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They also validated the measure versus other scores including the Tegner and Cincinnati scale. Responsiveness has not been confirmed. The utility of this score is perhaps its greatest asset. Marx designed it to be something that could be completed within one minute. With only four questions and four possible answers to the questions this scale is likely to be both patient friendly and clinician friendly. This scale fits well with the objective for content, methodology, and utility that we have defined. This combination may make it particularly useful in assessment of cartilage repair outcomes although its use has been limited for these purposes36 thus far. Wright14 observes that an additional strength of this scale is that it measures function rather than sports activity. One weakness may be that responsiveness has not been assessed.

13.3

Process-centered (objective) outcome measures

While patient-based outcome measures are arguably most important because they may give the best assessment of the patient's perception of outcome, distinct features of articular cartilage repair underline the importance of other methods of assessing outcomes. Specifically, for many articular cartilage repair techniques while patients may be subsequently improved, it remains controversial in many instances what quality of repair is actually being achieved. There is no debate over whether normal hyaline cartilage is the ideal surface and it is the standard that articular cartilage repairs are referenced against. However, controversy persists over how well these techniques actually replicate hyaline cartilage, if at all. Thus, objective measures of these repairs play an essential role in assessing outcome. The three major objective methods of outcome assessment are imaging or magnetic resonance imaging (MRI), to be specific, arthroscopic, and pathologic. Like subjective measures, these objective measures are subject to the same standards of methodology, while standards of content and utility are less well defined.

13.3.1 Magnetic resonance imaging MRI is rapidly becoming the most widely used objective outcome measure of articular cartilage repair. While it does not compare favorably to arthroscopy or histology as a measure of tissue integrity, its utility is greater because it is noninvasive. Continuous improvement in MRI technology has resulted in increased usefulness of this modality for assessment of repair. MRI is well established as a method of assessment of articular cartilage pathology37 and has more recently gained traction as a tool for assessment of cartilage repair tissue.38 The Articular Cartilage Imaging group of the International Cartilage Repair Society (ICRS) provided recommendations for MRI protocols for assessment of

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articular cartilage.39 While these recommendations are quite comprehensive, the most popular MRI techniques for visualizing articular cartilage are fatsuppressed three-dimensional spoiled T1 gradient-echo sequence (3D SPGR) and fast spin-echo (FSE) sequence. In general, detection of pathology is greater in the patellofemoral joint than other locations and higher grade lesions are detected with greater accuracy than lower grade lesions.40 Fat-suppressed three dimensional spoiled gradient technique and fast-spin echo have been validated in patients with 1 and 1.5 tesla magnets.37,40 MRI arthrography has also been confirmed to be an exceptional method of measuring injury of articular cartilage.41 Fat-suppressed 3D SPGR imaging is very useful in the assessment of osteochondral injury. This sequence provides special utility in evaluating the contour of actual defects.42 FSE scores more useful in the assessment of the actual signal changes within the cartilage. This sequence highlights pathology when actual defects may not be apparent. The combination of sequences proves especially useful in the pre-operative assessment of articular cartilage repair patients. Recently developed technology including 3 tesla high resolution 3D isotropic cartilage sequences may provide even more information for assessment of articular cartilage pathology.43 MRI in cartilage repair As mentioned previously, MRI has unique utility in assessment of articular cartilage repair. Unlike its two objective counterparts, arthroscopy and histology, MRI is a non-invasive assessment with few known health risks. MRI has considerable utility for the objective measurement of outcomes from a wide variety of cartilage repair interventions. Brittberg and Winalski39 point out that the goal of the Imaging Committee of the ICRS is to standardize the method used to evaluate MRI studies of cartilage repair. Important components of their suggested evaluation include assessment of: fill of the defect, integration of repair tissue, and signal changes in the articular cartilage and underlying base. MRI assessment of differing repair procedures will place emphasis on different areas of assessment. For example, fill of defect is perhaps more important to autologous chondrocyte implantation while evaluation of bony signal may have special relevance for osteochondral autograft. These authors suggest that defect fill can be approximated with MRI on the basis of several images as a percentage of the volume of the defect filled by repair tissue. Their guidelines for determination of integration include assessment of both the degree of integration with adjacent articular cartilage and with subchondral bone. As for osteochondral implants they suggest MRI assessment should include the amount of incorporation of bone plugs. Finally, they argue that the extent of abnormal marrow signal should be reported. They propose a system of grading the depth of signal change in the subchondral bone

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that spans from superficial (beneath the subchondral plate) to generalized (extending beyond the physeal scar). One scale for measuring MRI outcomes of articular cartilage repair was proposed by Henderson et al.30 This scale provided a scoring range from 1 to 4, 1 indicating the best result and 4 the worst. In this system fill of the defect with repair tissue was graded a 1 and a full thickness defect was received a 4. The signal at the repair site was graded from 1, identical to adjacent cartilage to 4, absent. Bone marrow edema was grade from 1 for absent to 4 severe. While this scale has been used in the study of articular cartilage repair it has not been validated. In fact, there has been minimal validation of MRI as a tool for assessment of articular cartilage repair. Perhaps the most extensive validation has been achieved with the magnetic resonance of cartilage repair tissue (MOCART) system.44 This system measures nine variables: (1) the degree of defect repair, (2) integration to border zone, (3) the surface of the repair tissue, (4) the structure of the repair tissue, (5) the signal intensity of the repair tissue, (6) subchondral lamina, (7) subchondral bone, (8) adhesions, and (9) effusion. Assessment of tissue was performed with both FSE and 3D FSGE. Marlovits et al. confirmed validity and reliability of this tool in measuring articular cartilage repair outcome.44 Roberts et al.45 also performed validation studies on MRI confirming its efficacy to measure outcome of articular cartilage repair. They proposed a system for semiquantitative assessment of repair scored with four features measured as either normal or abnormal. Normal received a score of 1 and abnormal a score of 0. The features assessed were surface integrity, cartilage signal, cartilage thickness, and changes in underlying bone. Their objective was to monitor articular cartilage repair, correlating MRI with histologic findings. They concluded that MRI provided useful information about the properties of the repairs in ACI. Other studies have shown relationships between MRI outcome and functional measures in articular cartilage repair.46,47 MRI results have also been compared to arthroscopic findings.46 While there is limited data on MRI as an outcome measure for articular cartilage repair, this tool is promising if for no other reason than its utility. Suggested utilization of this measurement includes routine MRI follow up of repair at 3 month, 1 year, and 2 years. Domayer et al.48 suggests that at 3 months early complications may be identified while at a year a sense of the quality of repair might provide guidance on how to counsel the patient regarding their level of activity. Additional development of MRI as an outcome measure is both necessary and eminent.

13.3.2 Arthroscopic evaluation Arthroscopic evaluation of articular cartilage injury has been prevalent for almost 50 years. Outerbridge49 provided the original scoring system for articular

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cartilage pathology in the patellofemoral joint. While this early system was described for patellar chondrolamacia assessed at arthroscopy, it remains the most commonly referenced system for arthroscopic appraisal of injury. Since Outerbridge there have been a number of scoring systems described to quantitate cartilage injury. Arthroscopy has long been considered the `gold standard' for assessment of intra-articular knee pathology.50 Arthroscopy has also been used as to monitor results of knee intervention for chondral problems for research purposes.51 While early measures of articular cartilage disease were made for the purposes of arthrotomy,49 a variety of other measures have been developed for arthroscopy.52,53 More recently a number of measures have been developed specifically for assessment of cartilage repair. Noyes and Stabler52 proposed one of the early arthroscopic grading systems for articular cartilage lesions. The scale uses an arbitrary point system to score depth and diameter of chondral lesions. It evaluates extent (depth), diameter, location, and the degree of flexion where the weight-bearing contact is made. This system has not been validated. Ayral et al.53 proposed two methods for scoring articular cartilage pathology that are among the most commonly used arthroscopic scales that are not specifically designed for articular cartilage repair. The first, the visual analog scale (VAS), is a subjective approach based on a clinician assessment. A score of 0 represents no disease while a score of 100 represents severe pathology. The second, the Socie te FrancË aise d'Arthroscopie (SFA) performed a multicenter study to to devise the SFA scoring system.54 The SFA score is graded between 0 and 100. It measures chondropathy with a relatively complex system designed to measure overall chondropathy employing a formula. It has been validated, and deemed reliable and responsive.55 However, its patient friendliness or acceptability is certainly of concern, since it involves an additional surgical procedure. Ayral coined the term `chondroscopy'.55 He proposed a unique outpatient procedure, performed under local anesthesia using a small glass lens scope and no tourniquet for the purposes of assessment of the chondral surfaces. In so doing, he sought to minimize the effect of an additional diagnostic procedure. While assorted scoring scales presented are useful for assessing chondral pathology, outcome measurement for articular cartilage repair may require special consideration. At least two major scoring systems have been designed for the purposes of measuring arthroscopic outcome of cartilage repair. These are the Oswestry Arthroscopy score and the ICRS scale. Oswestry arthroscopy score The Oswestry score was developed by the Oscell group in Oswestry, UK.46 The group agreed on the macroscopic findings that were relevant to determining the quality of cartilage repair and scores of 0, 1, or 2 points are given for five

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parameters: graft level, integration, appearance of the surface, color, and stiffness. A sum of the scores for each section is used to quantify success of the repair, with 10 being the maximum value. This scale has been validated and inter-observer reliability has been confirmed. However, data regarding other important features such as responsiveness and utility is not available. ICRS score The ICRS score was designed by Brittberg and Peterson.56 The scale measures three parameters: a degree of defect repair, integration of border zone, and macroscopic appearance. For each parameter a maximum score of 4 and a minimum score of 0 is allocated. An overall score of 12 is the maximum possible score and connotes a normal surface, while a score of 0 is an absent repair. This scale has been validated and inter-observer reliability has been confirmed. Like the Oswestry score, it has not been assessed for reliability. The utility of the ICRS scale, like the OAS, has yet to be determined. Both of these scales show promise for assessment of the outcomes of articular cartilage repair. Differences in these measures are relatively small although Smith et al.50 have suggested that the ICRS scale may be limited by the fact that there is no way to quantify hypertrophy, a fairly common problem after ACI, with this instrument. Van den Borne et al.57 also note that the ICRS score does not have provisions for measuring `stiffness', a widely held desirable characteristic of articular cartilage repair tissue. Arthroscopic assessment of cartilage repair is prevalent in the outcome database of articular cartilage repair.11,29,47,53 While there may well be an argument for arthroscopic as a routine clinical measure for assessment of cartilage repair outcomes, its feasibility is in question. The invasive nature of arthroscopy may preclude routine employment of arthroscopic measures of cartilage repair.

13.3.3 Histology Histologic evaluation has the distinction of being the most invasive method for assessing the outcome of articular cartilage repair. While it is especially important for the purpose of confirming what these interventions actually achieve, it is by definition less than patient friendly because it requires both an arthroscopy and direct injury to the repair site. It is widely held that the objective of articular cartilage repair is to achieve a repair comprising hyaline cartilage or its equivalent. The theoretical basis for this rationale is obvious, and there is also evidence to suggest that it is correct. For example, Knutsen's comparison of

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microfracture to ACI outcomes at 5 years24 found an overall failure rate of 23%, but none of the patients with a failure in their study had the `best quality cartilage'. While there are a number of histological scales that measure articular cartilage pathology and arthritis, there are very few scales specifically designed for the measurement of articular cartilage repair outcomes. Articular pathologic scoring systems The most widely recognized histologic grading system for articular cartilage pathology was introduced by Mankin et al. in 1971.58 The Histological Histochemical Grading System (HHGS) or Mankin system has long been the standard for grading articular cartilage pathology because it is comprehensive. It measures structure with specific reference to depth, cellularity, biochemical staining and tidemark integrity. This system has been widely used. Oostegaard et al.59 challenged the validity and reproducibility of this system. The Orthopaedic Research Society International (OARSI) developed a new system known as the cartilage histopathology assessment system (OOCHAS).60 Their goals were to develop a system that had five characteristics: simplicity, utility, scalability, extendability, and comparability. This system was initially presented in 2000. Increasing grade indicates more advanced disease. The score is a combined OA grade (0±6 points) and OA stage (0±4 points), resulting in a combined range of 24 points. Grades include 0 for an intact surface to grade 5 for denudation and grade 6 for deformation. Staging is based on the percent of joint involvement. This system has been correlated with the HHGS and deemed more reliable than the HHGS.61 Histology of cartilage repair Although there have been numerous reports which have precluded histologic assessment of articular cartilage repair tissue, outcome measures for histologic assessment of this tissue are few. The challenges faced with histologic grading of repair are numerous. First, biopsy is clearly the most invasive measure that we have discussed in this chapter. This certainly calls into question the utility of this measure from a standpoint of both patient and clinician friendliness. Second, the problem with any biopsy of a repair site is that heterogeneity in the repair tissue at the site can easily confound the results. Finally, while it is generally agreed that the optimal repair is hyaline articular cartilage, and absent repair or pure fibrous repair are clearly less than optimal; consensus about what `hyaline-like' means may be difficult to come by. In essence, in the event of a repair that approximates but does not achieve a hyaline repair, it may be difficult to determine which features of this substitute tissue actually correlate with a better result. Roberts et al.45 developed the OsScore, which assessed the predominant cartilage type, integrity and contour of the articulation surface, the degree of

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metachromasia with Safranin O, the degree of chondrocyte clustering, mineralization, vascularity, and basal integration. The scoring system is a scale ranging from 0 (which means deficient of repair or cartilage tissue) to 10 (which means nearly normal articular cartilage). This score was compared to the modified O'Driscoll score (MOD)58,62 that was devised for assessment of cartilage repair in rabbits. The authors chose this because there was no human standard for cartilage repair. They found this score comparable to the MOD, but in this study observers found the OsScore more easy to use. Although a histologic grading scale that is both widely accepted and validated for measurement of outcome of articular repair does not exist, there are measures that have been developed for these purposes. As noted previously, the actual attainment of a true hyaline cartilage repair remains the presumed optimal `outcome' of these procedures. Additional development and validation are clearly necessary for this essential measure of outcome to play its role in the assessment of the results articular cartilage repair. While it would not be considered a true measure of `outcome' as we know it, postmortem histologic assessment may prove particularly useful in determining the full extent of our success or failure of attempts to replicate hyaline cartilage.

13.4

Conclusions

Articular cartilage repair is among the most exciting innovations in orthopaedic surgery today. Outcome measurement will play a pivotal role in how this technology evolves. Both patient-reported or subjective outcome measures and process-centered or objective measures will play a pivotal role in this evolution. At the present, the vast numbers of tools employed in clinical research are not designed for or validated for the assessment outcomes of articular cartilage repair. Hence, both use of currently available measures and development of novel instruments are essential to ensuring that what we do in cartilage repair actually achieves our objective of replicating the functional and structural characteristics of hyaline articular cartilage.

13.5

References

1. Johnson RJ. Presidental Address of the American Orthopaedic Society for Sports Medicine. Am J Sports Med 1994; 22: 734. 2. Keller RB. Outcomes research in Orthopaedics. J Am Acad Orthop Surg 1993; 1: 122±9. 3. Suk M, Norvell DC, Hanson B, Dettori JR, Helfet D. Evidence-based orthopaedic surgery: what is evidence without the outcomes? J Am Acad Orthop Surg 2008 Mar; 16(3): 123±9. 4. Hoher J, Bach T, Munster A, Bouillon B, Tiling T. Does the mode of data collection change results in a subjective knee score? Self-administration. Am J Sports Med 1997 Sep±Oct; 25(5): 642±7.

Outcome measures of articular cartilage repair

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5. Lysholm J and Gillquist J. Evaluation of knee ligament surgery results with special emphasis or use of a scoring scale. Am J Sports Med 1982; 10(3): 150±4. 6. Larson RL and Smillie IS. Larson score. In: Smillie IS, ed. Diseases of the knee joint. Edinburgh, London: Churchill Livingstone, 1974, 28±31. 7. Kocher MS, Steadman JR, Briggs KK, Sterett WI, Hawkins RJ. Reliability, validity, and responsiveness of the Lysholm knee scale for various chondral disorders. J Bone Joint Surg Am 2004 Jun; 86-A(6): 1139±45. 8. Briggs KK, Lysholm J, Tegner Y, Rodkey WG, Kocher MS, Steadman JR. The reliability, validity, and responsiveness of the Lysholm score and Tegner activity scale for ligament injuries of the knee: 25 years later. Am J Sports Med 2009; 37(5): 890±7. 9. Marx RG, Jones EC, Allen AA, Altchek DW, O'Brien SJ, Rodeo SA, Williams RJ, Warren RF, Wickiewicz TL. Reliability, validity, and responsiveness of four knee outcome scales for athletic patients. J Bone Joint Surg Am 2001 Oct; 83-A(10): 1459±69. 10. Irrgang JJ, Snyder-Mackler L, Wainner RS, FU FH, Harner CD. Development of a patient-reported measure of function of the knee. J Bone Joint Surg Am 1998 Aug; 80(8): 1132±45. 11. Horas U, Pelinkovic D, Herr D, Aigner T, Schnettler R. Autologous chondrocyte implantation and osteochrondral cylinder transplantation in cartilage repair of the knee joint. JBJS Am 2003 Feb; 85-A(2): 185±92. 12. Asik M, Ciftci F, Sen C, Erdil M, Atalar A. The microfracture technique for the treatment of full-thickness articular cartilage lesions of the knee: midterm results. Arthroscopy 2008 Nov; 24(11): 1214±20. Epub 2008 Aug 28. 13. Sgaglione NA, Del Pizzo W, Fox JM, Friedman MJ. Critical analysis of knee ligament rating systems. Am J Sports Med 1995 Nov±Dec; 23(6): 660±7. 14. Wright RW. Knee injury outcomes measures. J Am Acad Orthop Surg 2009 Jan; 17(1): 31±9. 15. Tegner Y and Lysholm J. Rating Systems in the evaluation of knee ligament injuries, Clin Orthop Rel Res 1985; 198: 43±9. 16. Noyes FR, Mathes DS, Mooar PA, Grood ES. The symptomatic anterior cruciatedeficient knee. Part II: the results of rehabilitation, activity modification, andcounselin on functional disability. JBJS Am 1983; 65: 163±74. 17. Barber-Westin SD, Noyes FR, McCloskey JW. Rigorous statisyical reliability, validity, and responsiveness testing of the Cincinnatti Knee Rating System in 350 subjects with uninjured, injured, or anterior cruciate ligament-reconstructed knees. Am J Sports Med 1999; 27: 402±13. 18. Bartlett W, Skinner JA, Gooding CR, Carrington RWJ, Flanagan AM, Briss TWR, Bentley G. Autologous chondrocyte implantation versus matrix induced autologous chondrocyte implantation for osteochondral defects of the knee. JBJS Br 2005 May; 87(5): 640±5. 19. Krishnan SP, Skinner JA, Larrington RWJ, Flanagan AM, Briggs TWR, Bentley G. Collagen-covered autologous chondrocyte implantation for osteochondritis dissecans of the knee. JBJS Br, 2008 Feb; 88-B(2): 203±2. 20. Ware JE Jr, Sherbourne CD. The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med Care 1992 Jun; 30(6): 473±83. 21. Patel AA, Donegan D, Albert T. The 36-item short form. J Am Acad Orthop Surg 2007 Feb; 15(2): 126±34. 22. Sullivan M, Karlsson J, Ware JE. The Swedish SF-36 health survey. Soc Sci Med 1995; 41: 1349±58.

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Regenerative medicine and biomaterials in connective tissue repair

23. Pernegar TV, Leplege A, Etter JF, Rougemont A. Validation of a French language version of the MOS 36-item Short Form Health Survey (SF-36) item in young health adults. J Clinical Epid 1995; 48: 1051±60. 24. Knutsen G, Engelbretsen L, et al. Autologous chondrocyte implantation compared with microfracture: a randomized trial. JBJS, 2004; 86: 455±64. 25. Irrgang JJ, Anderson AF, Boland AL, Harner CD, Kurosaka M, Neyret P, Richmond JC, Shelborne KD. Development and validation of the International Knee Documentation Committee Subjective Knee Form. Am J Sports Med 2001 29: 600. 26. Higgins LD, Taylor MK, Park D, Ghodadra N, Marchant M, Pietrobon R, Cook C. Reliability and validity of the International Knee Documentation Committee (IDKC) Subjective Knee Form. Joint Bone Spine 2007; 74: 594±9. 27. Anderson AF, Irrgang JJ, Kocher MS, Mann BJ, Harrast JJ. IKDC Committee, The International Knee Documentation Form: normative data. Am J Sports Med 2006; 34(1): 128±35. 28. Tanner SM, Dainty KN, Marx RG, Kirkley A. Knee-specific quality of life instruments: which ones measure symptoms and diabilities most important to patients. Am J Sports Med 2007 Sept; 35(9), 1450±8. 29. Marcacci M, Berruto M, Brocchella D, Delcogliano A, Ghinelli B, Gobbi A, Kone E, Pederzini L, Rosa D, Sacchelli GL, Stefani G, Zarvasi S. Articular cartilage engineering with Hyalograft C. Clin Orth Rel Res 2005; 435: 96±105. 30. Henderson I, Francisco R, Oakes B, Cameron J. Autologous chondrocyte implantation for treatment of focal chondral defects of the knee ± a clinical, arthroscopic, MRI and histologic evaluation at 2 years. Knee 2005 Jun; 12(3): 209±16. 31. Roos EM, Roos HP, Lohmander LS, Ekdahl C, Beynnon BD. Knee injury and osteoarthritis outcome score (KOOS): development of a self-administered outcome measure. J Orthop Sp Phys Ther 1998, 28: 88±96. 32. Roos EM, Roos HP, Ekdahl C, Lohmander LS. Knee injury and Osteoarthritis Outcome Score (KOOS) ± validation of a Swedish version. Scan J Med Sci Sports 1998; 8: 439±48. 33. Roos EM and Lohmander LS. The knee injury and osteoarthritis score (KOOS): from joint injury to osteoarthritis. Health Qual Life Outcomes 2003: 1: 64. 34. Zaslav K, Cole B, Brewster R, Berardino T, Farr J, Fowler P, Nissen C. A prospective study of autologous chondrocyte implantation in patients with failed prior treatment for articular cartilage defect of the knee. Am J Sports Med 2009; 37(1): 42±53. 35. Marx RG, Stump TJ, Jones EC, Wickiewicz TL, Warren RF. Development and evaluation of an activity rating scale for disorders of the knee. Am J Sports Med 2001 Mar±Apr; 29(2): 213±18. 36. Mithoefer K, Williams RJ, Warren RK, Wickewicz TL, Marx RG. High-impact athletics after knee articular cartilage repair: a prospective evaluation of the microfracture technique. Am J Sports Med 2006, 34(9): 1413±18. 37. Potter HG et al. Magnetic resonance imaging of articular cartilage in the knee. JBJS 1998; 890A: 1276±84. 38. Recht M, Bobic V, Burstein D, Disler D, Gold G, Gray M, Kramer J, Lang P, McCauley T, Winalski C. Magnetic resonance imaging of articular cartilage. CORR 2001; 391: S379±95. 39. Brittberg M, Winalski CS. Evaluation of cartilage injuries and repair. J Bone Joint Surg Am 2003; 85-A Suppl 2: 58±69. 40. Disler DG, McCauley TR, Kelman CG, Fuchs MD, Ratner LM, Wirth CR, Hospodar PP. Fat suppressed 3D spoiled gradient echo MR imaging of hyaline cartilage

Outcome measures of articular cartilage repair

41. 42. 43.

44.

45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57.

58.

347

defects in the knee: comparison with standard MR imaging and arthroscopy. Am J Roentgenol 1996; 167: 127±32. Kramer J, Recht MP. MR arthrography of the lower extremity. Radiol Clin North Am 2002, 40: 1121±32. McCauley TR. MR imaging of chondral and osteochondral injuries of the knee. Radiol Clin North Am 2002 Sep; 40(5): 1095±107. Welsh GH, Mamish TC, Hughes T, Domayer S, Marlovits S, Trotting S. Advanced morphological and biochemical magnetic resonance imaging of cartilage repair procedures in thickness joint at 3 Tesla. Semin Musculoskel Rad 2008; 12(3): 196± 211. Marlovits S, Singer P, Zeller P, Mandl I, Haller J, Trattnig S. Magnetic resonance observation of cartilage repair tissue (MOCART) for the evaluation of autologous chondrocyte transplantation: determination of interobserver variability and correlation to clinical outcome after 2 years. Eur J Radiol 2006 Jan; 57(1): 16±23. Roberts S, McCall IW, Darby AJ, Menage J, Evans H, Harrison PE, Richardson JB. Autologous chondrocyte implantation for cartilage repair: monitoring its success by magnetic resonance imaging and histology. Arthritis Res Ther 2003; 5(1): R60±73. Henderson IJ, Tuy B, Oakes B, Hettwer WH. Prospective clinical study of autologous chondrocyte implantation and correlation with MRI at 23 and twelve months. JBJS Br 2003; 85-B: 1060±6. Robertson WB, Fick D, Wood DJ, Linklater JM, Zheng MH, Ackland TR. MRI and clinical evaluation of collagen-covered autologous chondrocyte implantation (CACI) at 2 years. The Knee 2007; 14: 117±27. Domayer SE, Welsch GH, Dorotka R, Mamisch TC, Marlovits S, Szomolanyi P, Trattnig S. MRI monitoring of cartilage repair in the knee: a review. Semin Musculoskelet Radiol 2008 Dec; 12(4): 302±17. Outerbridge RE. The etiology of chondro-malacia patella. JBJS 1961; 43B: 752±7. Smith GD, Taylor J, Alingvist KF, Erggelet C, Knutsen G, Garcia Portabella M, Smith T, Richardson JB. Arthroscopic assessment of cartilage repair; a validation study of 2 scoring systems. Arthroscopy 2006; 21: 1462±7. Fugisawa Y, Masuhara K, Shiomi S. The effects of high tibial osteotomy on OA of the knee. Orthopaedic Clinics of North America 1979; 10: 585±608. Noyes FR, Stabler CL. A system for grading articular cartilage lesions at arthroscopy. Am J Sports Med 1989; 17: 505±13. Ayral X, Dougados M, Listrat V, Bonvarlet JP, Simonnet J, Poiraudeau S, Amor B. Chondroscopy: a new method for scoring chondropathy. Semin Arthritis Rheum 1993 April; 22(5): 289±97. Dougados M et al. The SFA system for assessing articular lesions at arthroscopy of the knee. Arthroscopy YEAR?; 10: 69±77. Ayral X. Diagnostic and quantitative arthroscopy qualitative arthroscopy. Balliere's Clinical Rheumatology 1996 Aug; 10(3): 477±94. Brittberg M, Peterson L. Introduction of an articular cartilage classification. ICRS Newsletter 1998; 1: 5±8. Van den Borne MP, Raijmakers NJ, Vanlauwe J, Victor J, deJong SN, Belaman J, Saris DB. International Cartilage Repair Society (ICRS) and Oswestry macroscopic cartilage evaluation scores validated for use in autologous chondrocyte implantation (ACI) and microfracture. Osteoarthritis Cartilage 2007; 15: 1397±402. Mankin HJ, Dorfman H, Lippiello L, Zarins A. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips: II, correlation of morphology with biochemical and metabolic data, JBJS Am 1971, 53: 523±37.

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Regenerative medicine and biomaterials in connective tissue repair

59. Oostegaard K, Andersen CB, Petersen J, Bendtzen K, Salter DM. Validity of histopathological grading of articular from osteoarthritic knee joints. Ann Rheum Dis 1999 Apr; 58(4): 208±13. 60. Pritzker KP, Gay S, Jimenez SA, Ostergaad K, Pelletier JP, Revell PA, Salter D, van de Berg WB. Oseoarthritis cartilage histopathology: grading and staging Osteoarthritis Cartilage 2006; 14: 13±29. 61. Custers RJ, Creemers LB, Verbout AJ, van Rijen MH, Dhert WJ, Saris DB. Reliability, reproducibility and variability of the traditional Histologic/ Histochemical Grading System vs the new OARSI Osteoarthritis Cartilage Histopathology Assessment System. Osteoarthritis Cartilage 2007 Nov; 15(11): 1241±8. 62. O'Driscoll SW, Keeley FW, Salter RB. The chondrogenic potential of free autogenous periosteal grafts for biological resurfacing of major full-thickness defects in joint surfaces under the influence of continuous passive motion. An experimental investigation in the rabbit. J Bone Joint Surg Am 1986; 68: 1017±35.

14

The structure of tendons and ligaments M . B E N J A M I N , Cardiff University, UK

Abstract: Tendons and ligaments are not just mechanical ropes linking muscles to bone or bone to bone. They have a diversity of overlooked functions that are highly relevant to understanding the challenges faced by tissue engineers. This chapter highlights these functions before dealing with basic aspects of structure. Considerable emphasis is placed on key issues centering around `specialised regions' of tendons and ligaments ± notably their bony attachment sites (entheses), wrap-around regions and the muscle± tendon (myotendinous) junction. Mimicking the extraordinary complexity of many entheses is likely to be a limiting factor in the long-term success of tissue engineered tendons or ligaments. Key words: entheses, myotendinous junctions, wrap-around regions, tenocytes, stem cells.

14.1

Introduction

14.1.1 What are tendons and ligaments? Tendons are connective tissue structures that generally link muscles to bone. Occasionally, however, they connect one muscle belly to another ± as `intermediate tendons' exemplified in humans by the tendons of the omohyoid and digastric muscles. All tendons have a cross-sectional area (CSA) that is considerably smaller than that of their muscles. As a broad generalisation, thinner and longer tendons promote `precision' muscular movements, while thicker and shorter tendons are typical of the most powerful muscles. According to Ker et al. (1988), the ratio of muscle : tendon CSA is commonly 34, for this minimises the total mass of the musculotendinous unit and means that tendon stress is about 10 megapascals (MPs) when the muscle is in maximum isometric contraction. With this level of stress, there is a good intrinsic safety margin in the tendon, since typical stress-to-failure values exceed 100 MPs (Ker et al., 1988). Not all muscles have tendons and those that do are the ones that bring two bony points closer together by angulating the bones at a joint, rather than simply approximating origin to insertion (Wood Jones, 1944b). As Wood Jones (1944b) explains, the very act of angulation would kink highly vascular muscle tissue (leading to vessel occlusion), if tendon did not replace muscle at the sites of

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bending. Normal human tendons are flexible, soft tissues with a high tensile strength. As Keith (1925) points out, skeletal muscles differ from mechanical engines in having a `pulling' and not a `pushing' action. If human muscles were able to work like piston rods (i.e. with a pushing action), then their tendons would need to be rigid. This of course would reduce flexibility and conflict with the basic requirements for movement. It is important for the tissue engineer to recognise that tendons are part of a continuum of connective tissue between muscles and bone, so that tendons and their muscles are intimately `knitted' into each other. Some extend over the surface of muscle bellies (Fig. 14.1) and others pass into the muscle itself as intramuscular tendons that enable muscles to adopt a pennate form ± i.e. allow them to have a feathered arrangement of fibres that join the intramuscular tendon at an oblique angle. Highly pennate muscles (multipennate in particular) are geared towards maximising power, rather than contractile range. Ligaments typically connect one bone to another, but occasionally they link different regions of the same bone. They are thus generally shorter than tendons. However, like tendons, they also consist of dense connective tissue. Sometimes,

14.1 Close to the enthesis of iliopsoas, the tendon (T) covers the surface of the most distal region of the muscle belly (M). B, bone. Masson's trichrome. Scale bar ˆ 2 mm.

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it is difficult to decide whether to call a particular structure a tendon or a ligament. Thus, the bridging dense connective tissue linking the patella to the tibia is variably called the patellar tendon or the patellar ligament. `Patellar tendon' is favoured by some, because the patella itself is often viewed as a sesamoid bone, but `patellar ligament' is also logical because the structure links the patella to the tibia.

14.1.2 The diverse functions of tendons and ligaments In facing the considerable challenge of engineering tendons or ligaments, it is important to recognise that neither tendons nor ligaments simply act as passive mechanical ropes. Both have a diversity of functions that is sometimes overlooked and is particularly marked for tendons. Among the most informative and eloquent accounts of tendon function is undoubtedly that of the late Professor Frederic Wood Jones in his two seminal books on the anatomy of the hand and foot (Wood Jones, 1944a,b). These books were written at a time when scholarly works were far more discursive than they are today and it is difficult to do his ideas justice in a few sentences. Nevertheless, among the key points he made were that tendons (a) enable muscles to operate through confined spaces, e.g. the carpal tunnel, (b) are capable of changing the direction of muscle pull by wrapping around bony pulleys, (c) eliminate the need for any unnecessary length of muscle belly between origin and insertion and (d) focus or dissipate the action of muscles. More recently, the property of tendon compliance has attracted considerable attention. The ability of tendons to stretch and recoil means that not only can they act as shock absorbers for muscles, but that they can also economise on muscular effort (Wilson et al., 2001). This is of particular significance in limb muscles during running or galloping. To emphasise this point, Richardson et al. (2007a) distinguish between `positional tendons' such as those of the hand and `weight-bearing tendons' that function as springs during locomotion. The energy saving that comes from having limbs with long distal tendons that can act as springs is considerable. Ker et al. (1987) have calculated that the human Achilles tendon can store 35% of the kinetic and potential energy that is lost and recovered during a running step and the efficiency is even greater in horses. According to Minetti et al. (1999), it approaches 100% during galloping. It seems that the elastic spring-like mechanism provided by tendons, reduces the length changes that the fibres in the associated muscle must make during locomotion (Alexander, 1991). An extreme example is again apparent in horses. According to Dimery et al. (1986), the superficial digital flexor muscle has fibres that are about 3 mm long, yet the overall length change in the muscle± tendon unit that occur during galloping, beyond the point at which the musculotendinous unit becomes taut, is about 50±60 mm. Much of this length change comes from tendon recoil.

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Basic aspects of cell and extracellular matrix (ECM) structure

Both tendons and ligaments are dense fibrous connective tissues, i.e. they are dominated by collagen fibres which account for their high tensile strength. The collagen fibres are collections of fibrils and are themselves packaged as bundles of various sizes (sometimes called primary, secondary and tertiary ± or simply fascicles) that are separated by thin films of loose connective tissue constituting an endotenon (Fig. 14.2). The tendon or ligament as a whole is surrounded by further loose connective tissue called epitenon or epiligament. All the areolar tissue is important as a conduit for blood vessels, nerves and lymphatics and for allowing a degree of independent movement between fibre bundles. The ability of fibrils, fibres and/or fibre bundles to move relative to each other within tendons or ligaments is essential in allowing them to lengthen and/or change shape (Wood et al., 1998). The fibrils have a characteristic zig-zag appearance in longitudinal sections called `crimp' (Fig. 14.3). Crimp disappears under tensile load, and it has been suggested that it acts as a shock absorber, protecting against sudden load changes and that its presence accounts for the toe-region in a stress±strain curve (see Franchi et al., 2007, for further references). Although the predominant orientation of fibres in the mid-substance of tendons and ligaments is parallel to the overall long axis of the tendon or ligament, as Kannus (2000) points out, some fibres course in other directions. He argues that this protects against forces operating from a variety of angles. The older literature shows that there is considerable complexity and diversity

14.2 Tendon fascicles (F) separated from each other by endotenon (E) in a transverse section of a calf tendon. The tenocytes (T) can be recognised solely by their nuclei and in routine histology seem widely separated from each other by the collagen fibres (CF). Haematoxylin & Eosin. Scale bar ˆ 100 m.

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14.3 A longitudinal section of a calf tendon showing crimp, i.e. the zig-zag appearance of the collagen fibres (CF). Note that the tenocytes (T) are arranged in longitudinal rows between the collagen fibres. As in Fig. 14.2, they are only recognisable by their nuclei in routine sections. Haematoxylin & Eosin. Scale bar ˆ 100 m.

in the organisation of fascicles (Sick, 1965), but we know few more recent details because of the difficulty in tracing fibres and fibre bundles in three dimensions. Nevertheless, it is clear that one tendon or ligament differs from another, and/or varies regionally along its length. Thus, one may predict that there is likely to be a distinctive fascicular `fingerprint' for each tendon or ligament that will have some functional significance. The presence of fascicles at all, together with the subdivision of fibres into fibrils, means that there is a built-in safety mechanism against failure in any tendon or ligament. It offers the possibility that local failure can be isolated within one part of the cross-sectional profile of a tendon or ligament and not spread throughout its entire thickness. Fibril diameter may vary greatly between and within tendons and ligaments, as can the quantity of collagen (Parry, 1988). One possibility is that small diameter fibrils enhance resistance to creep because of the greater potential for stress transfer between small fibrils (with their greater surface area) and the surrounding matrix, and that larger fibrils promote tendon/ligament strength (Parry et al., 1978). Not surprisingly therefore, a given tendon may have a bimodal distribution of collagen fibrils that enables it to satisfy both requirements (Parry et al., 1978). Where tendons pass beneath retinacula, they are usually surrounded by synovial sheaths. These reduce the friction between the two contacting surfaces and allow tendons to move longitudinally. The sheaths develop during foetal life in response to movement and if this is prevented in chick embryos (by injecting D-tubocurarine into the air sac), then the sheaths fail to appear (Beckham et al.,

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1977). Occasionally, tendons that pass beneath retinacula, lack sheaths ± as evidenced by the common absence of a sheath around that part of the tibialis anterior tendon which passes beneath the inferior retinaculum in the region of the ankle (Wood Jones, 1944a). Some tendons can divide within their surrounding sheath, e.g. the tendon of extensor digitorum longus as it passes beneath the superior extensor retinaculum (Wood Jones, 1944a). Curiously, the tendons of extensor digitorum longus may have a second tendon sheath as each tendon cross the metatarsophalangeal joints (Lovell and Tanner, 1908). Their existence shows that tendon sheaths are not necessarily restricted to the regions where retinacula exist. Their occasional presence at the base of the toes is in marked contrast to the relationship between tendon sheaths and extensor tendons in the hand. Here, according to Wood Jones (1944b), synovial sheaths do not surround extensor tendons within the digits, because as a rule, fingers are not extended beyond the straightened position. Hence, there is little angulation between the back of the palm and the fingers. In contrast, the dorsiflexion at the metatarsophalangeal joints occurring at toe-off ensures that an angle change does happen at this location. The tendency for the tendons to `bowstring' must be resisted by the local deep fascia and it is the contact between fascia and tendon that must be the trigger for occasional sheath development.

14.2.1 The structure and function of the cells Fibroblasts The typical cell of a tendon or ligament is a fibroblast ± sometimes called a `tenocyte' in tendons (Figs 14.2 and 14.3). For several decades, they were more or less ignored, for the early emphasis of researchers was on the mechanical properties of tendons and ligaments and hence on their collagenous matrices. The matrix is far more conspicuous than the cells. However, it is now widely recognised that the cellular elements hold the key to understanding development, repair and the ability of tendons and ligaments to respond to changing mechanical load. Tenocytes are mechanosensitive cells that are hardwired in a way that allows them to deploy a tensegrity architectural system to detect changes in mechanical load via deformation of their cell membrane and cytoskeleton (Wang, 2006). Strain in the extracellular matrix (ECM) tenses cytoskeletal fibres via integrin receptors in the cell membrane, and this in turn is relayed to the cell nucleus so that gene expression can be altered. An important principle of the tensegrity system is fibre continuity between the ECM and the cells. As Myers et al. (2007) point out, the implication is that the existence of a continuous network of fibres (i.e. collagen fibres in the ECM; cytoskeletal fibres in the cells) means that stress on one part of one cell can be dissipated throughout the entire tissue. Consequently, individual cells are protected from damage and a small mechanical stimulus can potentially affect many cells

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(Ingber, 1997; Myers et al., 2007). It is also worth noting that an understimulation of tenocytes that results from an altered cell±ECM interaction consequent upon tendinopathy could down-regulate cell activity (Arnoczky et al., 2007). Thus, the tendon becomes weakened and more vulnerable to damage from mechanical overload. Although tenocytes and ligament fibroblasts are unremarkable cells in routine histological preparations and seemingly isolated within the ECM (Figs 14.2 and 14.3), they have a much more elaborate form when viewed by confocal microscopy (McNeilly et al., 1996). Each cell has numerous finger-like and sheet-like processes, extending around groups of collagen fibres and creating a 3D network of intercellular contacts throughout the tissue that fits well with the tensegrity model discussed above. The tenocytes are typically arranged in longitudinal rows, where the long axis is parallel to the tendon itself (Fig. 14.3). This reduces the risk of cell damage accompanying tendon or ligament loading. Adjacent cells communicate with each other via gap junctions ± structures that allow ions and small molecules to be rapidly exchanged between individual cells. Two types of gap junctions are present in tendons ± those expressing the protein connexin (cxn)32 and those expressing cxn 43 (McNeilly et al., 1996; Ralphs et al., 1998). It is notable that the distribution of these two connexins is distinctive and implies they have independent functions. Connexin 32 is only present between cells within a longitudinal row, whereas cxn 43 also links cells in adjacent rows. Consequently, gap junctions containing cxn 32 are oriented along the line of tensile loading of a tendon, but cxn 43 junctions link cells in all directions. Waggett et al. (2006) have since shown that collagen synthesis is inhibited in avian tendon cells subject to cyclic mechanical loading in vitro, via cxn 43 and stimulated via cxn 32. Thus, it seems likely that the interplay between these junctions in response to load changes in tendons and ligament is critical in allowing them to adapt their ECM to changing mechanical load (Waggett et al., 2006). The integrity of the cell junctions in a compliant tendon seems to be ensured by the presence of longitudinally oriented, actin stress fibres within the tenocytes, which are associated with adherens junctions that link cells within the same longitudinal row (Ralphs et al., 2002). Stem cells There is considerable interest in using stem cells to engineer tendons and ligaments and a number of cell sources and scaffolds have been explored, including mesenchymal stem cells (Lee et al., 2006). Thus Wang et al. (2005), for example, have transfected bone mesenchymal cell stems from rhesus monkeys with the BMP 12 gene and reported the subsequent differentiation of tenocytes. One of the most significant papers to address the issue of tendon/ ligament stem cells is that of Bi et al. (2007). These authors have identified and isolated multipotential stem cells from both human and mouse tendons and

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shown that their fate is determined by the local ECM, which contributes to the formation of a distinctive `stem-cell niche'. It seems that proteins in the ECM can regulate the fate of stem cells in the niche by modulating growth factor and cytokine activity. An interesting aspect of Bi et al.'s work is that stem cells isolated from human tendons have the ability to form tendinous tissue and even create an enthesis-like structure when the cells were transplanted into immunocompromised mice. However, a significant limitation (acknowledged by Bi et al., 2007) in using tendon stem cells for promoting tendon repair is the availability of autologous tendon tissue. Although bone marrow stromal cells can also differentiate into cells that can form tendon/ligament tissue, Bi et al. (2007) have highlighted the danger that such cells can form bone when transplanted into mice. This is clearly an undesirable outcome when the prime objective is to form a soft connective tissue. Other cells Where tendons or ligaments are subject to compression, fibrocartilage cells (sometimes called `chondroid cells'), replace the typical tenocytes (Figs 14.4 and 14.5). Such cells can synthesise a cartilage-like matrix that is rich in type II collagen and aggrecan (see Benjamin and Ralphs, 1998, for a review). Fibrocartilage cells are altogether plumper than tenocytes and thus clearly differ from them in routine histology sections. Typically, they have large numbers of vimentin-containing intermediate filaments, prominent areas of rough

14.4 The classical appearance of four zones of tissue at a fibrocartilaginous enthesis (that of the human Achilles tendon). The zones comprise: dense fibrous connective tissue (DT), uncalcified fibrocartilage (UF), calcified fibrocartilage (CF) and bone (B). Note the tidemark (T) at the interface between the calcified and non-calcified fibrocartilages. Scale bar ˆ 200 m.

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14.5 Higher power view of fibrocartilage at the human Achilles tendon enthesis. The fibrocartilage cells (FC) in the uncalcified tissue are arranged in longitudinal rows between parallel collagen fibres (CF). A tidemark (T) demarcates the calcification front, with tissue to its right in the figure being calcified and those to its left being uncalcified. In the calcified region, calcified fibrocartilage (CFC) can be distinguished from the thin, adjacent layer of bone (B) into which it knits via a highly irregular interface (arrows). Masson's trichrome. Scale bar ˆ 100 m.

endoplasmic reticulum and variable stores of glycogen granules. In contrast to tenocytes, they do not communicate with each other via gap junctions and thus any local intercellular communication hinges upon an indirect communication between the cells via integrins and the ECM ± as indeed is the case in articular cartilage (Benjamin et al., 1994). Tendon fibrocartilage cells have been shown to develop by the metaplasia of tenocytes or from synovial cells on a tendon surface in response to compressive load (Ralphs et al., 1992). Thus, if a tendon is re-routed surgically around a bony pulley so as to create a new area of compression, fibrocartilage appears at that site (see Benjamin and Ralphs, 1998, for a review). Equally, if compressive load is removed from an area of tendon that normally experiences it, fibrocartilage will regress (Malaviya et al., 2000). While fibrocartilage can certainly be a normal feature of local parts of a tendon or ligament, it can also develop pathologically (Khan et al., 1999). The pathological appearance of the tissue is sometimes referred to as indicating `chondroid metaplasia', e.g. Longo et al. (2007) or `mucoid degeneration', though the former term can also be also used to describe the normal differentiation of tenocytes into fibrocartilage cells (e.g. by Katzman et al. (1999). When authors use the term `chondroid metaplasia' to describe a pathological process, they often refer to a `mucinous change' in the ECM around the newly formed fibrocartilage (chondroid cells, e.g. Fowble et al., 2006). Though it is rarely stated explicitly as such, this mucin seems likely to be proteoglycan secreted by the fibrocartilage cells.

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Mast cells are occasionally documented in and around tendons, e.g. in the central tendon of the diaphragm (Sanchez-Mejorada and Alonso-deFlorida, 1992), the peritendinous tissue associated with the digital extensor tendons (Bade and Koebke, 1996), healing entheses (Kawamura et al., 2005) and the peritendinous fat associated with the Achilles tendon (Shaw et al., 2007). It has been suggested that they have a role in promoting neurogenic inflammation, which is hypothesised by Hart et al. (2005) to be a factor in the aetiology of at least some tendinopathies. Macrophages, neutrophils and lymphocytes may also migrate into tendons in inflammatory and/or degenerative conditions (Godbout et al., 2006; Kawamura et al., 2005).

14.2.2 The structure and formation of the extracellular matrix Collagens Although many collagen types have been demonstrated in tendons and ligaments (including types I±VI, XII and XIV; see Benjamin and Ralphs, 2000, and Riley, 2004, for further reviews), type I collagen is the most abundant and fibrils rich in it are of key importance for high tensile strength. Type I collagen molecules are rod-like structures that are aligned end-to-end in a quarter-staggered array to create heterotypic collagen fibrils that are 20±150 nm in diameter (see Kannus, 2000, for a review). The size varies with the tendon or ligament and increases from birth to maturity (Kannus, 2000). The other collagens with which type I is associated, include types III and V (see Riley, 2004, for a succinct review). Collagen molecules are synthesised within fibroblasts as procollagen, but the N and C propeptides are removed (both intra- and extracellularly) prior to fibril formation. Despite the clear importance of type I collagen in adult tendons and ligaments, it is not the first collagen to appear in development ± type III collagen precedes it in the annulus fibrosus of intervertebral discs (Hayes et al., 2001). The tendon/ligament cells themselves play a key role in controlling fibrillogenesis. Thanks to the pioneering work of Trelstad, Birk and co-workers on the development of tendons, it is no longer the view that fibril formation is a simple matter of self-assembly (Birk and Trelstad, 1986; Birk and Zycband, 1994; Trelstad et al., 1976). These authors showed that the presence of complex surface specialisations in tenocytes means that the cell membrane of individual fibroblasts is capable of cradling various hierarchies of fibrillar structures. These range from individual fibrils, to various sizes of fibril bundles. The seminal works of Trelstad, Birk and co-workers have clearly shown that the role of the cells in fibrillogenesis extends far beyond collagen synthesis. Further important insights have come from the studies of Canty et al. (2004). They demonstrated the existence of plasmalemmal organelles for which they have coined the term `fibripositors'. These receive the contents of membrane-bound `Golgi-toplasmalemmal carriers', in which the processing of procollagen occurs and

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fibrillogenesis is initiated. The fibripositors are polarised structures with a base that is deeply embedded in the cell (and is an active site of fibrillogenesis) and a tip, where fibrils are deposited into the ECM (typically in ones or twos). During the process of depositing a fibril in the ECM, the fibripositor retracts ± leaving the fibril in its extracellular location. Significant though this work is, it is unclear exactly how extensive fibripositor formation is at different developmental stages ± and indeed in different tendons and ligaments. Immature tendons and ligaments are highly cellular, but the cellularity decreases markedly as fibrillogenesis occurs. Nevertheless, the early alignment of cells in parallel longitudinal rows is important in ensuring that the collagen fibres subsequently formed by the cells, have a similar orientation. This cellular organisation is strikingly evident in the outer part of the developing annulus fibrosus in intervertebral discs. The alternating lamellae of parallel collagen fibres, which are arranged at right angles to each other in the outer annulus, are preceded by a similar organisation of the cells at an earlier stage of development (Hayes et al., 1999). The cell arrangement seems to be linked to the formation of adherens junctions and longitudinally arranged, actin stress fibres (Hayes et al., 1999). The importance of intact adherens junctions in maintaining cell alignment and shape (which in turn are critical to secreting a regular fibrillar matrix) has been recently confirmed by Richardson et al. (2007b). Knockdown of adherens junctions containing cadherin-11 resulted in a failure of tendon cells to produce an ECM with parallel collagen fibres (Richardson et al., 2007b). It is also intriguing to note that actin stress fibres within tenocytes are closely associated with cables of actin filaments and that cytochalasin treatment (which results in the dissolution of such filaments) not only leads to the disappearance of fibripositors, but also to an altered alignment of collagen fibrils in the ECM (Canty et al., 2006). Other molecules In addition to collagen, both tendons and ligaments contain numerous other molecules, including small and large proteoglycans (PGs), glycoproteins and small quantities of elastin. There are a wide variety of PGs documented, including decorin, biglycan, lumican, fibromodulin, aggrecan and versican (Milz et al., 2005; Riley, 2004; Vogel et al., 1993). The last two are both classed as large PGs and the remainder as small, leucine-rich PGs. Aggrecan and biglycan are characteristic of fibrocartilaginous regions of tendons or ligaments (Milz et al., 2005; Robbins and Vogel, 1994; Waggett et al., 1998), while versican and decorin are more typical of purely tensional areas (Milz et al., 2005). A multitude of functions have been suggested for PGs in general, including lubricating collagen fibrils, controlling interfibrillar spacing, regulating fibril diameter and determining the fluid content of the ECM. The use of knockoutmice suggests that the loss of decorin or biglycan has different effects on the

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mechanical properties of tendons, according to the location and function of the tendon (Robinson et al., 2005). Thus, the patellar tendon of decorin knockouts is affected to a greater extent than the tendon of flexor digitorum longus. This reinforces the growing view that individual tendons or ligaments have a unique molecular profile and/or histological organisation and cannot be treated as being essentially similar. Lumican and fibromodulin (which both bind to the same region of collagen molecules) play a role in regulating the assembly of collagen into fibrils. Studies on knockout mice by Jepsen et al. (2002) suggest that fibromodulin has an essential function in promoting the high tensile strength of tendons and determining the normal ultrastructure of the ECM, with lumican acting more as a modulator. The lower tensile strength of tendons in Fmodÿ/ÿ mice is associated with a larger number of small diameter collagen fibrils compared with wild types (Jepsen et al., 2002). However, the authors are careful to point out that the relationship between the tensile strength of tendons and their fibril diameter distribution is likely to be complex and influenced by factors as yet unknown. Decorin (so named because it `decorates' fibril surfaces) may be important in connecting fibrils to each other (Hedbom and Heinegard, 1993). The glycoproteins tenascin and tenomodulin are also present in tendons. Tenascin is typical of the myotendinous junction (Jarvinen et al., 2003) and of fibrocartilaginous regions, including numerous entheses (Milz et al., 2005). Mehr et al. (2000) have suggested that it has a role in maintaining the fibrocartilage phenotype by decreasing cell±matrix adhesion and Jarvinen et al. (2003) suggests that it gives tissues elasticity under load. According to Docheva et al. (2005), tenomodulin regulates the proliferation of tendon cells and is involved in controlling the maturation of collagen fibrils. As Ferdous and Grande-Allen (2007) have recognised, there is considerable potential for exploring the use of PGs in tissue engineering. The reader is referred to this useful article for views on how PGs could be integrated into the design of engineered tissues. The authors also consider the design of tissueengineered disease models in the light of what is currently known about PGs.

14.3

Specialised regions of tendons and ligaments

14.3.1 Entheses An enthesis is the region where a tendon or ligament (or joint capsule) attaches to the skeleton. In the adult, most tendons or ligaments attach to bone, but in earlier life they may attach to cartilage. At certain important locations in the body (notably the insertion of the Achilles tendon), entheses are parts of larger complexes that have been called `enthesis organs' (Benjamin and McGonagle, 2001; Benjamin et al., 2004). Such a unit comprises not only the enthesis itself, but also adjacent tissues that contribute to reducing local stress concentration. In

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the Achilles tendon (which has the archetypal enthesis organ), these include periosteal and sesamoid fibrocartilages that are located close to the enthesis itself, the retrocalcaneal bursa and the synovial-covered tip of Kager's fat pad. As a further extension of the enthesis organ idea, the term `synovio-entheseal complex' has also been coined in order to draw attention to the close juxtaposition of synovium at many attachment sites in the body to the enthesis itself (Benjamin and McGonagle, 2007; McGonagle et al., 2007). The synovium is frequently that of a subtendinous bursa. There are several recent review articles that expand greatly on many of the points made more briefly below and the interested reader is referred to those papers for further information (Benjamin et al., 2002, 2006; Shaw and Benjamin, 2007). The focus in this chapter will be on points that should be of special concern to tissue engineers. The presence of entheses (as with myotendinous junctions) poses considerable challenges for tissue engineering and is likely to be a limiting factor in the success of any newly constructed tissue. The primary functional demand that all entheses have to meet is for secure skeletal anchorage, coupled with a significant reduction in the stress concentration at the interface between two tissues. Stress concentration is a key issue because of the contrasting physical properties between soft and hard tissues. It is in order to reduce stress that adjacent entheses frequently merge imperceptibly with each other, so that stress is dissipated between them (Benjamin et al., 2008; Milz et al., 2004). Entheses have been classified in a number of different ways, but that favoured by the author is to distinguish simply between fibrous and fibrocartilaginous attachments. As the names suggest, the difference centres around the presence or absence of cartilage at the attachment site. Thus, at a fibrous enthesis, the tendon or ligament attaches via dense fibrous connective tissue, either directly to bone or indirectly to its periosteum. In a fibrocartilaginous enthesis, there is a transitional zone of fibrocartilage at the insertion site (Figs 14.4 and 14.5). The distinction is an oversimplification, for the reality is that many fibrocartilaginous entheses have some parts that are more cartilaginous than others. Thus, at the attachment of certain human tendons, the distal (superficial) part of the enthesis is predominantly fibrous and the proximal (deep) part is more fibrocartilaginous (Benjamin et al., 1986). In the context of the limbs, it is useful to know that most attachments that are near the ends of long bones (i.e. involving apophyses or epiphyses) are fibrocartilaginous, whereas tendons and ligaments that attach to long bone shafts are generally fibrous. There is an intriguing biomechanical basis underpinning the distribution that can best be understood by comparing the mechanical demands faced by the entheses of deltoid and supraspinatus ± two muscles that abduct the glenohumeral joint. The humeral head attachment of supraspinatus means that as abduction occurs, the insertional angle at which the tendon meets the bone changes substantially. As this is not the case at the mid-shaft attachment of deltoid, it means that the risk of wear and tear from tendon bending is far greater

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at the enthesis of supraspinatus than it is at the insertion of deltoid (Benjamin et al., 1986). The fibrocartilage (which is characteristic of supraspinatus but not deltoid) is thought to play a role similar to that of a grommet on an electrical plug (Schneider, 1956). Thus, it ensures that tendon bending stemming from joint movement is not all concentrated at the hard±soft tissue interface. It is presumably the stiffened matrix that is imparted by the presence of aggrecan in the fibrocartilage that enables the tissue to fulfil this function (Milz et al., 2005). It is uncalcified fibrocartilage that acts as a grommet, but calcified fibrocartilage is also present at a fibrocartilaginous enthesis (Figs 14.4 and 14.5). Hence such an attachment site classically has four tissue zones ± dense fibrous connective tissue, uncalcified fibrocartilage, calcified fibrocartilage and bone (Fig. 14.4). The two fibrocartilages are separated by a tidemark (Figs 14.4 and 14.5), which demarcates a calcification front ± as in articular cartilage. The tidemark is thus the mechanical boundary between hard and soft tissues, but is not the tissue boundary between tendon/ligament and bone (Hems and Tillmann, 2000). This is fractionally deeper and is a more jagged interface, i.e. that between the zone of calcified fibrocartilage and bone. A healthy tidemark is typically smooth. The mechanical and tissue boundaries are spatially distinct because of conflicting demands on their form. Thus, the tidemark needs to be straight and smooth so that the hard tissue does not damage the soft as insertional angles change. However, this is not the best way for a tendon or ligament to gain a firm foothold on the skeleton. This is better served by increasing the surface area of contact between the tissues ± hence the irregularity of the interface at the tissue boundary (Fig. 14.5). Clearly, mimicking these boundaries could be a limiting factor in tissue engineering. It is also important to appreciate the role of the cancellous bone at an attachment site in contributing to ensuring a secure skeletal anchorage. There is little, if any, compact bone at even the largest of fibrocartilaginous entheses (Benjamin et al., 2006). Hence, Sharpey's fibres (i.e. deeply penetrating, `nails' of collagen fibres riveted into bone) are more a feature of fibrous than fibrocartilaginous attachment sites (Benjamin et al., 2002). One must recognise, however, that the whole trabecular network around an enthesis contributes to anchoring the tendon/ligament to the bone (like the roots of a tree) and thus that an enthesis does not end at the edge of the bone (Benjamin et al., 2008). A point of further significance is that the thin, subchondral bone plate at a fibrocartilaginous enthesis is actually microscopically punctured at intervals at many attachment sites ± at least in elderly dissecting room cadavers (Benjamin et al., 2007). This means that a tendon or ligament is occasionally in direct contact with bone marrow ± and thus in proximity to mesenchymal stem cells. This may well relate to the presence of fibrocartilage repair tissue at entheses (Benjamin et al., 2007). Although the organisation of cells into longitudinal rows lying between parallel bundles of collagen fibres in the zone of uncalcified fibrocartilage may

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not always be as clear cut as readers expect, it is nevertheless easy to find examples of this architecture at numerous entheses (Fig. 14.5). It is a direct consequence of the developmental history of the tissue, for the fibrocartilage cells develop by metaplasia from fibroblasts in response to appropriate mechanical stimuli (Gao et al., 1996). Hence it seems likely that if a tissue engineered tendon starts off with an initial population of fibroblasts, then these cells would likely become fibrocartilaginous in response to compressive load at strategic sites within the tendon or ligament ± including the enthesis. Although it is likely to be tendons/ligaments with fibrocartilaginous entheses that attract the attention of tissue engineers (because these include many tendons in the hands and feet), there is a pertinent issue to be recognised in connection with tendons/ligaments with fibrous entheses that attach to the shaft of long bones. Because the growth in length of any long bone is promoted by growth plate(s), this means that a tendon/ligament attaching to the diaphysis or metaphysis must migrate along the bone as development proceeds, in order to maintain the same relative position of attachment. If this did not happen, then a tendon/ligament attaching to the metaphysis in early life would gradually assume an anchorage site that became more and more diaphyseal. This does not occur, because during the growing period, the attachment is to periosteum rather than directly to bone. The periosteum can grow interstitially, but the bone cannot ± hence by having an indirect attachment to the periosteum, the tendon or ligament can `creep' up the bone at a rate that matches the activity of the growth plate (Dorfl, 1980).

14.3.2 Wrap-around regions As briefly stated earlier, one of the many functions of tendons is to enable muscles to change their direction of pull. This is easiest to understand by considering tendons passing from the leg to the foot. Because the foot is at right angles to the leg in the standing position, then any muscle acting on the former, but located in the latter, must change its direction of pull by having a tendon that wraps around a bony pulley. Thus, the tendons of both peroneal (fibularis) muscles (longus and brevis) and the tendon of tibialis posterior for example, bend around the ankle malleoli. Indeed, the lateral malleolus is merely the first of a succession of three bony pulleys around which the peroneus longus tendon passes en route from the muscle belly to its insertion. Consequently, such wraparound regions of tendons are specialised to resist compression and shear ± the shear coming from the longitudinal excursion of the tendon that accompanies muscle contraction (Benjamin et al., 1995). Although there may be gross anatomical specialisations of tendons that wrap-around bony pulleys (e.g., flattening or the presence of fissures that enable tendons to change shape ± see review by Benjamin and Ralphs (1998) ± the most striking feature is the presence of fibrocartilage at wrap-around sites (Benjamin et al., 1995). It should be noted, however, that the fibrocartilage does not typically extend throughout

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the thickness of the tendon ± it disappears towards the convex surface that faces away from the bone. The phenotypic change is gradual, but the outer part of the tendon is commonly purely fibrous. Intriguingly, several authors have shown that the fibrocartilage in wrap-around regions disappears if the tendon is surgically translocated to another site so that the compressive force is removed (e.g. Malaviya et al., 2000). A principal adaptation for resisting shear in wrap-around regions is the presence of a synovial sheath. Such sheaths are typically described as having an inner visceral and an outer parietal layer separated from by a synovial cavity. However, the presence of a visceral layer on the side of the tendon that presses against the bone is variable. The absence relates to the compressive forces acting on both tissues in the region of tendon±bone contact. The pressure would occlude blood vessels ± which of course are a prominent feature of synovia. This is exactly the situation in synovial joints, where again there is no synovium over the articular cartilages themselves. Wrap-around regions of tendons are distinguished from `traction tendons', not only by the presence of fibrocartilage cells, but also by the arrangement of their collagen fibres. Commonly, the regular parallel arrangement of fascicles is less evident and the collagen fibres interweave extensively with each other in a basketweave fashion. This probably prevents the tendon from splaying out when it is pressed against the bone. But it is also important to recognise that one wraparound tendon may not be exactly like another. Thus, in some cases (e.g. the specimen of flexor hallucis longus illustrated in Benjamin et al. (1995), the fibrocartilaginous character of the tendon is primarily associated with the presence of fibrocartilage in the endotenon. Although wrap-around tendons are certainly better known, and more common than wrap-around ligaments, nevertheless the latter also exist and the mechanical factors that trigger fibrocartilage differentiation within them are similar ± i.e. compression and shear. Examples of wrap-around ligaments include the annular ligament that wraps around the head of the radius in the superior radioulnar joint and the transverse ligament that holds the dens against the anterior arch of the atlas.

14.3.3 Myotendinous junctions The myotendinous (or `muscle±tendon') junction (MTJ) is a site where forces are transmitted between muscle and tendon and also a site of sarcomere turnover. There are numerous papers demonstrating its plasticity ± it responds to changes in exercise levels, denervation and tenotomy (Abou Salem et al., 1993; Kannus et al., 1992; Tidball and Quan, 1992). This is in line with the pioneering work of Williams and Goldspink, 1971, 1973), who showed that the MTJ was the site where sarcomeres were added during the growing period or in response to changing mechanical load.

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14.6 The myotendinous junction of a thenar muscle in the human hand. Note the increased cellularity at the junction (nuclei indicated by arrowheads) and the terminal projections of the muscle fibres (arrows). M, muscle fibre, T, tendon. Masson's trichrome. Scale bar ˆ 50 m.

It is important to recognise that the MTJ does not just transmit tensile load from muscle to tendon, but also from tendon to muscle as a consequence of ground reaction forces (Benjamin et al., 2006). As at a fibrocartilaginous enthesis, the interface between the connecting tissues is complex, so that the surface area of contact between them is substantially increased (Fig. 14.6). Thus, it is a basic design of the MTJ that the terminal muscle fibres have many fingerlike extensions promoting an intimate mechanical linkage of myofilaments to tendon collagen via the basal lamina that surrounds muscle fibres (Tidball, 1991). According to Trotter et al. (1981), the actin filaments of the terminal muscle fibres are embedded into an electron-dense intracellular layer near the sarcolemma, that they later to refer to as the `internal domain' of the MTJ (Trotter et al., 1983). This in turn has filamentous links with the lamina densa of the basal lamina, external to the muscle fibre ± into which are anchored the collagen fibres of the tendon. Trotter et al. (1983) consider that each of the four domains (internal domain, connecting domain, lamina densa and matrix domain) they define at the MTJ has a distinctive location, molecular composition, 3D architecture and force-transmitting role. The development of the basal lamina is an early and critical step in the formation of MTJs during development ± the subsarcolemmal densities and thus indirect myofilament anchorage to the basal lamina, together with the muscle fibre folds that increase the contact area between muscle and tendon all follow later (Tidball and Lin, 1989). Although a number of authors have addressed various aspects of the molecular composition of the MTJ, the results are sometimes presented in studies that have a different primary focus. It can thus be difficult for the nonspecialist to build up an overall picture. On the tendon side of the junction, tenascin C is a particular feature of the rat gastrocnemius muscle and its presence at the MTJ contrasts with its absence in the muscle itself ± even when the muscle is loaded by strenuous treadmill running (Jarvinen et al., 2003). Type VI collagen is considered to be prominent on the tendon side of the junction

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(Senga et al., 1995), though it is also widespread in the rest of the tendon as well (Milz et al., 2005). Fibronectin and thrombospondin-1 are widely distributed in rat musculoskeletal tissues, but particularly strong immunolabelling is a feature of junctional regions, including the MTJ and entheses (Kannus et al., 1998). Equally, tetranectin is typical of interfaces in muscle tissue, for Wewer et al. (1998) have reported it at myofascial junctions as well as MTJs. It should also be noted that talin synthesis (talin is a large protein that links actin filaments to the sarcolemma) is upregulated at the MTJs of rat hind limb muscles in response to mechanical loading (Frenette and Tidball, 1998). There are at least two transmembrane receptors at MTJs ± dystrophinassociated protein complex and 7 1 integrin (Bao et al., 1993; IbraghimovBeskrovnaya et al., 1992). According to Miosge et al. (1999), 7 1 is a key molecule in providing for the linkage of muscle cells to tendon, while dystrophin complexes are more concerned with maintaining the integrity of the lateral aspect of skeletal muscle fibres. However, Grady et al. (2003) have detected dystrobrevin ± a cytoplasmic component of the dystrophin±glycoprotein complex at MTJs and utrophin (a homologous protein to dystrophin) has also been found at these sites (Taylor et al., 1997). McCullagh et al. (2007) have drawn our attention to the presence of syncoilin (an intermediate filament protein belonging to the dystrophin protein complex) at MTJs and Carlsson et al. (1999) and Vaittinen et al. (1999) have reported a further intermediate filament (nestin) at this site. Nestin has a particular association with sites where acetylcholine receptors are located, for it is also a feature of the neuromuscular junction (Vaittinen et al., 1999).

14.4

Conclusions

A fundamental `take-home message' of this chapter is that tendons/ligaments have a complexity that belies their initial appearance. Evidence increasingly points to their being unique structures, tailored to the functional demands of particular locations. Many do not attach to the skeleton in isolation, but are linked to each other, both at their insertions and via mutual attachments to fascia. They vary in structure and molecular composition, not only from one tendon (or ligament) to another, but also along their length. Thus, they may be flattened, rounded or oval, their collagen fibrils can be of different diameters, their fibre bundles of different shapes and sizes, their cells of varying phenotypes and their predominant collagens and PGs of different types. All this needs to be considered by those concerned with the tissue engineering of tendons and ligaments, as does the extraordinary complexity of enthesis architecture and the dynamic character of the myotendinous junction.

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14.5

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References

Abou Salem E A, Fujimaki N and Ishikawa H (1993), `Ultrastructural changes of myotendinous junctions in tenotomized soleus muscles of the rat', J Submicrosc Cytol Pathol, 25, 181±91. Alexander R M (1991), `Energy-saving mechanisms in walking and running', J Exp Biol, 160, 55±69. Arnoczky S P, Lavagnino M and Egerbacher M (2007), `The mechanobiological aetiopathogenesis of tendinopathy: is it the over-stimulation or the understimulation of tendon cells?', Int J Exp Pathol, 88, 217±26. Bade H and Koebke J (1996), `Histomorphology of the peritendinous tissue of the phalangeal extensor apparatus', Acta Anat (Basel), 156, 112±17. Bao Z Z, Lakonishok M, Kaufman S and Horwitz A F (1993), `Alpha 7 beta 1 integrin is a component of the myotendinous junction on skeletal muscle', J Cell Sci, 106, 579±89. Beckham C, Dimond R and Greenlee T K, Jr (1977), `The role of movement in the development of a digital flexor tendon', Am J Anat, 150, 443±59. Benjamin M and McGonagle D (2001), `The anatomical basis for disease localisation in seronegative spondyloarthropathy at entheses and related sites', J Anat, 199, 503± 26. Benjamin M and McGonagle D (2007), `Histopathologic changes at ``synovio-entheseal complexes'' suggesting a novel mechanism for synovitis in osteoarthritis and spondylarthritis', Arthritis Rheum, 56, 3601±9. Benjamin M and Ralphs J R (1998), `Fibrocartilage in tendons and ligaments ± an adaptation to compressive load', J Anat, 193, 481±94. Benjamin M and Ralphs J R (2000), `The cell and developmental biology of tendons and ligaments', Int Rev Cytol, 196, 85±130. Benjamin M, Evans E J and Copp L (1986), `The histology of tendon attachments to bone in man', J Anat, 149, 89±100. Benjamin M, Archer C W and Ralphs J R (1994), `Cytoskeleton of cartilage cells', Microsc Res Tech, 28, 372±7. Benjamin M, Qin S and Ralphs J R (1995), `Fibrocartilage associated with human tendons and their pulleys', J Anat, 187, 625±33. Benjamin M, Kumai T, Milz S, Boszczyk B M, Boszczyk A A and Ralphs J R (2002), `The skeletal attachment of tendons ± tendon ``entheses''', Comp Biochem Physiol A Mol Integr Physiol, 133, 931±45. Benjamin M, Moriggl B, Brenner E, Emery P, McGonagle D and Redman S (2004), `The ``enthesis organ'' concept: why enthesopathies may not present as focal insertional disorders', Arthritis Rheum, 50, 3306±13. Benjamin M, Toumi H, Ralphs J R, Bydder G, Best T M and Milz S (2006), `Where tendons and ligaments meet bone: attachment sites (``entheses'') in relation to exercise and/or mechanical load', J Anat, 208, 471±90. Benjamin M, Toumi H, Suzuki D, Redman S, Emery P and McGonagle D (2007), `Microdamage and altered vascularity at the enthesis±bone interface provides an anatomic explanation for bone involvement in the HLA-B27-associated spondylarthritides and allied disorders', Arthritis Rheum, 56, 224±33. Benjamin M, Kaiser E and Milz S (2008), `Structure-function relationships in tendons ± a review', J Anat, 212, 211±18. Bi Y, Ehirchiou D, Kilts T M, Inkson C A, Embree M C, Sonoyama W, Li L, Leet A I, Seo B M, Zhang L, Shi S and Young M F (2007), `Identification of tendon stem/

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progenitor cells and the role of the extracellular matrix in their niche', Nat Med, 13, 1219±27. Birk D E and Trelstad R L (1986), `Extracellular compartments in tendon morphogenesis: collagen fibril, bundle, and macroaggregate formation', J Cell Biol, 103, 231±40. Birk D E and Zycband E (1994), `Assembly of the tendon extracellular matrix during development', J Anat, 184, 457±63. Canty E G, Lu Y, Meadows R S, Shaw M K, Holmes D F and Kadler K E (2004), `Coalignment of plasma membrane channels and protrusions (fibripositors) specifies the parallelism of tendon', J Cell Biol, 165, 553±63. Canty E G, Starborg T, Lu Y, Humphries S M, Holmes D F, Meadows R S, Huffman A, O'Toole E T and Kadler K E (2006), `Actin filaments are required for fibripositormediated collagen fibril alignment in tendon', J Biol Chem, 281, 38592±8. Carlsson L, Li Z, Paulin D and Thornell L E (1999), `Nestin is expressed during development and in myotendinous and neuromuscular junctions in wild type and desmin knock-out mice', Exp Cell Res, 251, 213±23. Dimery N J, Alexander R M and Ker R F (1986), `Elastic extension of leg tendons in the locomotion of horses (Equus cabellus)', J Zool, 210, 415±25. Docheva D, Hunziker E B, Fassler R and Brandau O (2005), `Tenomodulin is necessary for tenocyte proliferation and tendon maturation', Mol Cell Biol, 25, 699±705. Dorfl J (1980), `Migration of tendinous insertions. I. Cause and mechanism', J Anat, 131, 179±95. Ferdous Z and Grande-Allen K J (2007), `Utility and control of proteoglycans in tissue engineering', Tissue Eng, 13, 1893±904. Fowble V A, Vigorita V J, Bryk E and Sands A K (2006), `Neovascularity in chronic posterior tibial tendon insufficiency', Clin Orthop Relat Res, 450, 225±30. Franchi M, Fini M, Quaranta M, De Pasquale V, Raspanti M, Giavaresi G, Ottani V and Ruggeri A (2007), `Crimp morphology in relaxed and stretched rat Achilles tendon', J Anat, 210, 1±7. Frenette J and Tidball J G (1998), `Mechanical loading regulates expression of talin and its mRNA, which are concentrated at myotendinous junctions', Am J Physiol, 275, C818±25. Gao J, Messner K, Ralphs J R and Benjamin M (1996), `An immunohistochemical study of enthesis development in the medial collateral ligament of the rat knee joint', Anat Embryol (Berl), 194, 399±406. Godbout C, Ang O and Frenette J (2006), `Early voluntary exercise does not promote healing in a rat model of Achilles tendon injury', J Appl Physiol, 101, 1720±6. Grady R M, Akaaboune M, Cohen A L, Maimone M M, Lichtman J W and Sanes J R (2003), `Tyrosine-phosphorylated and nonphosphorylated isoforms of alphadystrobrevin: roles in skeletal muscle and its neuromuscular and myotendinous junctions', J Cell Biol, 160, 741±52. Hart D A, Frank C B and Bary R C (2005), `Inflammatory processes in repetitive motion and overuse syndromes: potential role of neurogenic mechanisms in tendons and ligaments. In Repetitive Motion Disorders of the Upper Extremity, eds Gordon S L, Blair S J and Fine L J. American Academy of Orthopaedic Surgeons, Rosemont, pp. 247±63. Hayes A J, Benjamin M and Ralphs J R (1999), `Role of actin stress fibres in the development of the intervertebral disc: cytoskeletal control of extracellular matrix assembly', Dev Dyn, 215, 179±89. Hayes A J, Benjamin M and Ralphs J R (2001), `Extracellular matrix in development of the intervertebral disc', Matrix Biol, 20, 107±21.

The structure of tendons and ligaments

371

Hedbom E and Heinegard D (1993), `Binding of fibromodulin and decorin to separate sites on fibrillar collagens', J Biol Chem, 268, 27307±12. Hems T and Tillmann B (2000), `Tendon entheses of the human masticatory muscles', Anat Embryol (Berl), 202, 201±8. Ibraghimov-Beskrovnaya O, Ervasti J M, Leveille C J, Slaughter C A, Sernett S W and Campbell K P (1992), `Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix', Nature, 355, 696±702. Ingber D E (1997), `Tensegrity: the architectural basis of cellular mechanotransduction', Annu Rev Physiol, 59, 575±99. Jarvinen T A, Jozsa L, Kannus P, Jarvinen T L, Hurme T, Kvist M, Pelto-Huikko M, Kalimo H and Jarvinen M (2003), `Mechanical loading regulates the expression of tenascin-C in the myotendinous junction and tendon but does not induce de novo synthesis in the skeletal muscle', J Cell Sci, 116, 857±66. Jepsen K J, Wu F, Peragallo J H, Paul J, Roberts L, Ezura Y, Oldberg A, Birk D E and Chakravarti S (2002), `A syndrome of joint laxity and impaired tendon integrity in lumican- and fibromodulin-deficient mice', J Biol Chem, 277, 35532±40. Kannus P (2000), `Structure of the tendon connective tissue', Scand J Med Sci Sports, 10, 312±20. Kannus P, Jozsa L, Kvist M, Lehto M and Jarvinen M (1992), `The effect of immobilization on myotendinous junction: an ultrastructural, histochemical and immunohistochemical study', Acta Physiol Scand, 144, 387±94. Kannus P, Jozsa L, Jarvinen T A, Jarvinen T L, Kvist M, Natri A and Jarvinen M (1998), `Location and distribution of non-collagenous matrix proteins in musculoskeletal tissues of rat', Histochem J, 30, 799±810. Katzman B M, Klein D M, Garven T C, Caligiuri D A and Kung J (1999), `Comparative histology of the annular and cruciform pulleys', J Hand Surg [Br], 24, 272±4. Kawamura S, Ying L, Kim H J, Dynybil C and Rodeo S A (2005), `Macrophages accumulate in the early phase of tendon±bone healing', J Orthop Res, 23, 1425±32. Keith A (1925), The Engines of the Human Body, London, Williams and Norgate Ker R F, Bennett M B, Bibby S R, Kester R C and Alexander R M (1987), `The spring in the arch of the human foot', Nature, 325, 147±9. Ker R F, McNeill Alexander R and Bennett M B (1988), `Why are mammalian tendons so thick?' J Zool, 216, 309±324. Khan K M, Cook J L, Bonar F, Harcourt P and Astrom M (1999), `Histopathology of common tendinopathies. Update and implications for clinical management', Sports Med, 27, 393±408. Lee C H, Moioli E K and Mao J J (2006), `Fibroblastic differentiation of human mesenchymal stem cells using connective tissue growth factor', Conf Proc IEEE Eng Med Biol Soc, 1, 775±8. Longo U G, Franceschi F, Ruzzini L, Rabitti C, Morini S, Maffulli N, Forriol F and Denaro V (2007), `Light microscopic histology of supraspinatus tendon ruptures', Knee Surg Sports Traumatol Arthrosc, 15, 1390±4. Lovell A G and Tanner H H (1908), `Synovial membranes, with special reference to those related to the tendons of the foot and ankle', J Anat Physiol, 42, 415±32. Malaviya P, Butler D L, Boivin G P, Smith F N, Barry F P, Murphy J M and Vogel K G (2000), `An in vivo model for load-modulated remodeling in the rabbit flexor tendon', J Orthop Res, 18, 116±25. McCullagh K J, Edwards B, Poon E, Lovering R M, Paulin D and Davies K E (2007), `Intermediate filament-like protein syncoilin in normal and myopathic striated muscle', Neuromuscul Disord, 17, 970±9.

372

Regenerative medicine and biomaterials in connective tissue repair

McGonagle D, Lories R J, Tan A L and Benjamin M (2007), `The concept of a ``synovioentheseal complex'' and its implications for understanding joint inflammation and damage in psoriatic arthritis and beyond', Arthritis Rheum, 56, 2482±91. McNeilly C M, Banes A J, Benjamin M and Ralphs J R (1996), `Tendon cells in vivo form a three dimensional network of cell processes linked by gap junctions', J Anat, 189, 593±600. Mehr D, Pardubsky P D, Martin J A and Buckwalter J A (2000), `Tenascin-C in tendon regions subjected to compression', J Orthop Res, 18, 537±45. Milz S, Tischer T, Buettner A, Schieker M, Maier M, Redman S, Emery P, McGonagle D and Benjamin M (2004), `Molecular composition and pathology of entheses on the medial and lateral epicondyles of the humerus: a structural basis for epicondylitis', Ann Rheum Dis, 63, 1015±21. Milz S, Benjamin M and Putz R (2005), `Molecular parameters indicating adaptation to mechanical stress in fibrous connective tissue', Advances in Anatomy, 178, 1±71. Minetti A E, Ardig O L, Reinach E and Saibene F (1999), `The relationship between mechanical work and energy expenditure of locomotion in horses', J Exp Biol, 202, 2329±38. Miosge N, Klenczar C, Herken R, Willem M and Mayer U (1999), `Organization of the myotendinous junction is dependent on the presence of alpha7beta1 integrin', Lab Invest, 79, 1591±9. Myers K A, Rattner J B, Shrive N G and Hart D A (2007), `Hydrostatic pressure sensation in cells: integration into the tensegrity model', Biochem Cell Biol, 85, 543±51. Parry D A (1988), `The molecular and fibrillar structure of collagen and its relationship to the mechanical properties of connective tissue', Biophys Chem, 29, 195±209. Parry D A, Barnes G R and Craig A S (1978), `A comparison of the size distribution of collagen fibrils in connective tissues as a function of age and a possible relation between fibril size distribution and mechanical properties', Proc R Soc Lond B Biol Sci, 203, 305±21. Ralphs J R, Tyers R N and Benjamin M (1992), `Development of functionally distinct fibrocartilages at two sites in the quadriceps tendon of the rat: the suprapatella and the attachment to the patella', Anat Embryol (Berl), 185, 181±7. Ralphs J R, Benjamin M, Waggett A D, Russell D C, Messner K and Gao J (1998), `Regional differences in cell shape and gap junction expression in rat Achilles tendon: relation to fibrocartilage differentiation', J Anat, 193, 215±22. Ralphs J R, Waggett A D and Benjamin M (2002), `Actin stress fibres and cell±cell adhesion molecules in tendons: organisation in vivo and response to mechanical loading of tendon cells in vitro', Matrix Biol, 21, 67±74. Richardson L E, Dudhia J, Clegg P D and Smith R (2007a), `Stem cells in veterinary medicine ± attempts at regenerating equine tendon after injury', Trends Biotechnol, 25, 409±16. Richardson S H, Starborg T, Lu Y, Humphries S M, Meadows R S and Kadler K E (2007b), `Tendon development requires regulation of cell condensation and cell shape via cadherin-11-mediated cell±cell junctions', Mol Cell Biol, 27, 6218±28. Riley G (2004), `The pathogenesis of tendinopathy. A molecular perspective', Rheumatology (Oxford), 43, 131±42. Robbins J R and Vogel K G (1994), `Regional expression of mRNA for proteoglycans and collagen in tendon', Eur J Cell Biol, 64, 264±70. Robinson P S, Huang T F, Kazam E, Iozzo R V, Birk D E and Soslowsky L J (2005), `Influence of decorin and biglycan on mechanical properties of multiple tendons in knockout mice', J Biomech Eng, 127, 181±5.

The structure of tendons and ligaments

373

Sanchez-Mejorada G and Alonso-deFlorida F (1992), `Changes in mast-cell distribution in skeletal muscle after denervation', Muscle Nerve, 15, 716±19. Schneider H (1956), `Zur Struktur der Sehnenansatzzonen.', Z Anat Entwicklungsgesch, 119, 431±56. Senga K, Kobayashi M, Hattori H, Yasue K, Mizutani H, Ueda M and Hoshino T (1995), `Type VI collagen in mouse masseter tendon, from osseous attachment to myotendinous junction', Anat Rec, 243, 294±302. Shaw H M and Benjamin M (2007), `Structure±function relationships of entheses in relation to mechanical load and exercise', Scand J Med Sci Sports, 17, 303±15. Shaw H M, Santer R M, Watson A H and Benjamin M (2007), `Adipose tissue at entheses: the innervation and cell composition of the retromalleolar fat pad associated with the rat Achilles tendon', J Anat, 211, 436±43. Sick H (1965) L'adaptation des tendons a la reflexion. Strasbourg, Strasbourg University. Taylor J, Muntoni F, Dubowitz V and Sewry C A (1997), `The abnormal expression of utrophin in Duchenne and Becker muscular dystrophy is age related', Neuropathol Appl Neurobiol, 23, 399±405. Tidball J G (1991), `Myotendinous junction injury in relation to junction structure and molecular composition', Exerc Sport Sci Rev, 19, 419±45. Tidball J G and Lin C (1989), `Structural changes at the myogenic cell surface during the formation of myotendinous junctions', Cell Tissue Res, 257, 77±84. Tidball J G and Quan D M (1992), `Modifications in myotendinous junction structure following denervation', Acta Neuropathol, 84, 135±40. Trelstad R L, Hayashi K and Gross J (1976), `Collagen fibrillogenesis: intermediate aggregates and suprafibrillar order', Proc Natl Acad Sci USA, 73, 4027±31. Trotter J A, Corbett K and Avner B P (1981), `Structure and function of the murine muscle±tendon junction', Anat Rec, 201, 293±302. Trotter J A, Eberhard S and Samora A (1983), `Structural domains of the muscle±tendon junction. 1. The internal lamina and the connecting domain', Anat Rec, 207, 573± 91. Vaittinen S, Lukka R, Sahlgren C, Rantanen J, Hurme T, Lendahl U, Eriksson J E and Kalimo H (1999), `Specific and innervation-regulated expression of the intermediate filament protein nestin at neuromuscular and myotendinous junctions in skeletal muscle', Am J Pathol, 154, 591±600. Vogel K G, Ordog A, Pogany G and Olah J (1993), `Proteoglycans in the compressed region of human tibialis posterior tendon and in ligaments', J Orthop Res, 11, 68± 77. Waggett A D, Ralphs J R, Kwan A P, Woodnutt D and Benjamin M (1998), `Characterization of collagens and proteoglycans at the insertion of the human Achilles tendon', Matrix Biol, 16, 457±70. Waggett A D, Benjamin M and Ralphs J R (2006), `Connexin 32 and 43 gap junctions differentially modulate tenocyte response to cyclic mechanical load', Eur J Cell Biol, 85, 1145±54. Wang J H (2006), `Mechanobiology of tendon', J Biomech, 39, 1563±82. Wang Q W, Chen Z L and Piao Y J (2005), `Mesenchymal stem cells differentiate into tenocytes by bone morphogenetic protein (BMP) 12 gene transfer', J Biosci Bioeng, 100, 418±22. Wewer U M, Iba K, Durkin M E, Nielsen F C, Loechel F, Gilpin B J, Kuang W, Engvall E and Albrechtsen R (1998), `Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro', Dev Biol, 200, 247±59.

374

Regenerative medicine and biomaterials in connective tissue repair

Williams P E and Goldspink G (1971), `Longitudinal growth of striated muscle fibres', J Cell Sci, 9, 751±67. Williams P E and Goldspink G (1973), `The effect of immobilization on the longitudinal growth of striated muscle fibres', J Anat, 116, 45±55. Wilson A M, Mcguigan M P, Su A and Van Den Bogert A J (2001), `Horses damp the spring in their step', Nature, 414, 895±9. Wood M L, Lester G E and Dahners L E (1998), `Collagen fiber sliding during ligament growth and contracture', J Orthop Res, 16, 438±40. Wood Jones F (1944a), Structure and Function as Seen in the Foot, London, Bailliere, Tindall and Cox. Wood Jones F (1944b), The Principles of Anatomy as Seen in the Hand, London, Balliere, Tindall and Cox.

15

Tendon biomechanics

M . K J á R , Bispebjerg Hospital and University of Copenhagen, Denmark, S . P . M A G N U S S O N , University of Copenhagen, Denmark and A . M A C K E Y , Bispebjerg Hospital, Denmark

Abstract: Tendon connective tissue adapts to mechanical loading with increased synthesis and turnover of matrix proteins. Collagen formation and degradation increase with acute loading of tendon and skeletal muscle, and this is associated with local and systemic release of growth factors (e.g. IGF1, TGF-beta). Chronic loading of tissue, such as with physical training, leads to increased collagen turnover and a net collagen synthesis together with a modification of the mechanical properties of the tendon, including a reduction in tendon stress. The adaptation time to chronic loading is longer in tendon tissue than in contractile elements of skeletal muscle or heart, and only very prolonged loading will significantly change gross dimensions of the tendon. Mechanical signalling in the tissue leads to biochemical changes in the matrix that can be converted into adaptations in morphology, structure and biomechanical properties of the tendon. Key words: tendon connective tissue, chronic loading, collagen synthesis.

15.1

Introduction

Tendons, ligaments, bone and intramuscular connective tissue have been studied for decades, and the biomechanical properties of these tissues are well appreciated. However, at least for tendon and ligaments the structures have been considered to be relatively inert (Kjñr 2004). The methods available to study these collagen-rich tissues in vivo have in the past been somewhat limited, and often findings were based solely on studies of cadaver tissue properties, or relied on cell culture work. With the present methodological developments and renewed interest for metabolic, circulatory and tissue protein turnover in collagen tissue such as tendon ± in conjunction with a simultaneous determination of morphology and biomechanical tissue properties ± a greater insight into the way that mechanical loading influences both the cells and the matrix of tendon has been achieved. As an example, it has been shown that in response to mechanical loading the human tendon increases its blood flow, metabolic activity and substrate uptake (e.g. glucose) by 3±4-fold, acutely associated with exercise (for references see Kjñr 2004).

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Biochemical adaptation of tendon to loading

Matrix adaptation to loading involves several proteins, but the most mechanically important load-bearing structure in tendon tissue is collagen type I. Several indirect methods have been used so far to indicate changes of collagen turnover in connective tissue. Determination of increased pro-collagen mRNA in tissue indicates an upregulation of transcriptional activity, but does not guarantee for any mature collagen formation. Furthermore, activity in the intracellular biosynthesis pathway for collagen can be estimated by determination of enzymes such as prolyl-4-hydroxylase (P-4-H), galactosylhydroxy-lysylglucotransferase (GGT) or lysyl hydroxylase (LHy) and increased collagen synthesis has been estimated from such methods in animal experiments (Kovanen 1989). Collagen formation in the tendon is shown to rise with acute and chronic loading, as determined by the use of the microdialysis technique. With this technique, catheters are put into the region of interest (e.g. around or through a tendon) and the interstitial concentration of the pro-collagen propeptides that are cleaved off in the maturation from procollagen to collagen (PICP or PINP) are determined of both previously loaded and unloaded tendon (Langberg et al. 1999, 2001). Using this technique locally around a tendon provides the possibility to determine local changes of PICP or PINP in regions that otherwise would only contribute marginally to any change in these parameters globally (i.e. changes in concentrations of PINP or PICP in the circulating blood stream) (Langberg et al. 2000).

15.2.1 In vivo collagen turnover in tendon: stable isotopes In collagen-containing tissues such as tendon and bone, other methods such as the use of radioactive isotopes for incorporation of representative tracers into tissue protein have been used in animal experiments. More recently the use of non-radioactive stable isotopes also provided the use in human experiments. Given that representative samples of the tissue can be obtained, direct protein synthesis can be determined as incorporation of the tracer into the connective tissue (Babraj et al. 2005; Miller et al. 2006b). Tendon tissue sampling can be performed in humans by percutanous tendon biopsies, and has been used in protocols where protein turnover, mRNA transcription and collagen fibril diameter have been determined in both young and elderly, patients and healthy subjects (Miller et al. 2006b). The use of stable isotope techniques to study incorporation of labelled amino acids into tissue in order to study the kinetics of collagen has been tried in tendon, ligament and bone (Babraj et al. 2005). Briefly, the principle of the direct incorporation technique using the precursor± product approach applicable on tendon tissue is to label the amino acid, proline, e.g. L-13C-proline or L-15N-proline. Proline is abundant in collagen, and is incorporated directly into new collagen proteins. Newly synthesised pro-

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collagens are post-translationally hydroxylated at the proline residues (forming hydroxyproline) before assembling into the triplehelical structure. Thus, collagen-specific hydroxyproline will be labelled. By measuring the enrichment of hydroxyproline from a tendon sample, this provides a very specific synthesis measure of collagen protein in the tendon tissue (Babraj et al. 2005). Using this method, acute exercise has been shown to increase the fractional synthesis rate of collagen in the patella tendon from approximately 0.05%/hour to around 0.10%/hour within 24 hours after exercise, showing a significant rise 6 hours post-exercise (Miller et al. 2005). This corresponds to a collagen synthesis that on a 24 hour level increases from around 1% at rest to 2±3% after exercise. The collagen synthesis rate remains elevated for at least 2±3 days after acute exercise (Heinemeier et al. 2003). Similar to collagen synthesis, there is indication that protein degradation is activated after exercise, in that local levels of matrix metalloproteinases in tendon and muscle tissue are increased after acute exercise (Koskinen et al. 2004). Although no good determination of collagen degradation has been provided so far, it indirectly points towards an increased collagen turnover with exercise. It is difficult to state how much of this increased turnover is in fact turned into assembled load-bearing collagen, but there is reason to believe that at least a certain percentage of the newly formed collagen will end up as insoluble collagen type I in the final tendon structure (Laurent 1987). Within skeletal muscle, the collagen synthesis increases with acute exercise by 2±3-fold and over a time that resembles that of myofibrillar protein synthesis, suggesting a coordinated response with regards to structural proteins both in contractile elements and in the extracellular matrix of human skeletal muscle. Prolonged, repeated mechanical loading chronically elevates the collagen synthesis in tendon almost 3±4-fold (Langberg et al. 2001), and more recently it has been shown that inactivity for only 10 days resulted in a decrease in protein synthesis of both collagen and myofibrillar protein of around 20±25% (DeBoer et al. 2007). Interestingly, neither of these studies resulted in any dramatic morphological changes of the tendon when determined by ultrasound. Clearly, in none of the studies an accurate determination of protein degradation could be performed, and perhaps more importantly, it has been shown that determination of collagen synthesis per se does not always correlate with true incorporation of new load-bearing mature collagen into existing structures (Laurent 1987). Finally, it cannot clearly be stated from cross-measurements of a tendon diameter whether or not the total collagen content of the tendon has been changed.

15.2.2 Regulation of collagen adaptation to mechanical loading An important question in relation to increased collagen turnover is how tendon senses the external loading during muscular contraction and, more specifically,

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what factors are involved in this regulation. The first example of factors involved in collagen synthesis regulation comes from the observation that apparently a gender difference exists, in that females respond less than males with regards to the increase in collagen formation after exercise (Miller et al. 2006b). The expected increase in collagen synthesis to exercise was less pronounced in women than in men, and furthermore the basal collagen synthesis rate was also lower in women compared with men (Miller et al. 2006b). Further experiments in women who have varying levels of sex hormones (e.g. oral contraceptives) suggest that oestradiol may contribute to a diminished collagen synthesis response in women (Hansen et al. 2008). Interestingly, this finding of a lower collagen synthesis rate both at rest and in response to exercise in women vs men, is correlated to a demonstration of lower stress tolerance in patella tendons of women compared with men (Haraldsson et al. 2005). Overall this may contribute to our understanding with regards to the fact that women experience a larger number of soft tissue injuries in, for example, cruciate ligaments than men, and may also be important in understanding of how rapid humans of both sexes not only adapt to physical training but also how well they may resist prolonged periods of reduced activity. What exact mechanism lies behind this gender-specific response is not definitively answered, but the finding of a correlation between increased oestrogen levels and a reduction in collagen synthesis in humans, is supported by in vitro studies where it has been shown that oestradiol receptors are present in ligaments (Sciore et al. 1998) and the finding that oestradiol per se can exert an collagen synthesis inhibiting effect in tendons and ligaments (Liu et al. 1996; Yu et al. 2001). In addition to a suggested direct inhibiting effect of oestradiol upon collagen synthesis, it may also be that oestradiol levels exert an indirect effect by influencing other hormonal components of the human endocrine system. As an example, lately high levels of circulating oestradiol in women have been shown to be associated with low levels of circulating insulin-like growth factor (IGF-I), a substance that may be directly coupled to the degree of collagen synthesis rise with exercise. Thus the gender differences may not be an effect of estradiol directly, but rather via an influence of estradiol on IGF-I. An important regulating factor in collagen synthesis is the growth hormone (GH) ± insulin-like growth factor 1 (IGF-1) axis, where in vitro data have shown a role for collagen formation (Dùssing and Kjñr 2005). As an example, it has been shown that IGF-I (and IGF-II) administration in rabbits will accelerate the protein synthesis in tendons (Abrahamsson 1997), and likewise that the recovery after tendon injury was accelerated when IGF-I was administered (Kurtz et al. 1999). Although GH in skeletal muscle has been shown to exert an effect upon muscle growth in GH-deficient individuals, the effect of GH supplementation upon muscle protein synthesis is absent in both young and elderly humans (Lange et al. 2002). Despite this, it seems that GH/IGF-I influences connective tissue, and administration of GH over 3 weeks has been shown to elevate

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circulation blood levels of procollagen propeptides (Longobardi et al. 2000). Thus it may be tempting to speculate that GH/IGF-I has a stimulating effect upon tendon tissue. In dwarf rats, GH administration has been shown to increase the expression of both collagen type I and III in intramuscular fibroblasts (Wilson et al. 1995). Recently it has been shown that IGF-1 is present in human Achilles tendon linked directly to fibroblasts, and furthermore a detectable interstitial concentration has been demonstrated in human tendon (Olesen et al. 2006). Although this supports the possibility that IGF-I may play a role in human tendon, the question, however, remains: to what extent is IGF-1 upregulated with exercise? By analysing RNA extracted from whole rodent tendons, it has recently been shown that functional overloading of a single muscle attached to the tendon±muscle (by ablation of other muscle synergists) leads to increased expression of IGF-I within the tendon, and, furthermore, that short-term strength training induces the expression of both TGF- -1 and IGF-I in rat Achilles tendon (Heinemeier et al. 2006). Quantification of specific mRNA species has also been used as a tool for studying human tendon pathology, and by analysing RNA extracted from surgical tendon specimens several changes in expression of matrix proteins and enzymes have been identified in overused/damaged tendons compared with healthy tendons (Riley et al. 2002). Furthermore, increased expression in tendon of an IGF-1 isoform called MGF (mechano growth factor) was also found (Heinemeier et al. 2007). This is notable since until recently this isoform was believed to exist only in skeletal muscle cells. In the rodent overload study mentioned above (where synergist muscles were ablated), also an up-regulation of transforming growth factor beta 1 (TGF- 1) was found already after 2 days of stimulation, and it preceded the rise in collagen expression. As an indication that TGF- 1 could play a role in collagen synthesis (Heinemeier et al. 2007), the mRNA for connective tissue growth factor (CTGF) was increased similar to TGF- 1. CTGF is thought to mediate many of the effects of IGF-1 in relation to matrix proteins. A number of in vitro studies have demonstrated a coupling between mechanical loading of tissue and TGF-beta expression (Skutek et al. 2001), and furthermore it has been shown that mechanically induced type I collagen synthesis can be ablated by inhibiting TGF- activity (Lindahl et al. 2002). In human tendon, the presence of TGF- 1 has been demonstrated in association with fibroblasts, and in human skeletal muscle TGF- 1 has been demonstrated to be located mainly in the perimysium in relation to fibroblasts and endothelium (Heinemeier et al. 2007). Furthermore, after exercise of human skeletal muscle TGF- 1 was up-regulated by 2.5 hours after exercise followed by increased collagen synthesis 6 hours post-exercise. Using the microdialysis technique around the Achilles tendon in humans, it was found that both local and circulating levels of TGF- increased in response to running, and furthermore, the time relation between TGF- and indicators of local collagen type I synthesis supported a role of TGF- in regulation of local

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collagen type I synthesis of tendon-related connective tissue (Heinemeier et al. 2003). In a recent study, different types of muscular contraction were used in order to study responses of collagen synthesis. Both concentric and eccentric contractions were performed in two different experimental groups. When performing eccentric contractions, the loading of both muscle and tendon was higher than with concentric contractions, whereas strain was not assumed to be different to a major degree in the two situations (Heinemeier et al. 2006). The expression of IGF-1 and TGF- 1 in muscle increased the most during eccentric exercise, whereas the response in tendon was independent of type of contraction (Heinemeier et al. 2007). Likewise, the procollagen expression was similar in concentric and eccentric exercise, indicating that stress was not a major determiner in the magnitude of collagen synthesis response in tendon, and the findings suggests a more prominent role for strain in signalling a growth factor release and subsequent increase in collagen formation. Taken together, the data supports the view that TGF- plays a role in collagen synthesis of tendon with mechanical loading of collagen-rich tissue. In addition, it is important to note that whereas acute TGF- responses to mechanical loading most likely represents an important physiological response, a more prolonged and chronic elevation of TGF- in association with a variety of pathological situations more likely will result in an uncontrolled formation of fibrotic tissue (Leask et al. 2002).

15.2.3 Matrix and contracting skeletal muscle: playmates? Developing skeletal muscle clearly demonstrates a close coupling between myogenesis and development of the intramuscular extracellular matrix components. It is more unclear to what extent such an interplay exists in mature skeletal muscle, but given the important role that intramuscular collagen plays in force transmission, it would be relevant to assume some kind of coordinated response to loading. Experiments have shown that the myogenic stem cell in skeletal muscle, the satellite cell, is activated not only in pathological situations associated with skeletal muscle injury, but also in physiological situations associated with muscle hypertrophy development (Kadi et al. 2005), but to what extent the connective tissue within the skeletal muscle is involved in this activation of satellite cells is not known. When human muscle is subjected to eccentric loading, it will result in activation of satellite cells in the absence of major damage of the muscle cell itself (Crameri et al. 2004a). In fact, the activation of satellite cells was accompanied by increased staining intensity for the procollagen propeptide PINP and for tenacin-C, a marker for connective tissue and known to reinforce lateral adhesion of the myofibre to the surrounding endomysium (Crameri et al. 2004b). These observations suggest that activation of collagen synthesis in the intramuscular connective tissue is associated with

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satellite cell activation. Although this suggests a potential coupling between signalling to the myofibre that involves activation of the collagen-rich tissue within the muscle, it has to be stated that performing even more heavy eccentric exercise in association with electrical stimulation can result in a marked degree of myofibre injury and this will cause an even more pronounced activation of the satellite cells (Crameri et al. 2007). This illustrates that even if a coupling between connective tissue and the muscle cell exists in mature muscle, it certainly does not exclude a direct link between muscle cell injury and satellite cell activation. A further dissociation between muscle cells and connective tissue arises from a recent study that investigated protein synthesis in response to heavy resistance training and more prolonged low-resistance exercise, respectively. Whereas the exercise-induced rise in protein synthesis for myofibrilar proteins was dependent upon the exercise intensity, the rise in protein synthesis for collagen of the muscle was similar in the two situations where intensity was varied but the total work output was the same (Holm et al., unpublished). This may illustrate that muscle cells are sensing the exercise intensity, whereas the collagen-rich tissue does not have a similar detailed sensing of mechanical loading.

15.2.4 Stem cells and their importance for tendon adaptation The ability to harvest mesenchymal cells from the bone marrow, grow them in culture and re-introduce them into an injured tendon has been widely exploited in racehorses, despite a lack of controlled studies endorsing this treatment (Smith and Webbon 2005). In a recent controlled study, however, mesenchymal cells, extracted and treated in a similar way, were applied to severed rabbit tendons, where some positive findings support the use of these stem cells in this way, at least in the early stages of tendon healing (Chong et al. 2007). The most limiting factor in stem cell research in tendon tissue, as in all tissues, is the availability of suitable markers. Many of the traditional markers, used in cellsorting methods, stain endothelial cells, pericytes and inflammatory cells as well as stem cells, are rendered unsuitable for use in situ where these different cell types are present. In vitro studies do not suffer from this complication and recently cells derived from human hamstring tendons were observed to behave similarly to bone marrow-derived cells in vitro, suggesting the presence of a population of cells with differentiation potential in human tendon (de Mos et al. 2007). The temporal appearance of tendon-produced and circulation-derived cells during tendon healing was elegantly demonstrated with the aid of green fluorescent protein (GFP) chimeric rats (Kajikawa et al. 2007). There, circulation-derived GFP cells were observed in the tendon wound 24 hours after injury, whereas tendon-derived GFP cells were not evident until 3 days after the injury, but appeared to take over from the circulation-derived cells by day 7. The labelling and tracing method employed in this study opens up a new

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way of investigating tendon remodelling in response to injury. In vivo cell tracking using magnetic resonance imaging (MRI) of magnetically labelled cells is the latest non-invasive method for following the movement of cells around the body. The success of this method in tracking the migration of adult stem cells in the central nervous system has recently been reviewed (Sykowa and Jendelova 2007), and one can surmise that it is merely a matter of time before this method will be applied to the area of tendon research, which, together with the other methods mentioned here, will help enrich our understanding of the involvement of stem cells in adaptation of this tissue.

15.3

Biomechanics of human tendon

Human lower extremity tendons are subjected to appreciable loads during human locomotion. In fact, the Achilles and patellar tendons can be subjected to several times the body weight during running. Unfortunately, tendons are unable to adapt to certain loading conditions and therefore end up with pathology, including so-called overuse injuries and complete tendon ruptures (Kannus and Jozsa 1991; Maffulli et al. 1999). However, whether over-use injuries and ruptures are associated with predisposing morphological changes, or whether the tendon inadvertently exceeds a narrow safety margin remains unknown. Nevertheless, the mechanical properties of the tendon are generally thought to play a pivotal role. Because the aetiology of these injuries, and exactly how tendons adapt to physical activity or lack thereof, are at best incompletely understood, the ability of clinicians to provide optimal treatment and prevent injury remains circumscribed.

15.3.1 Does tendon adapt to loading by hypertrophy? Tendons subjected to considerable stress, including the Achilles tendon, are believed to operate as springs by storing and releasing elastic strain energy during locomotion (Alexander and Bennet-Clark 1977; Biewener and Roberts 2000). From this standpoint it is advantageous to have a thin (and long) tendon. On the other hand, a thicker tendon, which would yield less strain energy, would reduce the average stress (force/area) across the tendon and thereby provide a greater safety margin. For example, if the tendon were to increase in size (hypertrophy) as a function of physical activity/exercise, the average stress would be reduced for a given applied force (e.g. some multiple of the body weight). However, the time course and to what extent human tendon adapts by changing its material and/or undergo hypertrophy in response to exercise remain an enigma. Animal data show that tendon may undergo both qualitative (Buchanan and Marsh 2001), hypertrophic changes (Birch et al. 1999; Woo et al. 1982), or both (Woo et al. 1982) in response to endurance-type exercise and do therefore not provide a coherent picture.

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In humans cross-sectional data suggest that habitual long-distance running (over a time span of 5 years) is associated with a markedly greater crosssectional area (CSA; 22%) of the Achilles tendon compared with that of nonrunners (Rosager et al. 2002). However, a total training stimulus of ~9 months of running in previously untrained young subjects did not result in tendon hypertrophy of the Achilles tendon (Hansen et al. 2003). At the same time it has been shown that resistance training for 3 months induced remarkable changes in the material properties of human tendon in the absence of any tendon hypertrophy in older people (Reeves et al. 2003), while the opposite was demonstrated in young male subjects (Kongsgaard et al. 2007). It is possible that these apparent discrepancies may relate to the training mode (endurance versus resistance training), age and tendon type (Achilles vs patellar tendon). It is also possible that gender (hormonal milieu) may play a role (Miller et al. 2006a): recent cross-sectional data indirectly suggest that the ability of the patellar tendon to adapt in response to habitual loading such as running is attenuated in women as evidenced by the similar tendon CSA, stiffness and modulus in habitual runners and non-runners (Westh et al. 2008). Therefore, although it has been elegantly and repeatedly shown in a human model that there is an increased metabolic activity of human tendon tissue in response to an acute bout of loading (Bojsen-Moller et al. 2006; Langberg et al. 1999; Miller et al. 2005), it is not entirely understood to what extent this results in a larger structure or a different material. Finally, it may be that adaptation of tendon morphology to exercise just requires extremely long time, a notion that is recently supported by the finding that in athletes that load their two legs (and their patella tendons) differently, a significant difference could be demonstrated, in that the patella tendon that was subjected to repetitive high and eccentric muscle loading was thicker than the contralateral tendon (Couppe et al. 2008).

15.3.2 Tendon regional differences in the morphology Tendon stress is calculated commonly based on a single or average value of the tendon CSA. However, it appears that there is a large variation in the tendon CSA along the length of both the human Achilles and patellar tendon (Kongsgaard et al. 2005; Magnusson and Kjñr 2003; Rosager et al. 2002; Westh et al. 2008). In fact, both the Achilles and patellar tendon can have a CSA that differs by more than 50% along its length such that the most proximal segment is considerably smaller than the most distal tendon segment. Stress along the length of the tendon may consequently differ considerably. It is interesting to note that clinical conditions such as patellar and Achilles tendinopathy occur in the region with the greatest average stress for a given applied force, and the aetiology may therefore be somehow related to the stress in the region. Furthermore, if in fact there is tendon hypertrophy in response to increased

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loading (see above), there is some evidence that it may occur in a region-specific manner (Kongsgaard et al. 2007; Magnusson and Kjñr 2003).

15.3.3 Methods to study tendon in vivo: the ultrasonography method Much of the current knowledge regarding mechanical behaviour is based on data from experimental animal or cadaver tissue, while some more recent studies have studied mechanics on a microscopic level. Currently, the most widely used technique to investigate, in vivo, human tendon displacement during muscle contractions is B-mode ultrasonography, which was developed in the mid-1990s for measurements in the whole tendon±aponeurosis complex (Fukashiro et al. 1995) and late 1990s for measurements in isolated tendon (Maganaris and Paul 1999). The non-invasive approach of studying human tendons in vivo is attractive, and many limitations have been successfully addressed, but several details have to be considered. The method accounts for only two dimensions of structural deformation (i.e. in the sagittal plane). The technique was originally intended for the displacement of intramuscular fascicular structures, and therefore the resulting deformation does not represent that of the tendon per se, but rather the total deformation of the combined tendon and aponeurosis distal to the measurement site. This can, in some muscle± tendon complexes, but not all, be circumvented by identifying the very junction between the aponeurosis and free tendon (the myotendinous junction), or alternatively by introducing a visible landmark such as a needle (Magnusson et al. 2003). For the patellar tendon the problem can be overcome by having two bony landmarks (tibia and patella) (Hansen et al. 2006). The technique is most commonly employed during `isometric' conditions in which small amounts of joint rotation or body movement may take place that can affect the displacement measurements, which need to be accounted for (Magnusson et al. 2003). A true resting length of the tendon (i.e. 0% strain) may be difficult to obtain in vivo, but may be defined as that corresponding to zero net joint moment. The current time resolution of ultrasonography (frequency of sampling) and of video recording apparatus is a limiting factor that precludes analysis of faster contraction velocities. However, the technique still lacks an associated accurate measurement of tendon force. Notwithstanding these methodological difficulties, the technique has provided recent insights into human, in vivo, tendon behaviour.

15.3.4 Aponeurosis shear in human muscle±tendon complex The facts that the CSA of tendon differs along its length and that the stress seen by the tendon therefore also differs considerably for a given applied force prompt questions as to how force is transmitted in general throughout the tendon. In this regard the human Achilles tendon is of interest because of its

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unique anatomy and because it is often implicated clinically. The human triceps surae muscle±tendon structure is composed of three separate muscle compartments that merge via their aponeuroses into one common tendon to ultimately insert on the calcaneus. To what extent activation of the individual muscles of the triceps surae complex influences aponeurosis and tendon strain is uncertain. An in vitro study demonstrated that differences in medial and lateral forces in the Achilles tendon can be observed when single muscles of the triceps surae were subjected to force (Arndt et al. 1999). Non-uniform tendon force would theoretically result in intratendinous shear strain and cause sliding between planes of tissue layers parallel to the acting forces. Because the triceps surae includes the gastrocnemii muscles that cross both the ankle and knee joints, and the soleus muscle that crosses the ankle joint alone, the relative contribution of these muscles to the tendon force will be influenced by the degree of knee flexion (Cresswell et al. 1995). It has been shown that during maximal isometric contractions with the plantarflexor muscles, there is a differential displacement between the soleus and gastrocnemius aponeuroses proximal to the junction with the Achilles tendon (Bojsen-Moller et al. 2004). When the knee joint was extended, displacement of the medial gastrocnemius aponeurosis exceeded that of the soleus aponeurosis, whereas the converse occurred when the knee joint was flexed. These differences in aponeurosis displacement created a `shear' effect with a direction that was governed by the knee joint position. Since the collagen fibres of the aponeurosis fuse distally to become free tendon structures, any difference in displacement at the level of the aponeuroses may be manifested as shear strain at the level of the tendon, although this has yet to be confirmed. The difference in displacement between separate aponeuroses within the triceps surae amounted to more than 30% of the maximal observed displacement, indicating that a considerable `shear potential' exists during movement tasks. The observed differential aponeurosis displacement was likely caused by differences in force output of the medial gastrocnemius and soleus muscles.

15.3.5 Mechanical properties of individual human tendon fascicles Patellar tendinopathy chiefly concerns the proximal and posterior portion of the patellar tendon (Johnson et al. 1996), and this poses the question as to whether the mechanical properties differ by region within a whole tendon. Data on strain properties of the anterior and posterior portion of the human patellar tendon is sparse and conflicting. In cadaver knees of older persons it has been shown that during quadriceps loading tensile strain was uniform in the anterior and posterior region with the knee in full extension, but tensile strain increased on the anterior side and decreased on the posterior with knee flexion (Almekinders et al. 2002). It has also been shown, however, that quadriceps loading in flexion caused a

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greater strain on the posterior compared to the anterior side of the patellar tendon (Basso et al. 2002). An alternative way to examine if the mechanical properties differ by region in the human patellar tendon is to test the isolated portions of the tendon. It was recently possible to obtain human tissue from healthy young men in association with elective anterior cruciate ligament reconstruction (Haraldsson et al. 2005). Thin collagen bundles that were ~35 mm in length and ~3.5 mm in diameter were obtained from the anterior and posterior portion of the harvested tendon. From these collagen bundles, individual strands of fascicles ( ~ 300 m) were dissected and tested for mechanical properties. The data showed that tendon fascicles from the anterior portion of the human patellar tendon in young men displayed substantially greater peak and yield stress and tangent modulus compared with the posterior portion of the tendon. This observation suggests that regions of the patellar tendon may have markedly different mechanical properties. It is not surprising since there may be regional differences when it comes to the structural components of the tendon. In the horse there may be region-specific differences with respect to collagen fibril diameter (PattersonKane et al. 1997), and it has also been shown that there may be a site-specific loss of larger size collagen fibrils in the core of ruptured human Achilles tendons (Magnusson et al. 2002). It remains to be established if the above noted differences in mechanical properties of the patellar tendon can be attributed to factors such as fibril size and density (Parry et al. 1978), cross-links (Thompson and Czernuszka 1995), fibril length (Redaelli et al. 2003) and components of the extracellular matrix (Kjñr 2004). It is also unknown to what extent training regimes with varying degrees of knee flexion may influence the region-specific material properties of the tendon, and if such exercises can reduce the sitespecific patellar tendinopathy. There is some understanding of force transmission in skeletal muscle, but relatively little is known about force transmission in tendon. Skeletal muscle fibres do not always extend from one tendon plate to the other, which requires that contractile force can be transmitted laterally to adjacent fibres to initiate movement, which was deomonstrated elegantly by Street (1983). Force is transmitted from the muscle fibres to the aponeurosis via the myotendinous junction, and can also be transmitted to parallel adjacent structures via the aponeurosis (Huijing and Jaspers 2005). The aponeurosis, which has different mechanical properties than free tendon (Magnusson et al. 2003), may be loaded heterogeneously during skeletal muscle contraction (Finni et al. 2003), although how this affects tendon force transmission and to what extent lateral force transmission exists in human free tendon remains unknown. The tendon is a hierarchical structure (Kastelic et al. 1978), and to understand how force is transmitted within the whole tendon it is necessary to examine the mechanical properties at the various levels. A disparity in magnitude of strain at different hierarchical levels has been observed, and while the exact mechanism

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for such a hierarchy of strain distribution within the tendon structure remains unknown, it suggests that `gliding' processes may be involved (Mosler et al. 1985; Puxkandl et al. 2002). Studies on force pathways in tendons have largely focused on molecular strain and intermolecular force transmission (Mosler et al. 1985; Puxkandl et al. 2002; Sasaki and Odajima 1996), fibril±fibroblast mechanotransduction (Arnoczky et al. 2002) and contributing factors to interfibrillar force transmission (Scott 2003; Vesentini et al. 2005). The tendon fascicle, however, is a distinct entity within the tendon hierarchy. The ability of the fascicle to transmit force to the adjacent parallel fascicle was recently examined in human patellar and Achilles tendon tissue obtained during surgery. From each of the tendon specimens, two adjoining strands of collagen fascicles enclosed in an intact fascicular membrane were carefully dissected and special care was taken to ensure that the fascicular membrane between adjoining fascicles was left intact. The specimens were subjected to a series of load±displacement cycles to a given displacement (~3% strain). The first load±displacement cycle was performed with both fascicles and fascicular membrane intact. The second load±displacement cycle was performed with one fascicle transversally cut while the other fascicle and the fascicular membrane remained intact. The third load±displacement cycle was performed with both fascicles transversally cut on opposite ends while the fascicular membrane was kept intact. Theoretically, if there is no or inconsequential lateral transfer of force, a reduction in the CSA of a structure by 50% (one of two fascicles of similar size) should reduce the stiffness of the structure by half. Severing one fascicle from the patellar tendon reduced the material stiffness by ~40% while the same procedure reduced the material stiffness by more than 50% in the Achilles tendon, indicating that lateral force transmission between adjacent human tendon fascicles was marginal. Cycle 3 represented two adjacent fascicles that could potentially transmit force only in parallel rather than in series, and these data confirm the findings of the comparison between cycle 1 and 2 by demonstrating that the lateral force transmission between fascicles was relatively small or negligible. Of course, these data do not show to what extent fascicles are functionally `independent' in whole tendon in vivo. It appears that the magnitude of strain at the various hierarchical levels differ such that strain of the whole tendon exceeds that of the strain at the molecule and fibril level (Mosler et al. 1985; Puxkandl et al. 2002; Sasaki and Odajima 1996). Specifically, it has been suggested that during loading the triple helix itself may elongate, that the gap region between longitudinally adjoining molecules increases, that there is relative slippage between laterally adjacent molecules, and that fibrils themselves may slide relative to one another. At the level of the molecule, strain will be taken up by cross-links (Puxkandl et al. 2002), while the strain between fibrils may be prevented by components of the extracellular matrix (Scott, 2003). Intuitively, this means that some of the force is `transferred' to the adjacent structure by

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means of lateral shear forces. The fascicle cutting experiment adds to the existing knowledge of tendon mechanical properties by demonstrating that gliding processes can take place freely between adjacent fascicles. However, the present data also suggests that the aforementioned mechanisms of lateral shear force are largely limited to within the fascicle.

15.4

References and further reading

Abrahamsson SO. (1997). Similar effect of recombinant human insulin-like growth factor-I and II in cellular activities in flexor tendons of young rabbits: experimental studies in vitro. J Orthop Res 15: 256±262. Alexander RM and Bennet-Clark HC. (1977). Storage of elastic strain energy in muscle and other tissues. Nature 265: 114±117. Almekinders LC, Vellema JH, and Weinhold PS. (2002). Strain patterns in the patellar tendon and the implications for patellar tendinopathy. Knee Surg Sports Traumatol Arthrosc 10: 2±5. Arndt AN, Bruggemann GP, Koebke J, and Segesser B. (1999). Asymmetrical loading of the human tricpes surae: I. Mediolateral force difference in the Achilles tendon. Foot Ankle Int 20: 445±449. Arnoczky SP, Lavagnino M, Whallon JH, and Hoonjan A. (2002). In situ cell nucleus deformation in tendons under tensile load; a morphological analysis using confocal laser microscopy. J Orthop Res 20: 29±35. Babraj J, Cuthbertson D, Smith J, Langberg H, Miller BF, Krogsgaard M, Kjñr M, and Rennie MJ. (2005). Collagen synthesis in human musculoskeletal tissues and skin. Am. J. Physiol. 99: 986±994. Basso O, Amis AA, Race A, and Johnson DP. (2002). Patellar tendon fiber strains: their differential responses to quadriceps tension. Clin Orthop 400: 246±253. Biewener AA and Roberts TJ. (2000). Muscle and tendon contributions to force, work, and elastic energy savings: a comparative perspective. Exerc Sport Sci Rev 28: 99± 107. Birch HL, McLaughlin L, Smith RK, and Goodship AE. (1999). Treadmill exerciseinduced tendon hypertrophy: assessment of tendons with different mechanical functions. Equine Vet J Suppl 30: 222±226. Bojsen-Moller J, Hansen P, Aagaard P, Svantesson U, Kjñr M, and Magnusson SP. (2004). Differential displacement of the human soleus and medial gastrocnemius aponeuroses during isometric plantar flexor contractions in vivo. J Appl Physiol 97: 1908±1914. Bojsen-Moller J, Kalliokoski KK, Seppanen M, Kjñr M, and Magnusson SP. (2006). Low-intensity tensile loading increases intratendinous glucose uptake in the Achilles tendon. J Appl Physiol 101: 196±201. Buchanan CI and Marsh RL. (2001). Effects of long-term exercise on the biomechanical properties of the Achilles tendon of guinea fowl. J Appl Physiol 90: 164±171. Chong, A.K., et al. (2007), Bone marrow-derived mesenchymal stem cells influence early tendon-healing in a rabbit Achilles tendon model. J Bone Joint Surg Am 89: 74±81. Couppe C, Kongsgaard M, Aagaard P, Hansen P, Bojsen-Moller J, Kjñr M, and Magnusson SP. (2008). Habitual loading results in tendon hypertrophy and increased stiffness of the patellar tendon. J Appl Physiol 105: 805±810. Crameri R, Langberg H, Jensen CH, Teisner B, Schrùder HD, and Kjñr M. (2004a).

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Activation of satellite cells in human skeletal muscle after a single bout of exercise. J Physiol 558: 333±340. Crameri R, Langberg H, Teisner B, Magnusson P, Olesen JL, Koskinen S, Suetta C, and Kjñr M. (2004b). Synchronous disruption of the extracellular matrix and mechanical tenderness in skeletal muscle after a single bout of eccentric loading in humans. Matrix Biol 23: 259±264. Crameri R, Aagaard P, Qvortrup K, Langberg H, Olesen JL, and Kjñr M. (2007). Myofibre damage in human skeletal muscle: Effects of electrical stimulation vs voluntary contraction. J Physiol 583: 365±380. Cresswell AG, Loscher WN, and Thorstensson A. (1995). Influence of gastrocnemius muscle length on triceps surae torque development and electromyographic activity in man. Exp Brain Res 105: 283±290. DeBoer M, Maganaris CN, Seynnes OR, Rennie MJ, and Narici MV (2007). Time course of muscular, neural and tendinous adaptation to 23 days unilateral lower-limb suspension in young men. J Physiol 583: 1079±1091. de Mos M, et al. (2007). Intrinsic differentiation potential of adolescent human tendon tissue: an in-vitro cell differentiation study. BMC Musculoskelet Disord 8: 16±22. Dùssing S, and Kjñr M. (2005). Growth hormone and connective tissue in exercise. Scand J Med Sci Sports 15: 202±210. Finni T, Hodgson JA, Lai AM, Edgerton VR, and Sinha S. (2003). Nonuniform strain of human soleus aponeurosis±tendon complex during submaximal voluntary contractions in vivo. J Appl Physiol 95: 829±837. Fukashiro S, Itoh M, Ichinose Y, Kawakami Y, and Fukunaga T. (1995). Ultrasonography gives directly but noninvasively elastic characteristic of human tendon in vivo. Eur J Appl Physiol 71: 555±557. Hansen M, Miller BF, Holm L, Doessing S, Petersen SG, Skovgaard D, Frystyk J, Flyvbjerg A, Koskinen SO, Pingel J, Kjñr M, and Langberg H (2008). Effect of administration of oral contraceptives in vivo on collagen synthesis in tendon and muscle connective tissue in young women. J Appl Physiol 586: 3005±3016. Hansen P, Aagaard P, Kjñr M, Larsson B, and Magnusson SP. (2003). The effect of habitual running on human Achilles tendon load-deformation properties and crosssectional area. J Appl Physiol 95: 2375±2380. Hansen P, Bojsen-Moller J, Aagaard P, Kjñr M, and Magnusson SP. (2006). Mechanical properties of the human patellar tendon, in vivo. Clin Biomech 21: 54±58. Haraldsson BT, Aagaard P, Krogsgaard M, AlKjñr T, Kjñr M, and Magnusson SP. (2005). Region-specific mechanical properties of the human patella tendon. J Appl Physiol 98: 1006±1012. Heinemeier K, Langberg H, Olesen JL, and Kjñr M. (2003). Role of transforming growth factor beta in relation to exercise induced type I collagen synthesis in human tendinous tissue. J Appl Physiol 95: 2390±2397. Heinemeier KM, Olesen JL, Schjerling P, Haddad F, Langberg H, Baldwin KM, and Kjñr M. (2006). Strength training and the expression of myostatin- and IGF-I splice variants in rat muscle and tendon: differential effects of specific contraction types. J Appl Physiol 102: 573±581. Heinemeier KM, Olesen JL, Haddad F, Langberg H, Kjñr M, Baldwin KM, and Schjerling P. (2007). Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types. J Physiol 582: 1303± 1306. Huijing PA and Jaspers RT. (2005). Adaptation of muscle size and myofascial force transmission: a review and some new experimental results. Scand J Med Sci Sports

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15: 349±380. Johnson DP, Wakeley CJ, and Watt I. (1996). Magnetic resonance imaging of patellar tendonitis. J Bone Joint Surg Br 78: 452±457. Kadi F, Charifi N, Denis C, Lexell J, Andersern JL, Schjerling P, Olsen S, and Kjñr M (2005). The behaviour of satellite cells in response to exercise: what have we learned from human studies? PfuÈgers Arch 451: 319±327. Kajikawa, Y., et al. (2007). GFP chimeric models exhibited a biphasic pattern of mesenchymal cell invasion in tendon healing. J Cell Physiol 210: 684±691. Kannus P and Jozsa L. (1991). Histopathological changes preceding spontaneous rupture of a tendon. A controlled study of 891 patients. J Bone Joint Surg Am 73: 1507± 1525. Kastelic J, Galeski A, and Baer E. (1978). The multicomposite structure of tendon. Connect Tissue Res 6: 11±23. Kjñr M. (2004). Role of extracellular matrix in adaptation of tendon and skeletal muscle to mechanical loading. Physiol Rev 84: 649±698. Kongsgaard M, Aagaard P, Kjñr M, and Magnusson SP. (2005). Structural Achilles tendon properties in athletes subjected to different exercise modes and in Achilles tendon rupture patients. J Appl Physiol 99: 1965±1971. Kongsgaard M, Reitelseder S, Pedersen TG, Holm L, Aagaard P, Kjñr M, and Magnusson SP. (2007). Region specific patellar tendon hypertrophy in humans following resistance training. Acta Physiol 191: 111±121. Koskinen SO, Heinemeier KM, Olesen JL, Langberg H, and Kjñr M. (2004). Physical exercise can influence local levels of matrix metalloproteinases and their inhibitors in tendon related connective tissue. J Appl Physiol 96: 861±864. Kovanen V (1989). Effects of ageing and physical training on rat skeletal muscle. Acta Physiol Scand 135, suppl 577: 1±56. Kurtz CA, Loebig TG, Anderson DD, Demeo PJ and Cambell PG (1999). Insulin-like growth factor I accelerates functional recovery from Achilles tendon injury in a rat model. Am J Sports Med 27: 363±369. Langberg H, Skovgaard D, Petersen LJ, Bulow J, and Kjñr M. (1999). Type I collagen synthesis and degradation in peritendinous tissue after exercise determined by microdialysis in humans. J Physiol 521 Pt 1: 299±306. Langberg H, Skovgaard D, BuÈlow J, and Kjñr M (2000). Time pattern of exerciseinduced changes in type-I collagen turnover after prolonged endurance exercise in humans. Calcif Tissue Int 67: 41±44. Langberg H, Rosendal L, and Kjñr M. (2001). Training induced changes in peritendinous type I collagen turnover determined by microdialysis in humans. J Physiol 534: 297±302. Langberg H, Ellingsgaard H, Madsen T, Jansson J, Magnusson SP, Aagaard P, and Kjñr M. (2007). Eccentric rehabilitation exercise increases peritendinous type I collagen synthesis in humans with Achilles tendinosis. Scand J Med Sci Sports 17: 61±68. Lange KH, Andersen JL, Beyer N, Isaksson F, Larsson B, Rasmussen MH, Juul A, BuÈlow J, and Kjñr M. (2002). GH administration changes myosin heavy chain isoforms in skeletal muscle but does not augment muscle strength or hypertrophy, either alone or combined with resistance exercise training in healthy elderly men. J Clin Endocrinol Metab 87: 513±523. Laurent GJ. (1987). Dynamic state of collagen pathways of collagen degradation in vivo and their possible role in regulation of collagen mass. Am J Physiol 252: C1±9. Leask A, Holmes A, and Abraham DJ (2002). Connective tissue growth factor: a new and important player in the pathogenesis of fibrosis. Curr Rheumatol Rep 4: 136±142.

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Lindahl GE, Chambers RC, Papakrivopoulou J, Dawson SJ, Jacobsen MC, Bishop JE, and Laurent GJ. (2002). Activation of fibroblast procollagen alpha I(I) transcription by mechanical strain is transforming growth factor beta dependent and involves increased binding of CCAAT-binding factor (CBF/NF-y) at the proximal promotor. J Biol Chem 277: 6153±6161. Liu SH, al Shaikh R, Panossian V, Yang RS, Nelson SD, Soleiman N, Finerman GA and Lane JM (1996). Primary immunolocalization of estrogen and progesterone target cells in the human anterior cruciate ligament. J Orthop Res 14: 526±533. Longobardi S, Keay N, Ehrnborg C, Cittadini A, Rosen T, Dall R, Boroujerdi MA, Bassett EE, Healy ML, Pentecost C, Wallace JD, Powrie J, Jorgensen JO, and Sacca L. (2000). Growth hormone (GH) effects on bone and collagen turnover in healthy adults and its potential as a marker of GH abuse in sports: a double blind, placebocontrolled study. The GH-2000 Study Group. J Clin Endocrinol Metab 85, 1505± 1512. Maffulli N, Waterston SW, Squair J, Reaper J, and Douglas AS. (1999). Changing incidence of Achilles tendon rupture in Scotland: a 15-year study. Clin J Sport Med 9: 157±160. Maganaris CN and Paul JP. (1999). In vivo human tendon mechanical properties. J Physiol (Lond) 521: 307±313. Magnusson SP and Kjñr M. (2003). Region-specific differences in Achilles tendon crosssectional area in runners and non-runners. Eur J Appl Physiol 90: 549±553. Magnusson SP, Qvortrup K, Larsen JO, Rosager S, Hanson P, Aagaard P, Krogsgaard M, and Kjñr M. (2002). Collagen fibril size and crimp morphology in ruptured and intact Achilles tendons. Matrix Biol 21: 369±377. Magnusson SP, Hansen P, Aagaard P, Brond J, Dyhre-Poulsen P, Bojsen-Moller J, and Kjñr M. (2003). Differential strain patterns of the human gastrocnemius aponeurosis and free tendon, in vivo. Acta Physiol Scand 177: 185±195. Miller BF, Olesen JL, Hansen M, Dossing S, Crameri RM, Welling RJ, Langberg H, Flyvbjerg A, Kjñr M, Babraj JA, Smith K, and Rennie MJ. (2005). Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise. J Physiol 567: 1021±1033. Miller B, Hansen M, Olesen JL, Flyvbjerg A, Schwarz P, Babraj JA, Smith K, Rennie MJ, and Kjñr M. (2006a). No effect of menstrual cycle on myofibrillar and connective tissue synthesis in contracting skeletal muscle. Am J Physiol 290: E163±E168. Miller BF, Hansen M, Olesen JL, Schwarz P, Babraj JA, Smith K, Rennie MJ, and Kjñr M. (2006b). Tendon collagen synthesis at rest and after exercise in women. J Appl Physiol 102: 542±547. Mosler E, Folkhard W, Knorzer E, Nemetschek-Gansler H, Nemetschek T, and Koch MH. (1985). Stress-induced molecular rearrangement in tendon collagen. J Mol Biol 182: 589±596. Olesen JL, Heinemeier KM, Langberg H, Magnusson SP, Kjñr M, and Flyvbjerg A (2006). Expression, content and localization of IGF-1 in human Achiles tendon. Conn Tissue Res 47: 200±206. Olesen JL, Langberg H, Heinemeier K, Flyvbjerg A, and Kjñr M. (2007a). Exercisedependent IGF-I, IGFBPs, and type I collagen changes in human peritendinous connective tissue determined by microdialysis. J Appl Physiol 102: 214±20. Olesen JL, Heinemeier KM, Haddad F, Langberg H, Flyvbjerg A, Kjñr M, and Baldwin KM (2007b). Expression of insulin-like growth factor I, insulin-like growth factor binding proteins, and collagen mRNA in mechanically loaded plantaris tendon. J Appl Physiol 101: 183±188.

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Parry DA, Barnes GR, and Craig AS. (1978). A comparison of the size distribution of collagen fibrils in connective tissues as a function of age and a possible relation between fibril size distribution and mechanical properties. Proc R Soc Lond B: Biol Sci 203: 305±321. Patterson-Kane JC, Wilson AM, Firth EC, Parry DA, and Goodship AE. (1997). Comparison of collagen fibril populations in the superficial digital flexor tendons of exercised and nonexercised thoroughbreds. Equine Vet J 29: 121±125. Erratum in Equine Vet J 1998; 30(2): 176. Puxkandl R, Zizak I, Paris O, Keckes J, Tesch W, Bernstorff S, Purslow P, and Fratzl P. (2002). Viscoelastic properties of collagen: synchrotron radiation investigations and structural model. Philos Trans R Soc Lond B Biol Sci 357: 191±197. Redaelli A, Vesentini S, Soncini M, Vena P, Mantero S, and Montevecchi FM. (2003). Possible role of decorin glycosaminoglycans in fibril to fibril force transfer in relative mature tendons ± a computational study from molecular to microstructural level. J Biomech 36: 1555±1569. Reeves ND, Maganaris CN, and Narici MV. (2003). Effect of strength training on human patella tendon mechanical properties of older individuals. J Physiol 548: 971±981. Riley GP, Curry V, DeGroot J, van El B, Verzijl N, Hazleman B, and Bank RA. (2002). Matrix metalloproteinase activities and their relationship with collagen remodelling in tendon pathology. Matrix Biol 21: 185±195. Rosager S, Aagaard P, Dyhre-Poulsen P, Neergaard K, Kjñr M, and Magnusson SP. (2002). Load±displacement properties of the human triceps surae aponeurosis and tendon in runners and non-runners. Scand J Med Sci Sports 12: 90±98. Sasaki N and Odajima S. (1996). Elongation mechanism of collagen fibrils and force-strain relations of tendon at each level of structural hierarchy. J Biomech 29: 1131±1136. Sciore P, Frank CB, and Hart DA. (1998). Identification of sex hormone receptors in human and rabbit ligaments of the knee by reverse transcription-polymerase chain reaction: evidence that receptors are present in tissue from both male and female subjects. J Orthop Res 16: 604±610. Scott JE. (2003). Elasticity in extracellular matrix 'shape modules' of tendon, cartilage, etc. A sliding proteoglycan-filament model. J Physiol 553: 335±343. Skutek M, Van Griensven M, Zeichen J, Brauer N, and Bosch U (2001). Cyclic mechanical stretching modulates secretion pattern of growth factors in human fibroblasts. Eur J Appl Physiol 86: 48±52. Smith RK and Webbon PM. (2005). Harnessing the stem cell for the treatment of tendon injuries: heralding a new dawn? Br J Sports Med 39: 582±584. Street SF. (1983). Lateral transmission of tension in frog myofibers: a myofibrillar network and transverse cytoskeletal connections are possible transmitters. J Cell Physiol 114: 346±364. Sykova E and Jendelova P. (2007). Migration, fate and in vivo imaging of adult stem cells in the CNS. Cell Death Differ 14(7): 1336±1342. Thompson JI and Czernuszka JT. (1995). The effect of two types of cross-linking on some mechanical properties of collagen. Biomed Mater Eng 5: 37±48. Vesentini S, Redaelli A, and Montevecchi FM. (2005). Estimation of the binding force of the collagen molecule-decorin core protein complex in collagen fibril. J Biomech 38: 433±443. Westh E, Kongsgaard M, Bojsen-Moller J, Aagaard P, Hansen M, Kjñr M, and Magnusson SP. (2008). Effect of habitual exercise on the structural and mechanical properties of human tendon, in vivo, in men and women. Scand J Med Sci Sports 18: 23±30.

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Wilson VJ, Rattray M, Tomas CR, Moreland BH, and Schulster D (1995). Growth hormone increases IGF-I, collagen type I and collagen II gene expression in dwarf rat skeletal muscle. Mol Cell Endocrinol 115: 187±197. Woo SL, Gomez MA, Woo YK, and Akeson WH. (1982). Mechanical properties of tendon and ligaments. The relationship of immobilization and exercise on tissue remodeling. Biorheology 19: 397±408. Yu WD, Panossian V, Hatch JD, Liu SH, and Finerman GA (2001). Combined effects of estrogen and progesterone on the anterior cruciate ligament. Clin Orthop 21: 268± 281.

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Tendon injury and repair mechanics N . M A F F U L L I , Barts and The London School of Medicine and Dentistry, UK and U . G . L O N G O , P . S H A R M A and V . D E N A R O , Campus Biomedico University, Italy

Abstract: Tendon injuries give rise to substantial morbidity, and current understanding of the mechanisms involved in tendon injury and repair is limited. This chapter discusses tendon injury and intrinsic repair. The chapter first reviews the current knowledge on aetiology and pathophysiology of tendon injuries (tendinopathies and ruptures). Mechanisms of tendon healing are then discussed. Key words: tendon injuries, tendon rupture, tendinopathy, intrinsic repair, aetiology, pathophysiology.

16.1

Introduction: tendon injury

Tendon injuries can be acute or chronic, and can be caused by intrinsic or extrinsic factors, either alone or in combination. In acute trauma, extrinsic factors predominate, whilst in chronic cases intrinsic factors also play a role.

16.2

Tendinopathy

In chronic tendon disorders, interaction between intrinsic and extrinsic factors is common (Williams, 1993; Ames et al., 2008). Intrinsic factors such as alignment and biomechanical faults are claimed to play a causative role in two-thirds of athletes with Achilles tendon disorders (Kvist, 1994). In particular, hyperpronation of the foot has been linked with an increased incidence of Achilles tendinopathy. Excessive loading of tendons during vigorous physical training is regarded as the main pathological stimulus for the failed healing response typically observed in these lesions. In the presence of intrinsic risk factors, excessive loading may carry a greater risk of inducing tendinopathy. Tendons respond to repetitive overload beyond physiological threshold by either inflammation of their sheath, a failed healing response of their body, or a combination of both. Different stresses may induce different responses. Active repair of fatigue damage must occur, or tendons would weaken and eventually rupture. The repair mechanism is probably mediated by resident tenocytes, which maintain a fine balance between extracellular matrix (ECM) production and

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degradation. Tendon damage may even occur from stresses within physiological limits, as frequent cumulative microtrauma may not allow enough time for repair. Microtrauma can also result from non-uniform stress within tendons, producing abnormal load concentrations and frictional forces between the fibrils, resulting in localised fibre damage. The aetiology of tendinopathy remains unclear, and many causes have been theorised. Hypoxia, ischaemic damage, oxidative stress, hyperthermia, impaired apoptosis, inflammatory mediators, fluoroquinolones and matrix metalloproteinase imbalance have all been implicated as mechanisms of tendon degeneration. Failure to adapt to recurrent excessive loads results in the release of cytokines leading to further modulation of cell activity (Leadbetter, 1992). Tendon damage may even occur from stresses within physiological limits, as frequent cumulative microtrauma may not allow enough time for repair (Selvanetti et al., 1997). Microtrauma can also result from non-uniform stress within tendons, producing abnormal load concentrations and frictional forces between the fibrils, with localised fibre damage (Leadbetter, 1992). Free radical damage occurring on reperfusion after ischaemia, hypoxia, hyperthermia and impaired tenocyte apoptosis have been linked with tendinopathy (Bestwick and Maffulli, 2004). In animal studies, local administration of cytokines and inflammatory agents such as prostaglandins has resulted in tendinopathy (Sullo et al., 2001). Fluoroquinolones have also been implicated in the pathogenesis of tendinopathy. Ciprofloxacin causes enhanced interleukin-1 mediated MMP3 release, inhibits tenocyte proliferation and reduces collagen and matrix synthesis. Degenerative tendinopathy is the most common histological finding in spontaneous tendon ruptures. Tendon degeneration may lead to reduced tensile strength and a predisposition to rupture. Indeed, ruptured tendons have more advanced intratendinous changes than tendinopathic tendons (Tallon et al., 2001). In Achilles tendinopathy, changes in the expression of genes regulating cell±cell and cell±matrix interactions have been reported, with down-regulation of matrix metalloproteinase 3 (MMP 3) mRNA (Sharma and Maffulli, 2006b). Significantly higher levels of type I and type III collagen mRNAs have been reported in tendinopathic samples compared to normal samples. Imbalance in MMP activity in response to repeated injury or mechanical strain may result in tendinopathy (Magra and Maffulli, 2005a,b, 2006; Magra et al., 2007a,b). The cells in a normal tendon are well organised (Longo et al., 2008d). Tenocytes and tenoblasts form up to 95% of the cellular element of the tendon (Ippolito et al., 1980). Specialised fibroblasts, the tenocytes, appear in transverse sections as stellate cells, possibly due to the uniform centrifugal secretion of collagen. Tenoblasts have variable shapes and sizes, and are arranged in long parallel chains (Ippolito et al., 1980). Collagen constitutes about 90% of tendon protein, or approximately 70% of the dry weight of a tendon (JoÂzsa and Kannus, 1997b). The collagen fibres are

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tightly packed in parallel bundles (Khan et al., 1999). Type I collagen is the commonest; it forms 95% of tendon collagen, and is held in parallel bundles by small proteoglycan molecules (Maffulli et al., 1998a). Elastin accounts for only about 2% of the dry mass of tendon (JoÂzsa and Kannus, 1997b) and can undergo up to 200% strain before failure. Ageing significantly decreases tendon glycosaminoglycans and increases collagen concentration (Vailas et al., 1985). Acute exercise increases type I collagen formation in peritendinous tissue (Langberg et al., 1999). The essence of tendinopathy is a failed healing response, with haphazard proliferation of tenocytes, some evidence of degeneration in tendon cells and disruption of collagen fibres, and subsequent increase in non-collagenous matrix (Longo et al., 2009c). Macroscopically, the affected portions of the tendon lose their normal glistening white appearance and become grey-brown and amorphous. Tendon thickening, which can be diffuse, fusiform or nodular, occurs (Khan et al., 1999). A histopathological picture compatible with tendinopathy is often clinically silent, and its only manifestation may be a rupture, but it may also co-exist with symptomatic paratendinopathy. Tendinopathic lesions affect both collagen matrix and tenocytes (Maffulli and Longo, 2008a,b). The parallel orientation of collagen fibres is lost, and there is a decrease in collagen fibre diameter and in the overall density of collagen. Collagen microtears may also occur, and may be surrounded by erythrocytes, fibrin and fibronectin deposits. Normally, collagen fibres in tendons are tightly bundled in a parallel fashion. In tendinopathic samples, there is unequal and irregular crimping, loosening and increased waviness of collagen fibres, with an increase in Type III (reparative) collagen (Jarvinen et al., 1997; Waterston et al., 1997; Maffulli and Kader, 2002; Maffulli et al., 2003a; Carmont and Maffulli, 2007). Frank inflammatory lesions and granulation tissue are infrequent, and are mostly associated with tendon ruptures (Maffulli et al., 2000a, 2007). At electron microscopy, various types of degeneration have been described, namely (a) hypoxic degeneration, (b) hyaline degeneration, (c) mucoid or myxoid degeneration, (d) fibrinoid degeneration, (e) lipoid degeneration, (f) calcification, (g) fibrocartilaginous and bony metaplasia (Perugia et al., 1986; JoÂzsa and Kannus, 1997a; Longo et al., 2006). All can coexist, depending on the anatomical site and the nature of their causal insult. Therefore, tendinopathy can be considered the end result of a number of etiologic processes with a relatively narrow spectrum of histopathological features (Maffulli et al., 1998c, 2004a, 2006). In tendinopathic tendons, tenocytes are abnormally plentiful in some areas (Sharma and Maffulli, 2005c; Magra and Maffulli, 2007a; Maffulli et al., 2008b). They have rounded nuclei, and there is ultrastructural evidence of increased production of proteoglycan and protein which gives them a chondroid appearance. Other areas may contain fewer tenocytes than normal with small, pyknotic nuclei (Jarvinen et al., 1997), with occasional infiltration of

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lymphocytes and macrophage type cells, possibly part of a healing process (Jarvinen et al., 1997) associated with proliferation of capillaries and arterioles. Collagen fibres that are thinner than normal, and large interfibrillar mucoid patches and vacuoles are seen (Maffulli et al., 2004b). There is an increase in the Alcian-blue-staining ground substance. The characteristic hierarchical structure is also lost (Burry and Pool, 1973; Puddu et al., 1976; Harms et al., 1977; Merkel et al., 1982). Vascularity is typically increased, and blood vessels are randomly oriented, sometimes perpendicular to collagen fibres (Clancy et al., 1976; Williams, 1993; Knobloch et al., 2008a,b). Inflammatory lesions (Fox et al., 1975; Clancy et al., 1976; Denstad and Roaas, 1979; Williams, 1993) and granulation tissue (Fox et al., 1975; Denstad and Roaas, 1979) are infrequent, and, when found, are associated with partial ruptures. Inflammatory cells and acellular necrotic areas are exceptional, and probably not typical of the degenerative process (Astrom and Rausing, 1995; Maffulli et al., 1997, 2004b; Sharma and Maffulli, 2006b). On the other hand, mucoid degeneration, fibrosis and vascular proliferation with an inflammatory infiltrate may be found in the paratenon (Clancy et al., 1976; Puddu et al., 1976; Harms et al., 1977; Kvist et al., 1985; Williams, 1993; Maffulli et al., 2000a). Using common staining techniques, light microscopic degeneration was not a feature of tendons from healthy, older persons. Type I collagen is the main collagen in tendons; type III collagen is present in small amounts. We used an in vitro model to determine whether tenocytes from ATs that were ruptured, nonruptured, tendinopathic, and foetal show different behaviour (Maffulli et al., 2000b). In cultures from ruptured and tendinopathic tendons, there was increased production of type III collagen. Athletic participation places excess stress on the AT, which could potentially lead to areas of microtrauma within the tendon. These areas may heal by the production of type III collagen, an abnormal healing response. Accumulation of such episodes of microtrauma could result in a critical point where the resistance of the tissue to tensile forces is compromised and tendon rupture occurs.

16.2.1 Metalloproteases in tendinopathy Tendon matrix constantly remodels, with higher rates of turnover at sites exposed to high levels of strain. Matrix metalloproteases (MMPs), a family of zinc and calcium-dependent endopeptidases active at a neutral pH, are involved in the remodel ling of ECM through their broad proteolytic capability (Sharma and Maffulli, 2005a). Degradation of collagen in tendon ECM is initiated by MMPs. Twenty-three human MMPs have been identified, with a wide range of extracellular substrates (Sharma and Maffulli, 2005b). MMPs can be subdivided into four main groups: collagenases, which cleave native collagen types I, II and III; gelatinases, which cleave denatured collagens and type IV collage; stromelysins, which degrade proteoglycans, fibronectin, casein, collagen types

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III, IV and V; membrane type MMPs. The activity of MMPs is inhibited reversibly by tissue inhibitors of metalloproteinase (TIMPs) in a non-covalent fashion in a 1:1 stoichiometry. There are four types of TIMP: TIMP1, TIMP2, TIMP3 and TIMP4. The balance between the activities of MMPs and TIMPs regulates tendon remodelling, and an imbalance produces collagen disturbances in tendons. MMP3 may play a major role in regulation of tendon ECM degradation and tissue remodelling. An increased expression of MMP3 may be necessary for appropriate tissue remodelling and prevention of tendinopathic changes (Sharma and Maffulli, 2006b). The timing of MMP3 production is probably also critical in this process. MMP3 and TIMP1, TIMP2, TIMP3 and TIMP4 are downregulated in tendinopathic tendons (Sharma and Maffulli, 2006a). Decreased MMP3 expression may therefore lead to tendinopathic changes in tendons. The expression of MMP2 can be upregulated in Achilles tendinopathy. Physical exercise can influence local MMP and TIMP activities in human tendo Achillis with a pronounced increase in local levels of pro-MMP9 after exercise (Sharma and Maffulli, 2006b). MMP9 may well have a role in a potential inflammation reaction in human tendo Achillis induced by intensive exercise. Also, exercise causes a rapid increase in serum MMP9, a probable result of increased leucocytes in the circulation (Sharma and Maffulli, 2006b). MMPs and their inhibitors are crucial to ECM remodelling, and a balance exists between them in normal tendons. Alteration of MMP and TIMP expression from basal levels leads to alteration of tendon homoeostasis. Tendinopathic tendons have an increased rate of matrix remodelling, leading to a mechanically less stable tendon which is more susceptible to damage (Kumar et al., 2000; Kader et al., 2002; Maffulli et al., 2004a; Magra et al., 2007a,b, Magra and Maffulli, 2005a,b, 2006, 2007b, 2008).

16.3

Genetics

A genetic component has been implicated in tendinopathies (Mokone et al., 2005, 2006; Lippi et al., 2009). An underlying genetic factor as a contributing cause to tendon injury was originally proposed because of an association between the ABO blood group and the incidence of AT ruptures or chronic Achilles tendinopathy evident in Hungarian and Finnish populations with blood group O (JoÂzsa et al., 1989a). The distribution of the ABO and Rh blood groups was determined in 832 patients with a tendon rupture. Among these, the frequency of blood group O (52.8%) was significantly higher than in the general population of Hungary (31.1%) and the frequency of group A was significantly lower. Of the 83 cases of multiple ruptures or re-rupture, 57 patients (68.7%) had group O blood. The dominance of group O was found for all sites of tendon rupture, but there was no significant association with the Rh groups. Individuals with blood group O appeared to have an increased risk of tendon rupture (JoÂzsa et al., 1989a).

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The distribution of the ABO blood groups was studied in Finland in 917 patients with specific musculoskeletal diagnoses. The ABO blood group distribution of patients with rupture of the AT (P ˆ 0:030) and of patients with chronic Achilles tendinopathy (P ˆ 0:10) differed from the controls. The ABO blood group distribution was not associated with other musculoskeletal injuries studied. The blood group A/O ratio was 1.42 in the control population. In the group with rupture of the AT this ratio was 1.0, and in the group with Achilles tendinopathy it was 0.70 (Kujala et al., 1992). These studies implied that ABO or closely linked genes on the tip of the long arm of chromosome 9 could be associated with tendinopathy or tendon injuries. The gene for ABO on chromosome 9q34 encode for transferases which, apart from determining the structure of glycoprotein antigens on red blood cells, may also determine the structure of some proteins of the extracellular matrix of tendons (JoÂzsa et al., 1989a). To test whether the association between blood groups and AT rupture reported in Finland and in Hungary was present in Scotland, the distribution of ABO blood groups of 78 patients was compared with that found in 24 501 blood donors typed at the Blood Transfusion Centre during the same period. Overall, 47 of 78 (60%) of patients with an AT rupture belonged to blood group O, compared with 51% of the population as a whole. Only 22 (28%) of the AT rupture patients belonged to blood group A, whereas 35% of the general population were members of this group (NS). The A/O ratio was 0.47 for the tendon rupture patients, compared with 0.68 for the general population. The authors could not demonstrate any significant association between the proportions of ABO blood groups and ATR in the Grampian Region of Scotland (Maffulli et al., 2000c). The findings in other studies could be due to peculiarities in the distribution of the ABO groups in genetically segregated populations (Maffulli et al., 2000c). Polymorphisms within the COL5A1 and tenascin-C (TNC) genes have been associated with AT injuries in a physically active population (Mokone et al., 2005, 2006). The COL5A1 gene, which is in close proximity to the ABO genes on chromosome 9q34 (Caridi et al., 1992) encodes for a structural component of type V collagen which forms heterotypic fibres with type I collagen in tendons and possibly plays an important role in regulating fibrillogenesis and, therefore, tendon strength (Silver et al., 2003; Riley, 2004). COL5A1 gene has a role in the pathogenesis of Achilles tendinopathy and it has been observed that South African individuals with the A2 allele of this gene are less likely to develop AT (Mokone et al., 2006). Although no direct link with COL5A1 gene has been demonstrated, the genes encoding for collagen I and III, namely COL1A1 and COL3A1, show relatively high but variable levels of expression in normal tendon, and significantly increased expression of both genes in painful tendinopathy (Riley, 2005). This correlation needs to be investigated further. TenascinC is a modular and multifunctional ECM glycoprotein that is exquisitely regulated during embryonic development and in adult tissue remodelling (Jones and Jones, 2000b). The TNC gene, which encodes for the ECM glycoprotein

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tenascin-C in tendons, is also closely linked to the ABO genes on chromosome 9q32±q34 (Rocchi et al., 1991). The TNC gene is expressed in regions of the tendon predominantly responsible for transmitting high levels of mechanical force, such as the myotendinous and osteotendinous junctions (Jarvinen et al., 2003), and is regulated by mechanical loading in a dose-dependent manner (Jarvinen et al., 1999, 2003). The tenascin-C protein is involved in regulating cell±matrix interactions (Jones and Jones, 2000a). It interacts with fibronectin, aggrecan, versican, brevican, neurocan, integrins, cell adhesion molecules (CAM) and annexin II (Mokone et al., 2005). Allele distribution of the guanine±thymine (GT) dinucleotide repeat polymorphism in the TNC gene is associated with AT injury (Mokone et al., 2005). Alleles containing 12 and 14 GT repeats were significantly higher in patients with AT injuries, while alleles containing 13 and 17 GT repeats were higher in the asymptomatic controls (Mokone et al., 2005). A possible biological explanation for tenascin-C involvement in the aetiology of AT injuries could be explained by abnormal mechanical loading leading to altered synthesis of TNC (Chiquet et al., 2003), which could disrupt the regulation of cell±matrix interactions in the tendon (Jones and Jones, 2000a), with onset of apoptotic changes in the tenocytes (Murrell, 2002). The exact role of COL5A1 and TNC genes in the pathogenesis of tendinopathy is still debated, and the current evidence does not allow to clarify whether COL5A1 and TNC genes are ideal markers of tendinopathy (Mokone et al., 2006). A negative association of a particular gene, such as with the ABO system (Maffulli et al., 2000c), does not necessarily mean that there is absolutely no association with that particular gene(s) at that locus. The fact that certain studies have found an association with the ABO system and tendon injuries (Leppilahti et al., 1996; Maffulli et al., 2000c) warrants further investigation at that particular locus. It is also possible that other genes, yet to be determined, may contribute towards the pathogenesis of tendinopathy, which could be a polygenic condition, given the multitude of the genes involved in maintaining normal tendon homeostasis. Based on current evidence, it is difficult to conceive that only a single gene and not multiple genes are involved in the pathogenesis of Achilles tendinopathy. Thus, additional investigation needs to be performed to identify these genes (Smith et al., 2002; Mokone et al., 2005, 2006).

16.4

Tendon rupture

Tendon rupture is an acute injury in which extrinsic factors predominate, although intrinsic factors are also important. In rotator cuff tears we showed that normal, but in the high range of normal, increasing plasma glucose levels may be a risk factor for rotator cuff tear (Longo et al., 2009a). We were not able to demonstrate the same association of rotator cuff tears with triglycerides and total serum cholesterol (Longo et al., 2009b).

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In another study (Longo et al., 2007b), we investigated supraspinatus tendon samples obtained from patients undergoing arthroscopic repair of a rotator cuff tear to examine the distribution of tendinopathic changes associated with this condition. At arthroscopy, a full thickness supraspinatus tendon biopsy was harvested close to the tear edge. We found more frequent tendon changes on the articular side of the rotator cuff. We found also more cartilage-like changes in patients affected by rotator cuff tears, but not in our control group. Recent biomechanical data suggest that the stress-shielded and transversely compressed side of the enthesis has a distinct tendency to develop cartilage-like or atrophic changes in response to the lack of tensile load (Maffulli et al., 1998c, 2004a, 2006; Gardner et al., 2008). Over a long period, this process may develop into a failed healing response lesion in that area of the tendon. This may explain why tendinopathy is not always clearly activity-related, but more strongly correlated with age. In this manner, it could almost be considered an `underuse' injury rather than an overuse injury as a result of stress-shielding (Maffulli et al., 1998c, 2004a, 2006). The formation of cartilage-like changes in the enthesis in many ways can be considered a physiological adaptation to the compressive loads (Maffulli et al., 2002, 2003b, 2004b). It may not allow the tendon to maintain its ability to withstand high tensile loads in that region of the tendon. As the stress-shielding may have led to tensile weakening over time, an injury may occur more easily in this region. In this manner, insertional tendinopathy could be considered an overuse injury, but predisposed by pre-existing weakening of the tendon (Maffulli et al., 2002, 2003b, 2004b). In another study (Longo et al., 2008a) to evaluate the histopathological features of macroscopic intact tendon portion of patients with rotator cuff tears, we demonstrated that the supraspinatus tendons of patients undergoing arthroscopic repair for a rupture show profound histopathologic changes, while the tendons of aged persons with no known tendon abnormalities have, as a group, little histological evidence of pathological changes. Moreover, tendon changes are not only localised at the site of rupture, but also in the macroscopic intact tendon portion. We found the same histopathological features in tendon samples obtained from patients with lesions of the tendon of the long head of the biceps brachii (Longo et al., 2007b). Relatively few studies have tried to quantify the histopathological findings of tendinopathy, and the histopathological changes are currently described in a subjective or at best semiquantitative fashion (Astrom and Rausing, 1995; Movin et al., 1997; Maffulli et al., 2000a,b; Khan et al., 1999). This may result in uncertainty about the histopathological findings of tendinopathy, and has produced a lack of diagnostic uniformity among surgical pathologists. Ideally, the pathological diagnoses in different studies should follow an accepted classification scheme, thus allowing data comparison and combination. We are aware of two scoring systems that can be used for classification of the histopathological findings of tendinopathy: the Movin score (Movin et al.,

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1997) with its validated modifications (Maffulli et al., 2000a,b, Longo et al., 2008a) and the Bonar score (Cook et al., 2004). We compared the reliability of these two different histopathological evaluation scores of tendon tissue of rotator cuff tears (Maffulli et al., 2008a). Movin's and Bonar's scores have a high correlation, and assess similar characteristics and variables of tendon pathology. Macroscopically intact supraspinatus tendon may show profound light microscopy changes. These changes may be the pathogenic precursor to a subsequent rotator cuff tear In Achilles tendon rupture, an acceleration/deceleration mechanism has been reported in up to 90% of sports-related injuries (Soldatis et al., 1997). Malfunction of the normal protective inhibitory pathway of the musculo-tendinous unit may result in injury (Inglis et al., 1976). The aetiology of tendon rupture remains unclear. Degenerative tendinopathy is the most common histological finding in spontaneous tendon ruptures. Arner et al. first reported degenerative changes in all their patients with Achilles tendon rupture, and hypothesised that these changes were due to intrinsic abnormalities present before the rupture (Arner et al., 1959). Kannus and JoÂzsa found degenerative changes in 865 of 891 (97%) spontaneous tendon ruptures, whilst degenerative changes were only seen in 149 of 445 (34%) of control tendons (Kannus and JoÂzsa, 1991). Tendon degeneration may lead to reduced tensile strength and a predisposition to rupture. Indeed, ruptured Achilles tendons have a more advanced histological picture of failed healing response than chronically painful tendons from overuse injuries (Tallon et al., 2001). There is little agreement with regard to aetiology of Achilles tendon ruptures (Maffulli et al., 1998b), which have been attributed to many factors, including poor tendon vascularity and collagen disruption (Williams, 1993; Maffulli, 1995), gastrocnemius-soleus dysfunction, a suboptimally conditioned musculotendinous unit, age, gender, changes in training pattern, poor technique, previous injuries and footwear (Inglis and Sculco, 1981; Clain and Baxter, 1992). They have also been associated with a multitude of conditions, such as inflammatory and autoimmune conditions, hyperuricaemia, genetically determined collagen abnormalities (Dent and Graham, 1991), infectious diseases, neurological conditions (Maffulli, 1996), hyperthyroidism, renal insufficiency and arteriosclerosis (Myerson, 1999). High serum lipid concentrations have been reported in patients with complete ruptures of the Achilles tendon (Mathiak et al., 1999; Ozgurtas et al., 2003). Although there is uptake and excretion of sterols by the enzyme sterol 27-hydroxylase (CYP27A1) in the Achilles tendon (von Bahr et al., 2002), histopathological evidence of lipomatosis was only found in 6% of specimens from Achilles tendon ruptures (Kannus and JoÂzsa, 1991). Further, patients with familiar hypercholesterolemia and Achilles tendon xanthomata do not appear to be at greater risk of ruptures, although it should be acknowledged that their activity levels are probably not high.

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Two main theories are advocated, the `degenerative theory' and the `mechanical theory'. According to the degenerative theory, chronic degeneration of the tendon leads to a rupture without excessive loads being applied. Degenerative changes can result from several factors, including physiological alterations in the tendon, chronic over-loading with microtraumata, pharmacological treatment and in association with other diseases.

16.4.1 Degenerative theory The events leading to a rupture are unclear (Carden et al., 1987; Campani et al., 1990; Karousou et al., 2008). Normal tendons would not rupture when subjected to large strains (Carden et al., 1987). Arner et al. (1959) first reported degenerative changes in all their 74 patients with Achilles tendon rupture, and hypothesised that these changes were primary abnormalities present before the rupture. However, nearly two-thirds of the specimens were obtained more than 2 days post-rupture. Davidsson and Salo (1969) reported marked degenerative changes in two Achilles tendon rupture patients operated on the day of injury. The changes should therefore be regarded as having developed prior to the rupture (JoÂzsa et al., 1991). In our own series, all tendons operated within 24 hours from the injury showed marked degenerative changes and collagen disruption (Jarvinen et al., 1997; JoÂzsa and Kannus, 1997a). Several authors detected degenerative intratendinous alterations in the spontaneously ruptured tendons from all sites studied. Most of these abnormalities had no aetiological explanation (JoÂzsa and Kannus, 1997a). Probably, alterations in blood flow with subsequent hypoxia and impaired metabolism were factors in the development of the degenerative changes observed (Kannus and JoÂzsa, 1991). Alternating exercise with inactivity could produce the histopathological changes observed in tendons. Sport places additional stress on tendons, leading to accumulation of microtrauma, which, although below the threshold for frank rupture, could lead to secondary intratendinous degenerative changes (Fox et al., 1975). Kannus and JoÂzsa evaluated biopsy specimens of patients with spontaneous Achilles tendon ruptures harvested at the time of repair. Only one-third of control tendons had similar changes, but significantly less frequently (Kannus and JoÂzsa, 1991). They also noted that only relatively small proportion of the patients had symptoms prior to the rupture. They suggested that there are clear indications that, at least in urban populations, tendinopathic changes are common in the tendons of subjects older than 35 and that these changes can be associated with spontaneous rupture (Kannus and JoÂzsa, 1991). In our centre, in the 176 Achilles tendon ruptures treated from January 1990 to December 1996, we found that only 9 (5%) patients had had previous symptoms (Waterston et al., 1997). Failure of the cellular matrix may also lead to intratendinous degeneration. JoÂzsa et al. observed fibronectin on the torn surfaces of ruptured Achilles

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tendons. Fibronectin is normally located in basement membranes, is present in a soluble form in plasma, and binds more readily to denatured collagen than to normal collagen, indicating pre-existing collagen denaturation (JoÂzsa et al., 1989b; Lehto et al., 1990). We demonstrated that tenocytes from ruptured and tendinopathic tendons show increased production of type III collagen, which disturbs the tissue architecture, and makes the tissue less resistant to tensile forces (Waterston et al., 1997).

16.4.2 The mechanical theory Damage to the tendon can occur even though the tendon itself is stressed within the physiological threshold when the frequent cumulative microtrauma applied do not leave enough time for repair (Selvanetti et al., 1997; Hayes et al., 2003; Longo et al., 2008b). Barfred (1971) demonstrated that complete ruptures can occur in a healthy tendon, the greatest risk being present when the tendon was obliquely loaded at a short initial length with maximum muscle contraction. Such factors are probably all present in movements occurring in many sports which require rapid push-off. A healthy tendon may rupture after a violent muscular strain in the presence of certain functional and anatomical conditions, including incomplete synergism of agonist muscle contractions, inefficient action of the plantaris muscle acting as a tensor of the Achilles tendon, and a discrepancy in the thickness quotient between muscle and tendon. Inglis and Sculco (1981) suggested that a malfunction in the inhibitory mechanism that prevents excessive or uncoordinated muscle contractions could cause a rupture of an otherwise normal tendon. Athletes who return to training too quickly after a period of inactivity seem to be at greater risk of a rupture due to this mechanism. The risk of rupture of the Achilles tendon is further increased if an oblique stress is applied during inversion or eversion of the subtalar joint. Participation in sports plays a major rule in the development of problems with the Achilles tendon, and training errors are a major factor (Clain and Baxter, 1992). The flared heel on most sport shoes forces the hindfoot into pronation when the heel strikes the ground, and the heel tabs on some shoes may play a similar role. In a study of 109 runners, Clement showed that Achilles tendon injury may result from structural or dynamic disturbances in normal lower leg mechanics such as overtraining, functional overpronation, and gastrocnemius/ soleus insufficiency. They also speculated that repeated microtrauma produced by the eccentric loading of fatigued muscle may play an important role in tendon injury (Clement et al., 1984). Complete rupture is the consequence of multiple micro-ruptures which lead to failure of the tendon after reaching a critical point (Knorzer et al., 1986).

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16.4.3 Drug-related tendon rupture Anabolic steroids and fluoroquinolones have been related to tendon rupture. Both drugs cause dysplasia of collagen fibrils, which decreases tendon tensile strength. Systemic and local corticosteroids have been widely implicated in tendon rupture (Dickey and Patterson, 1987; Newnham et al., 1991). However, studies on the patella tendon show that normal tendon is not damaged by intratendinous injection of steroids (Matthews et al., 1974). Most of the available evidence suggests that intratendinous or peritendinous injection of corticosteroids into an injured tendon may precipitate a rupture (Unverferth and Olix, 1973; Matthews et al., 1974). The aetiological role of corticosteroids in tendon rupture has not been fully clarified. However, available evidence discourages prolonged oral administration and repeated peritendinous administration of corticosteroids. The antiinflammatory and analgesic properties of corticosteroids may mask the symptoms of tendon damage (DiStefano and Nixon, 1973), inducing individuals to maintain their high activity levels even when the tendon is damaged. Corticosteroids interfere with healing, and intratendinous injection of corticosteroids results in weakening of the tendon for up to 14 days post-injection. The disruption is directly related to collagen necrosis, and restoration of tendon strength is attributable to the formation of a cellular amorphous mass of collagen. For these reasons, vigorous activity should be avoided for at least 2 weeks following injection of corticosteroids in the vicinity of a tendon (Kennedy and Willis, 1976). Fluoroquinolones (4-quinolone) antibiotics such as ciprofloxacin have recently been implicated in the aetiology of tendon rupture. Pefloxacin, ofloxacin, levofloxacin, norfloxacin and ciprofloxacin are the fluoroquinolones most often associated with tendon disorders. Nalidixic acid is a quinolone not as toxic to the tendon as the fluorinated quinolones. In contrast to fluoroquinolones, nalidixic acid does not alter the mytocondrial activity in tenocytes (BernardBeaubois et al., 1998). In France, between 1985 and 1992, 100 patients taking fluoroquinolones suffered tendon disorders, including 31 ruptures (Royer et al., 1994). Many of them had also received corticosteroids, making it difficult to solely implicate the fluoroquinolones. Szarfman et al. (1995) reported animal studies with fluoroquinolone doses close to those administered to humans, and showed disruption of the ECM of cartilage, chondrocyte necrosis and depletion of collagen. The abnormalities seen in animals might also occur in humans. These authors recommended updating the labelling on fluoroquinolone packing to include a warning about the possibility of tendon rupture. Recently, laboratory evidence for the direct deleterious effects of fluoroquinolones on tenocytes has been produced (Bernard-Beaubois et al., 1998).

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16.4.4 Hyperthermia and tendon rupture Up to 10% of the elastic energy stored in tendons may be released as heat (Ker, 1981). Wilson and Goodship (1994) evaluated in vivo the temperatures generated within equine superficial digital flexor tendons during exercise. A peak temperature of 45 ëC, at which tenocytes can be damaged, was measured within the core of the tendon after just 7 minutes of trotting (Arancia et al., 1989). Exercise-induced hyperthermia may therefore contribute to tendon degeneration. As good blood supply to a tissue should prevent overheating, tissues such as the tendon, with relatively avascular areas, may be more susceptible to the effects of hyperthermia.

16.5

Pain in tendinopathy

Classically, pain in tendinopathy has been attributed to inflammation. However, chronically painful Achilles and patellar tendons show no evidence of inflammation, and many tendons with intratendinous pathology detected on magnetic resonance imaging (MRI) or ultrasound are not painful (Khan et al., 1999). Pain may originate from a combination of mechanical and biochemical causes (Khan et al., 1999). Tendon degeneration with mechanical breakdown of collagen could theoretically explain the pain, but clinical and surgical observations challenge this view (Khan et al., 1999). Chemical irritants and neurotransmitters may generate pain in tendinopathy. Microdialysis sampling revealed a two-fold increase in lactate levels in tendinopathic tendons compared with controls (Alfredson et al., 2002). Patients with chronic Achilles tendinopathy and patellar tendinopathy show high concentrations of the neurotransmitter glutamate, with no statistically significant elevation of the proinflammatory prostaglandin PG E2 (Alfredson et al., 2002). However, the levels of PG E2 were consistently higher in tendinopathic tendons compared with controls, and the results possibly lacked statistical significance owing to the small sample size of the study. Substance P functions as a neurotransmitter and neuromodulator, and is found in small unmyelinated sensory nerve fibres (Zubrzycka and Janecka, 2000). A network of sensory innervation is present in tendons (Ackermann et al., 1999). Sensory nerves transmit nociceptive information to the spinal cord, and increased levels of substance P correlate with pain levels in rotator cuff disease and medial and lateral epicondylopathy (Gotoh et al., 1998). An opioid system exists in the Achilles tendon of rats (Ackermann et al., 2001b). Under normal conditions, a balance probably exists between nociceptive and anti-nociceptive peptides (Brodin et al., 1983). However, this balance may be altered in pathological conditions (Brodin et al., 1983).

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407

Tendon healing following acute injuries

Tendon healing studies have predominantly been performed on transected animal tendons or ruptured human tendons, and their relevance to human tendinopathy with its associated healing failure response remains unclear (Sharma and Maffulli, 2005a). Tendon healing occurs in three overlapping phases. In the initial inflammatory phase, erythrocytes and inflammatory cells, particularly neutrophils, enter the site of injury. In the first 24 hours, monocytes and macrophages predominate, and phagocytosis of necrotic materials occurs. Vasoactive and chemotactic factors are released with increased vascular permeability, initiation of angiogenesis, stimulation of tenocyte proliferation, and recruitment of more inflammatory cells. Tenocytes gradually migrate to the wound, and type III collagen synthesis is initiated. After a few days, the remodelling stage begins. Synthesis of type III collagen peaks during this stage, which lasts for a few weeks. Water content and glycosaminoglycan concentrations remain high during this stage. After approximately 6 weeks, the modelling stage commences. During this stage, the healing tissue is resized and reshaped. A corresponding decrease in cellularity, collagen and glycosaminoglycan synthesis occurs. The modelling phase can be divided into a consolidation and maturation stage. The consolidation stage commences at about 6 weeks and continues up to 10 weeks. In this period, the repair tissue changes from cellular to fibrous. Tenocyte metabolism remains high during this period, and tenocytes and collagen fibres become aligned in the direction of stress. A higher proportion of type I collagen is synthesised during this stage (Abrahamsson, 1991). After 10 weeks, the maturation stage occurs, with gradual change of fibrous tissue to scar-like tendon tissue over the course of a year. During the latter half of this stage, tenocyte metabolism and tendon vascularity decline (Amiel et al., 1983). Tendon healing can occur intrinsically, via proliferation of epitenon and endotenon tenocytes, or extrinsically, by invasion of cells from the surrounding sheath and synovium (Gelberman et al., 1986). Epitenon tenoblasts initiate the repair process through proliferation and migration (Manske et al., 1985). Healing in severed tendons can be performed by cells from the epitenon alone, without relying on adhesions for vascularity or cellular support (Gelberman et al., 1984). Internal tenocytes contribute to the intrinsic repair process and secrete larger and more mature collagen than epitenon cells (Field, 1971). Despite this, fibroblasts in the epitenon and tenocytes synthesise collagen during repair, and different cells probably produce different collagen types at different time points. Initially, collagen is produced by epitenon cells, with endotenon cells later synthesising collagen (Ingraham et al., 2003). The relative contribution of each cell type may be influenced by the type of trauma sustained, anatomical position, presence of a synovial sheath, and the amount of stress induced by motion after repair has taken place (Koob, 2002). Tenocyte function may vary

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depending on the region of origin. Cells from the tendon sheath produce less collagen and GAG than epitenon and endotenon cells. However, fibroblasts from the flexor tendon sheath proliferate more rapidly. The variation in phenotypic expression of tenocytes has not been extensively investigated, and this information may prove useful for optimising repair strategies. Intrinsic healing results in improved biomechanics and fewer complications. In particular, a normal gliding mechanism within the tendon sheath is preserved (Koob, 2002). In extrinsic healing, scar tissue results in adhesion formation which disrupts tendon gliding. Different healing patterns may predominate in particular locations, and, for example, extrinsic healing tends to prevail in torn rotator cuffs (Uhthoff and Sarkar, 1991).

16.6.1 Remodelling responses The histopathological process at the basis of the clinical manifestations of tendinopathy then can be viewed as a failure of cell matrix adaptation to a variety of stresses, due to an imbalance between matrix degeneration and synthesis (Leadbetter, 1992). Remodelling plays an important role in responding to microtrauma from repetitive loading. This repair mechanism is probably mediated by resident tenocytes, which maintain a fine balance between ECM production and degradation. Modelling is also involved in the physiological response of tendon to resistance training. In such situations, modelling adapts the tendon to the mechanical loads placed on it, and prevents the tendons from incurring injuries. An increase in the tendon mass and cross-sectional area occurs during modelling.

16.6.2 Modulators of healing MMPs are important regulators of ECM remodelling, and their levels are altered during tendon healing. In a rat flexor tendon laceration model, the expression of MMP-9 and MMP-13 (collagenase-3) peaked between days 7 and 14. MMP-2, MMP-3 and MMP-14 (MT1-MMP) levels increased after surgery, and remained high until day 28 (Oshiro et al., 2003). These findings suggest that MMP-9 and MMP-13 participate only in collagen degradation, whereas MMP-2, MMP-3 and MMP-14 participate in both collagen degradation and collagen remodelling. Wounding and inflammation also provoke the release of growth factors and cytokines from platelets, polymorphonuclear leucocytes, macrophages and other inflammatory cells (Evans et al., 1990). These growth factors induce neovascularisation and chemotaxis of fibroblasts and tenocytes and stimulate fibroblast and tenocytes proliferation and synthesis of collagen (Molloy et al., 2003). Nitric oxide is a short-lived free radical, with many biological functions: it is bactericidal, can induce apoptosis in inflammatory cells, and causes

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angiogenesis and vasodilation (Ziche et al., 1994). Nitric oxide may play a role in several aspects of tendon healing. Nitric oxide synthase is responsible for synthesising nitric oxide from L-arginine. Experimental studies have shown that levels of nitric oxide synthase peak after 7 days and return to baseline 14 days after tenotomy of rat Achilles tendons (Murrell et al., 1997). Inhibition of nitric oxide synthase reduced healing and resulted in decreased cross-sectional area and a reduced failure load (Murrell et al., 1997; Longo et al., 2008c). In that study, the specific isoforms of nitric oxide synthase were not identified. More recently, the same group has demonstrated a temporal expression of the three isoforms of nitric oxide synthase (Lin et al., 2001). The inducible isoform peaks at day 4, the endothelial isoform peaks at day 7, and the neuronal isoform peaks at day 21 (Lin et al., 2001). Interestingly, in a rat Achilles tendon rupture model, peak nerve fibre formation occurred between weeks 2 and 6, in concert with peak levels of the neuronal isoform of nitric oxide synthase (Ackermann et al., 1999, 2001a). These nerve fibres presumably deliver neuropeptides, which act as chemical messengers and regulators, and may play an important role in tendon healing. Substance P and calcitonin generelated peptide (CGRP) are pro-inflammatory and cause vasodilation and protein extravasation (Ackermann et al., 1999, 2001a,b). In addition, Substance P enhances cellular release of prostaglandins, histamines and cytokines. Peak levels of substance P and CGRP occur during the proliferative phase, suggesting a possible role during this phase.

16.6.3 Limitations of healing in acute tendon injuries Adhesion formation after intrasynovial tendon injury poses a major clinical problem (Manske et al., 1984, 1985). Synovial sheath disruption at the time of injury or surgery allows granulation tissue and tenocytes from surrounding tissue to invade the repair site. Exogenous cells predominate over endogenous tenocytes, allowing the surrounding tissues to attach to the repair site, resulting in adhesion formation. Despite remodelling, the biochemical and mechanical properties of healed tendon tissue never match those of intact tendon. In spontaneously healed transected sheep Achilles tendons, rupture force was only 56.7% of normal at 12 months (Bruns et al., 2000). One possible reason for this may be the absence of mechanical loading during the period of immobilisation. The degree and extent of adhesions have been classified by Siegler et al. (1980): Grade 0, complete absence of adhesions; grade I, thin avascular, filmy, and easily separable; grade II, thick, avascular, and limited to the site of anastomosis; grade III, thick, vascular, and extensive. This classification has been used for evaluation of severity of adhesion formation in various human and animal models. With increasing information available concerning the nature of the scar tissue responsible for the peritendinous adhesions, along with changes in surgical and

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post-operative rehabilitation techniques, several modalities, such as modulation of inflammatory response and growth factors that promote scarring by various pharmacological agents, introduction of mechanical barriers between the tendons and the proliferating tissue, use of ultrasound and electromagnetic therapy and, recently, gene therapy are being explored. To date, the only thing that appears clinically justified in adhesion prevention is the need for early postoperative mobilisation of digits after tendon injury or repair but the best method of mobilisation remains controversial (Khanna et al., 2009).

16.7

Conclusions

Tendon injuries give rise to substantial morbidity, and current understanding of the mechanisms involved in tendon injury and repair is limited. Further research is required to improve our knowledge of tendon healing. This will enable specific treatment strategies to be developed (Sharma and Maffulli, 2005b).

16.8

References

Abrahamsson, S. O. (1991) Matrix metabolism and healing in the flexor tendon. Experimental studies on rabbit tendon. Scand J Plast Reconstr Surg Hand Surg Suppl, 23, 1±51. Ackermann, P. W., Finn, A. and Ahmed, M. (1999) Sensory neuropeptidergic pattern in tendon, ligament and joint capsule. A study in the rat. Neuroreport, 10, 2055±60. Ackermann, P. W., Li, J., Finn, A., Ahmed, M. and Kreicbergs, A. (2001a) Autonomic innervation of tendons, ligaments and joint capsules. A morphologic and quantitative study in the rat. J Orthop Res, 19, 372±8. Ackermann, P. W., Spetea, M., Nylander, I., Ploj, K., Ahmed, M. and Kreicbergs, A. (2001b) An opioid system in connective tissue: a study of achilles tendon in the rat. J Histochem Cytochem, 49, 1387±95. Alfredson, H., Bjur, D., Thorsen, K., Lorentzon, R. and Sandstrom, P. (2002) High intratendinous lactate levels in painful chronic Achilles tendinosis. An investigation using microdialysis technique. J Orthop Res, 20, 934±8. Ames, P. R., Longo, U. G., Denaro, V. and Maffulli, N. (2008) Achilles tendon problems: not just an orthopaedic issue. Disabil Rehabil, 30, 1646±50. Amiel, D., Akeson, W. H., Harwood, F. L. and Frank, C. B. (1983) Stress deprivation effect on metabolic turnover of the medial collateral ligament collagen. A comparison between nine- and 12-week immobilization. Clin Orthop Relat Res, 172, 265±70. Arancia, G., Crateri Trovalusci, P., Mariutti, G. and Mondovi, B. (1989) Ultrastructural changes induced by hyperthermia in Chinese hamster V79 fibroblasts. Int J Hyperthermia, 5, 341±50. Arner, O., Lindholm, A. and Orell, S. R. (1959) Histologic changes in subcutaneous rupture of the Achilles tendon; a study of 74 cases. Acta Chir Scand, 116, 484±90. Astrom, M. and Rausing, A. (1995) Chronic Achilles tendinopathy. A survey of surgical and histopathologic findings. Clin Orthop Relat Res, 151±64. Barfred, T. (1971) Experimental rupture of the Achilles tendon. Comparison of various types of experimental rupture in rats. Acta Orthop Scand, 42, 528±43.

Tendon injury and repair mechanics

411

Bernard-Beaubois, K., Hecquet, C., Hayem, G., Rat, P. and Adolphe, M. (1998) In vitro study of cytotoxicity of quinolones on rabbit tenocytes. Cell Biol Toxicol, 14, 283± 92. Bestwick, C. S. and Maffulli, N. (2004) Reactive oxygen species and tendinopathy: do they matter? Br J Sports Med, 38, 672±4. Brodin, E., Gazelius, B., Panopoulos, P. and Olgart, L. (1983) Morphine inhibits substance P release from peripheral sensory nerve endings. Acta Physiol Scand, 117, 567±70. Bruns, J., Kampen, J., Kahrs, J. and Plitz, W. (2000) Achilles tendon rupture: experimental results on spontaneous repair in a sheep-model. Knee Surg Sports Traumatol Arthrosc, 8, 364±9. Burry, H. C. and Pool, C. J. (1973) Central degeneration of the achilles tendon. Rheumatol Rehabil, 12, 177±81. Campani, R., Bottinelli, O., Genovese, E., Bozzini, A., Benazzo, F., Barnabei, G., Jelmoni, G. P. and Carella, E. (1990) [The role of echotomography in sports traumatology of the lower extremity]. Radiol Med (Torino), 79, 151±62. Carden, D. G., Noble, J., Chalmers, J., Lunn, P. and Ellis, J. (1987) Rupture of the calcaneal tendon. The early and late management. J Bone Joint Surg Br, 69, 416± 20. Caridi, G., Pezzolo, A., Bertelli, R., Gimelli, G., Di Donato, A., Candiano, G. and Ghiggeri, G. M. (1992) Mapping of the human COL5A1 gene to chromosome 9q34.3. Hum Genet, 90, 174±6. Carmont, M. R. and Maffulli, N. (2007) Less invasive Achilles tendon reconstruction. BMC Musculoskelet Disord, 8, 100. Chiquet, M., Renedo, A. S., Huber, F. and Fluck, M. (2003) How do fibroblasts translate mechanical signals into changes in extracellular matrix production? Matrix Biol, 22, 73±80. Clain, M. R. and Baxter, D. E. (1992) Achilles tendinitis. Foot Ankle, 13, 482±7. Clancy, W. G. J., Neidhart, D. and Brand, R. L. (1976) Achilles tendonitis in runners: A report of cases. Am J Sports Med, 4, 46±57. Clement, D. B., Taunton, J. E. and Smart, G. W. (1984) Achilles tendinitis and peritendinitis: etiology and treatment. Am J Sports Med, 12, 179±84. Cook, J., Feller, J., Bonar, S. and Khan, K. (2004) Abnormal tenocyte morphology is more prevalent than collagen disruption in asymptomatic athletes' patellar tendons. J Orthop Res, 22, 334±8. Davidsson, L. and Salo, M. (1969) Pathogenesis of subcutaneous tendon ruptures. Acta Chir Scand, 135, 209±12. Denstad, T. F. and Roaas, A. (1979) Surgical treatment of partial Achilles tendon rupture. Am J Sports Med, 7, 15±17. Dent, C. M. and Graham, G. P. (1991) Osteogenesis imperfecta and Achilles tendon rupture. Injury, 22, 239±40. Dickey, W. and Patterson, V. (1987) Bilateral Achilles tendon rupture simulating peripheral neuropathy: unusual complication of steroid therapy. J R Soc Med, 80, 386±7. Distefano, V. J. and Nixon, J. E. (1973) Ruptures of the achilles tendon. J Sports Med, 1, 34±7. Evans, E. J., Benjamin, M. and Pemberton, D. J. (1990) Fibrocartilage in the attachment zones of the quadriceps tendon and patellar ligament of man. J Anat, 171, 155±62. Field, P. L. (1971) Tendon fibre arrangement and blood supply. Aust N Z J Surg, 40, 298± 302.

412

Regenerative medicine and biomaterials in connective tissue repair

Fox, J. M., Blazina, M. E., Jobe, F. W., Kerlan, R. K., Carter, V. S., Shields, C. L., Jr and Carlson, G. J. (1975) Degeneration and rupture of the Achilles tendon. Clin Orthop Relat Res, 107, 221±4. Gardner, K., Arnoczky, S. P., Caballero, O. and Lavagnino, M. (2008) The effect of stress-deprivation and cyclic loading on the TIMP/MMP ratio in tendon cells: An in vitro experimental study. Disabil Rehabil, 30(20-2), 1523±9. Gelberman, R. H., Manske, P. R., Vande Berg, J. S., Lesker, P. A. and Akeson, W. H. (1984) Flexor tendon repair in vitro: a comparative histologic study of the rabbit, chicken, dog, and monkey. J Orthop Res, 2, 39±48. Gelberman, R. H., Manske, P. R., Akeson, W. H., Woo, S. L., Lundborg, G. and Amiel, D. (1986) Flexor tendon repair. J Orthop Res, 4, 119±28. Gotoh, M., Hamada, K., Yamakawa, H., Inoue, A. and Fukuda, H. (1998) Increased substance P in subacromial bursa and shoulder pain in rotator cuff diseases. J Orthop Res, 16, 618±21. Harms, J., Biehl, G. and Von Hobach, G. (1977) Pathologie der Paratenonitis achillea bei Hochleistungssportlern. Arch Orthop Unfallchir, 88, 65±74. Hayes, T., McClelland, D. and Maffulli, N. (2003) Metasynchronous bilateral Achilles tendon rupture. Bull Hosp Jt Dis, 61, 140±4. Inglis, A. E. and Sculco, T. P. (1981) Surgical repair of ruptures of the tendo Achillis. Clin Orthop Relat Res, 156, 160±9. Inglis, A. E., Scott, W. N., Sculco, T. P. and Patterson, A. H. (1976) Ruptures of the tendo achillis. An objective assessment of surgical and non-surgical treatment. J Bone Joint Surg Am, 58, 990±3. Ingraham, J. M., Hauck, R. M. and Ehrlich, H. P. (2003) Is the tendon embryogenesis process resurrected during tendon healing? Plast Reconstr Surg, 112, 844±54. Ippolito, E., Natali, P. G., Postaccinni, F., Accinni, L. and De Martino, C. (1980) Morphological, immunochemical, and biochemical study of rabbit achilles tendon at various ages. J Bone Joint Surg Am, 62, 583±98. Jarvinen, M., JoÂzsa, L., Kannus, P., Jarvinen, T. L., Kvist, M. and Leadbetter, W. (1997) Histopathological findings in chronic tendon disorders. Scand J Med Sci Sports, 7, 86±95. Jarvinen, T. A., JoÂzsa, L., Kannus, P., Jarvinen, T. L., Kvist, M., Hurme, T., Isola, J., Kalimo, H. and Jarvinen, M. (1999) Mechanical loading regulates tenascin-C expression in the osteotendinous junction. J Cell Sci, 112 Pt 18, 3157±66. Jarvinen, T. A., JoÂzsa, L., Kannus, P., Jarvinen, T. L., Hurme, T., Kvist, M., PeltoHuikko, M., Kalimo, H. and Jarvinen, M. (2003) Mechanical loading regulates the expression of tenascin-C in the myotendinous junction and tendon but does not induce de novo synthesis in the skeletal muscle. J Cell Sci, 116, 857±66. Jones, F. S. and Jones, P. L. (2000a) The tenascin family of ECM glycoproteins: structure, function, and regulation during embryonic development and tissue remodeling. Dev Dyn, 218, 235±59. Jones, P. L. and Jones, F. S. (2000b) Tenascin-C in development and disease: gene regulation and cell function. Matrix Biol, 19, 581±96. JoÂzsa, L. and Kannus, P. (1997a) Histopathological findings in spontaneous tendon ruptures. Scand J Med Sci Sports, 7, 113±18. JoÂzsa, L. and Kannus, P. (1997b) Human Tendon: Anatomy, physiology and pathology. Human Kinetics, Champaign, IL. JoÂzsa, L., Balint, J. B., Kannus, P., Reffy, A. and Barzo, M. (1989a) Distribution of blood groups in patients with tendon rupture. An analysis of 832 cases. J Bone Joint Surg Br, 71, 272±4.

Tendon injury and repair mechanics

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JoÂzsa, L., Lehto, M., Kannus, P., Kvist, M., Reffy, A., Vieno, T., Jarvinen, M., Demel, S. and Elek, E. (1989b) Fibronectin and laminin in Achilles tendon. Acta Orthop Scand, 60, 469±71. JoÂzsa, L., Kannus, P., Balint, J. B. and Reffy, A. (1991) Three-dimensional ultrastructure of human tendons. Acta Anat (Basel), 142, 306±12. Kader, D., Saxena, A., Movin, T. and Maffulli, N. (2002) Achilles tendinopathy: some aspects of basic science and clinical management. Br J Sports Med, 36, 239±49. Kannus, P. and JoÂzsa, L. (1991) Histopathological changes preceding spontaneous rupture of a tendon. A controlled study of 891 patients. J Bone Joint Surg Am, 73, 1507±25. Karousou, E., Ronga, M., Vigetti, D., Passi, A. and Maffuli, N. (2008) Collagens, proteoglycans, MMP-2, MMP-9 and TIMPs in human achilles tendon rupture. Clin Orthop Relat Res, 466, 1577±82. Kennedy, J. C. and Willis, R. B. (1976) The effects of local steroid injections on tendons: a biomechanical and microscopic correlative study. Am J Sports Med, 4, 11±21. Ker, R. F. (1981) Dynamic tensile properties of the plantaris tendon of sheep (Ovis aries). J Exp Biol, 93, 283±302. Khan, K. M., Cook, J. L., Bonar, F., Harcourt, P. and Astrom, M. (1999) Histopathology of common tendinopathies. Update and implications for clinical management. Sports Med, 27, 393±408. Khanna, A., Friel, M., Gougoulias, N., Longo, U. G. and Maffulli, N. (2009) Prevention of adhesions in surgery of the flexor tendons of the hand: what is the evidence? Br Med Bull, 90, 85±109. Knobloch, K., Schreibmueller, L., Longo, U. G. and Vogt, P. M. (2008a) Eccentric exercises for the management of tendinopathy of the main body of the Achilles tendon with or without an AirHeel Brace. A randomized controlled trial. B: Effects of compliance. Disabil Rehabil, 30, 1692±6. Knobloch, K., Schriebmueller, L., Longo, U. G. and Vogt, P. M. (2008b) Eccentric exercises for the management of tendinopathy of the main body of the Achilles tendon with or without the AirHeel Brace. A randomized controlled trial. A: effects on pain and microcirculation. Disabil Rehabil, 30, 1685±91. Knorzer, E., Folkhard, W., Geercken, W., Boschert, C., Koch, M. H., Hilbert, B., Krahl, H., Mosler, E., Nemetschek-Gansler, H. and Nemetschek, T. (1986) New aspects of the etiology of tendon rupture. An analysis of time-resolved dynamic-mechanical measurements using synchrotron radiation. Arch Orthop Trauma Surg, 105, 113± 20. Koob, T. J. (2002) Biomimetic approaches to tendon repair. Comp Biochem Physiol A Mol Integr Physiol, 133, 1171±92. Kujala, U. M., Jarvinen, M., Natri, A., Lehto, M., Nelimarkka, O., Hurme, M., Virta, L. and Finne, J. (1992) ABO blood groups and musculoskeletal injuries. Injury, 23, 131±3. Kumar, K., Sharma, V. K., Sharma, R. and Maffulli, N. (2000) Correction of cubitus varus by French or dome osteotomy: a comparative study. J Trauma, 49, 717±21. Kvist, M. (1994) Achilles tendon injuries in athletes. Sports Med, 18, 173±201. Kvist, M., JoÂzsa, L., Jarvinen, M. and Et, A. (1985) Fine structural alterations in chronic Achilles paratenonitis in athletes. Path Res Pract, 180, 416±423. Langberg, H., Skovgaard, D., Petersen, L. J., Bulow, J. and Kjñr, M. (1999) Type I collagen synthesis and degradation in peritendinous tissue after exercise determined by microdialysis in humans. J Physiol, 521 Pt 1, 299±306. Leadbetter, W. B. (1992) Cell±matrix response in tendon injury. Clin Sports Med, 11, 533±78.

414

Regenerative medicine and biomaterials in connective tissue repair

Lehto, M., JoÂzsa, L., Kvist, M., Jarvinen, M., Balint, B. J. and Reffy, A. (1990) Fibronectin in the ruptured human Achilles tendon and its paratenon. An immunoperoxidase study. Ann Chir Gynaecol, 79, 72±7. Leppilahti, J., Puranen, J. and Orava, S. (1996) ABO blood group and Achilles tendon rupture. Ann Chir Gynaecol, 85, 369±71. Lin, J. H., Wang, M. X., Wei, A., Zhu, W., Diwan, A. D. and Murrell, G. A. (2001) Temporal expression of nitric oxide synthase isoforms in healing Achilles tendon. J Orthop Res, 19, 136±42. Lippi, G., Longo, U. G. and Maffulli, N. (2009) Genetics and sports. Br Med Bull, Feb 9 [Epub ahead of print]. Longo, G., Ripalda, P., Denaro, V. and Forriol, F. (2006) Morphologic comparison of cervical, thoracic, lumbar intervertebral discs of cynomolgus monkey (Macaca fascicularis). Eur Spine J, 15, 1845±51. Longo, U. G., Franceschi, F., Ruzzini, L., Rabitti, C., Morini, S., Maffulli, N. and Denaro, V. (2007a) Characteristics at Haematoxylin and Eosin staining of ruptures of the long head of the biceps tendon. Br J Sports Med, 43(8), 603±7. Longo, U. G., Franceschi, F., Ruzzini, L., Rabitti, C., Morini, S., Maffulli, N., Forriol, F. and Denaro, V. (2007b) Light microscopic histology of supraspinatus tendon ruptures. Knee Surg Sports Traumatol Arthrosc, 15, 1390±4. Longo, U. G., Franceschi, F., Ruzzini, L., Rabitti, C., Morini, S., Maffulli, N. and Denaro, V. (2008a) Histopathology of the supraspinatus tendon in rotator cuff tears. Am J Sports Med, 36, 533±8. Longo, U. G., Garau, G., Denaro, V. and Maffulli, N. (2008b) Surgical management of tendinopathy of biceps femoris tendon in athletes. Disabil Rehabil, 30, 1602±7. Longo, U. G., Olivia, F., Denaro, V. and Maffulli, N. (2008c) Oxygen species and overuse tendinopathy in athletes. Disabil Rehabil, 30, 15±71. Longo, U. G., Ramamurthy, C., Denaro, V. and Maffulli, N. (2008d) Minimally invasive stripping for chronic Achilles tendinopathy. Disabil Rehabil, 30, 1709±13. Longo, U. G., Franceschi, F., Ruzzini, L., Spiezia, F., Maffulli, N. and Denaro, V. (2009a) Higher fasting plasma glucose levels within the normoglycaemic range and rotator cuff tears. Br J Sports Med, 43, 284±7. Longo, U. G., Franceschi, F., Spiezia, F., Forriol, F., Maffulli, N. and Denaro, V. (2009b) Triglycerides and total serum cholesterol in rotator cuff tears: do they matter? Br J Sports Med, Apr 8 [Epub ahead of print]. Longo, U. G., Rittweger, J., Garau, G., Radonic, B., Gutwasser, C., Gilliver, S. F., Kusy, K., Zielinski, J., Felsenberg, D. and Maffulli, N. (2009c) No influence of age, gender, weight, height, and impact profile in Achilles tendinopathy in masters track and field athletes. Am J Sports Med, 37(7), 1400±5. Maffuli, N. (1995) Achilles tendon rupture. Br J Sports Med, 29, 279±80. Maffuli, N. (1996) Clinical tests in sports medicine: more on Achilles tendon. Br J Sports Med, 30, 250. Maffulli, N. and Kader, D. (2002) Tendinopathy of tendo achillis. J Bone Joint Surg Br, 84, 1±8. Maffulli, N. and Longo, U. G. (2008a) Conservative management for tendinopathy: is there enough scientific evidence? Rheumatology (Oxford), 47, 390±1. Maffulli, N. and Longo, U. G. (2008b) How do eccentric exercises work in tendinopathy? Rheumatology (Oxford), 47, 1444±5. Maffulli, N., Testa, V., Capasso, G., Bifulco, G. and Binfield, P. M. (1997) Results of percutaneous longitudinal tenotomy for Achilles tendinopathy in middle- and longdistance runners. Am J Sports Med, 25, 835±40.

Tendon injury and repair mechanics

415

Maffulli, N., Binfield, P. M. and King, J. B. (1998a) Tendon problems in athletic individuals. J Bone Joint Surg Am, 80, 142±4. Maffulli, N., Irwin, A. S., Kenward, M. G., Smith, F. and Porter, R. W. (1998b) Achilles tendon rupture and sciatica: a possible correlation. Br J Sports Med, 32, 174±7. Maffulli, N., Khan, K. M. and Puddu, G. (1998c) Overuse tendon conditions: time to change a confusing terminology. Arthroscopy, 14, 840±3. Maffulli, N., Barrass, V. and Ewen, S. W. (2000a) Light microscopic histology of achilles tendon ruptures. A comparison with unruptured tendons. Am J Sports Med, 28, 857±63. Maffulli, N., Ewen, S. W., Waterston, S. W., Reaper, J. and Barrass, V. (2000b) Tenocytes from ruptured and tendinopathic achilles tendons produce greater quantities of type III collagen than tenocytes from normal achilles tendons. An in vitro model of human tendon healing. Am J Sports Med, 28, 499±505. Maffulli, N., Reaper, J. A., Waterston, S. W. and Ahya, T. (2000c) ABO blood groups and Achilles tendon rupture in the Grampian Region of Scotland. Clin J Sport Med, 10, 269±71. Maffulli, N., Waterston, S. W. and Ewen, S. W. (2002) Ruptured Achilles tendons show increased lectin stainability. Med Sci Sports Exerc, 34, 1057±64. Maffulli, N., Kenward, M. G., Testa, V., Capasso, G., Regine, R. and King, J. B. (2003a) Clinical diagnosis of Achilles tendinopathy with tendinosis. Clin J Sport Med, 13, 11±15. Maffulli, N., Wong, J. and Almekinders, L. C. (2003b) Types and epidemiology of tendinopathy. Clin Sports Med, 22, 675±92. Maffulli, N., Sharma, P. and Luscombe, K. L. (2004a) Achilles tendinopathy: aetiology and management. J R Soc Med, 97, 472±6. Maffulli, N., Testa, V., Capasso, G., Ewen, S. W., Sullo, A., Benazzo, F. and King, J. B. (2004b) Similar histopathological picture in males with Achilles and patellar tendinopathy. Med Sci Sports Exerc, 36, 1470±5. Maffulli, N., Reaper, J., Ewen, S. W., Waterston, S. W. and Barrass, V. (2006) Chondral metaplasia in calcific insertional tendinopathy of the Achilles tendon. Clin J Sport Med, 16, 329±34. Maffulli, N., Ajis, A., Longo, U. G. and Denaro, V. (2007) Chronic rupture of tendo Achillis. Foot Ankle Clin, 12, 583±96. Maffulli, N., Longo, U. G., Franceschi, F., Rabitti, C. and Denaro, V. (2008a) Movin and Bonar scores assess the same characteristics of tendon histology. Clin Orthop Relat Res, 466, 1605±11. Maffulli, N., Testa, V., Capasso, G., Oliva, F., Panni, A. S., Longo, U. G. and King, J. B. (2008b) Surgery for chronic Achilles tendinopathy produces worse results in women. Disabil Rehabil, 10, 1±7. Magra, M. and Maffulli, N. (2005a) Matrix metalloproteases: a role in overuse tendinopathies. Br J Sports Med, 39, 789±91. Magra, M. and Maffulli, N. (2005b) Molecular events in tendinopathy: a role for metalloproteases. Foot Ankle Clin, 10, 267±77. Magra, M. and Maffulli, N. (2006) Nonsteroidal antiinflammatory drugs in tendinopathy: friend or foe? Clin J Sport Med, 16, 1±3. Magra, M. and Maffulli, N. (2007a) Genetics: does it play a role in tendinopathy? Clin J Sport Med, 17, 231±3. Magra, M. and Maffulli, N. (2007b) Genetic aspects of tendinopathy. J Sci Med Sport, 11(3), 243±7. Magra, M. and Maffulli, N. (2008) Genetic aspects of tendinopathy. J Sci Med Sport, 11, 243±7.

416

Regenerative medicine and biomaterials in connective tissue repair

Magra, M., Caine, D. and Maffulli, N. (2007a) A review of epidemiology of paediatric elbow injuries in sports. Sports Med, 37, 717±35. Magra, M., Hughes, S., El Haj, A. J. and Maffulli, N. (2007b) VOCCs and TREK-1 ion channel expression in human tenocytes. Am J Physiol Cell Physiol, 292, C1053±60. Manske, P. R., Gelberman, R. H., Vande Berg, J. S. and Lesker, P. A. (1984) Intrinsic flexor-tendon repair. A morphological study in vitro. J Bone Joint Surg Am, 66, 385±96. Manske, P. R., Gelberman, R. H. and Lesker, P. A. (1985) Flexor tendon healing. Hand Clin, 1, 25±34. Mathiak, G., Wening, J. V., Mathiak, M., Neville, L. F. and Jungbluth, K. (1999) Serum cholesterol is elevated in patients with Achilles tendon ruptures. Arch Orthop Trauma Surg, 119, 280±4. Matthews, L. S., Sonstegard, D. A. and Phelps, D. B. (1974) A biomechanical study of rabbit patellar tendon: effects of steroid injection. J Sports Med, 2, 349±57. Merkel, K. H., Hess, H. and Kunz, M. (1982) Insertion tendinopathy in athletes. A light microscopic, histochemical and electron microscopic examination. Pathol Res Pract, 173, 303±9. Mokone, G. G., Gajjar, M., September, A. V., Schwellnus, M. P., Greenberg, J., Noakes, T. D. and Collins, M. (2005) The guanine±thymine dinucleotide repeat polymorphism within the tenascin-C gene is associated with Achilles tendon injuries. Am J Sports Med, 33, 1016±21. Mokone, G. G., Schwellnus, M. P., Noakes, T. D. and Collins, M. (2006) The COL5A1 gene and Achilles tendon pathology. Scand J Med Sci Sports, 16, 19±26. Molloy, T., Wang, Y. and Murrell, G. (2003) The roles of growth factors in tendon and ligament healing. Sports Med, 33, 381±94. Movin, T., Gad, A., Reinholt, F. and Rolf, C. (1997) Tendon pathology in long-standing achillodynia. Biopsy findings in 40 patients. Acta Orthop Scand, 68, 170±5. Murrell, G. A. (2002) Understanding tendinopathies. Br J Sports Med, 36, 392±3. Murrell, G. A., Szabo, C., Hannafin, J. A., Jang, D., Dolan, M. M., Deng, X. H., Murrell, D. F. and Warren, R. F. (1997) Modulation of tendon healing by nitric oxide. Inflamm Res, 46, 19±27. Myerson, M. S. (1999) Achilles tendon ruptures. Instr Course Lect, 48, 219±30. Newnham, D. M., Douglas, J. G., Legge, J. S. and Friend, J. A. (1991) Achilles tendon rupture: an underrated complication of corticosteroid treatment. Thorax, 46, 853±4. Oshiro, W., Lou, J., Xing, X., Tu, Y. and Manske, P. R. (2003) Flexor tendon healing in the rat: a histologic and gene expression study. J Hand Surg [Am], 28, 814±23. Ozgurtas, T., Yildiz, C., Serdar, M., Atesalp, S. and Kutluay, T. (2003) Is high concentration of serum lipids a risk factor for Achilles tendon rupture? Clin Chim Acta, 331, 25±8. Perugia, L., Postaccchini, F. and Ippolito, E. (1986) The Tendons. Biology, pathology, clinical aspects. Editrice Kurtis s.r.l., Milan. Puddu, G., Ippolito, E. and Postacchini, F. (1976) A classification of Achilles tendon disease. Am J Sports Med, 4, 145±50. Riley, G. (2004) The pathogenesis of tendinopathy. A molecular perspective. Rheumatology (Oxford), 43, 131±42. Riley, G. P. (2005) Gene expression and matrix turnover in overused and damaged tendons. Scand J Med Sci Sports, 15, 241±51. Rocchi, M., Archidiacono, N., Romeo, G., Saginati, M. and Zardi, L. (1991) Assignment of the gene for human tenascin to the region q32±q34 of chromosome 9. Hum Genet, 86, 621±3.

Tendon injury and repair mechanics

417

Royer, R. J., Pierfitte, C. and Netter, P. (1994) Features of tendon disorders with fluoroquinolones. Therapie, 49, 75±6. Selvanetti, A., Cipolla, M. and Puddu, G. (1997) Overuse tendon injuries: Basic science and classification. Operative Techniques in Sports Medicine, 5, 110±7. Sharma, P. and Maffulli, N. (2005a) Basic biology of tendon injury and healing. Surgeon, 3, 309±16. Sharma, P. and Maffulli, N. (2005b) The future: rehabilitation, gene therapy, optimization of healing. Foot Ankle Clin, 10, 383±97. Sharma, P. and Maffulli, N. (2005c) Tendon injury and tendinopathy: healing and repair. J Bone Joint Surg Am, 87, 187±202. Sharma, P. and Maffulli, N. (2006a) Biology of tendon injury: healing, modeling and remodeling. J Musculoskelet Neuronal Interact, 6, 181±90. Sharma, P. and Maffulli, N. (2006b) Understanding and managing achilles tendinopathy. Br J Hosp Med (Lond), 67, 64±7. Siegler, A. M., Kontopoulos, V. and Wang, C. F. (1980) Prevention of postoperative adhesions in rabbits with ibuprofen, a nonsteroidal anti-inflammatory agent. Fertil Steril, 34, 46±9. Silver, F. H., Freeman, J. W. and Seehra, G. P. (2003) Collagen self-assembly and the development of tendon mechanical properties. J Biomech, 36, 1529±53. Smith, R. K., Birch, H. L., Goodman, S., Heinegard, D. and Goodship, A. E. (2002) The influence of ageing and exercise on tendon growth and degeneration ± hypotheses for the initiation and prevention of strain-induced tendinopathies. Comp Biochem Physiol A Mol Integr Physiol, 133, 1039±50. Soldatis, J. J., Goodfellow, D. B. and Wilber, J. H. (1997) End-to-end operative repair of Achilles tendon rupture. Am J Sports Med, 25, 90±5. Sullo, A., Maffulli, N., Capasso, G. and Testa, V. (2001) The effects of prolonged peritendinous administration of PGE1 to the rat Achilles tendon: a possible animal model of chronic Achilles tendinopathy. J Orthop Science, 6, 349±57. Szarfman, A., Chen, M. and Blum, M. D. (1995) More on fluoroquinolone antibiotics and tendon rupture. N Engl J Med, 332, 193. Tallon, C., Maffulli, N. and Ewen, S. W. (2001) Ruptured Achilles tendons are significantly more degenerated than tendinopathic tendons. Med Sci Sports Exerc, 33, 1983±90. Uhthoff, H. K. and Sarkar, K. (1991) Surgical repair of rotator cuff ruptures. The importance of the subacromial bursa. J Bone Joint Surg Br, 73, 399±401. Unverferth, L. J. and Olix, M. L. (1973) The effect of local steroid injections on tendon. J Sports Med, 1, 31±7. Vailas, A. C., Pedrini, V. A., Pedrini-Mille, A. and Holloszy, J. O. (1985) Patellar tendon matrix changes associated with aging and voluntary exercise. J Appl Physiol, 58, 1572±6. Von Bahr, S., Movin, T., Papadogiannakis, N., Pikuleva, I., Ronnow, P., Diczfalusy, U. and Bjorkhem, I. (2002) Mechanism of accumulation of cholesterol and cholestanol in tendons and the role of sterol 27-hydroxylase (CYP27A1). Arterioscler Thromb Vasc Biol, 22, 1129±35. Waterston, S. W., Maffulli, N. and Ewen, S. W. (1997) Subcutaneous rupture of the Achilles tendon: basic science and some aspects of clinical practice. Br J Sports Med, 31, 285±98. Williams, J. G. (1993) Achilles tendon lesions in sport. Sports Med, 16, 216±20. Wilson, A. M. and Goodship, A. E. (1994) Exercise-induced hyperthermia as a possible mechanism for tendon degeneration. J Biomech, 27, 899±905.

418

Regenerative medicine and biomaterials in connective tissue repair

Ziche, M., Morbidelli, L., Masini, E., Amerini, S., Granger, H. J., Maggi, C. A., Geppetti, P. and Ledda, F. (1994) Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest, 94, 2036±44. Zubrzycka, M. and Janecka, A. (2000) Substance P: transmitter of nociception (Minireview). Endocr Regul, 34, 195±201.

17

Tissue engineering for ligament and tendon repair M . L E E and B . M . W U , University of California, Los Angeles, USA

Abstract: Tendon and ligament injuries are one of the most common injuries of the knee with chronic or acute pain conditions. This chapter reviews functional tissue engineering approaches for ligament and tendon reconstruction focusing on various cell sources, biomaterial scaffolds, and biochemical/mechanical environments mimicking the normal ligaments and tendons. This chapter then reviews biological and functional performance of regenerated ligaments and tendons. Key words: ligament and tendon repair, functional tissue engineering, growth factors, mechanical stimulation.

17.1

Introduction

Tendon and ligament injuries are among the most common injuries of the knee with accompanying chronic or acute pain (Koh and Dietz, 2005). The principal causes of tendon and ligament injuries include intrinsic factors such as age, gender, systemic disease, or extrinsic factors like physical load, environment, occupation, and training (Frank and Jackson, 1997). The anterior cruciate ligament (ACL) is most frequently injured in the knee and rupture of ACL causes pain, joint instability, and eventually degenerative joint diseases (Fetto and Marshall, 1980; Barrack et al., 1990; Caborn and Johnson, 1993). The native ligament has a poor healing capacity because it has a poor blood supply and is surrounded by synovial fluid. Therefore, surgical intervention is frequently required to restore knee stability. An estimated over 100 000 ACL reconstructions are performed in the United States annually (Owings and Kozack, 1998). Current therapeutic options to replace the native ligament consist of using biological substitutes such as autograft or allograft. Autograft reconstruction using the patellar tendon or hamstring tendons harvested from the patient has produced satisfactory long-term outcomes and become the most popular surgical procedure. Nevertheless, donor site morbidity remains a major concern and it has often been associated with anterior knee pain, decreased range of motion, and weakness (Bach et al., 1998a,b). The use of allograft is limited by the possibility of immunogenic response, the risk of disease transmission, and an inadequate number of donor organs (Jackson et al., 1993; Crawford et al., 2005).

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A variety of synthetic ligament replacements have been studied for human implantation to address the shortcomings of existing strategies. Carbon-based prostheses and the Leeds-Keio (polyethylene terephthalate; Neoligaments Ltd, UK), Gore-tex (polytetrafluroethylene; W. L. Gore & Associates, Flagstaff, AZ), Stryker (polyethylene terephthalate±polypropylene; Stryker, Kalamazoo, MI) prostheses emerged and these grafts had high initial sustained strength. However, the use of these synthetic grafts was limited because of foreign body inflammation, particulate-induced synovitis, and graft rupture (Bolton and Bruckman, 1985; Richmond et al., 1992; Mody et al., 1993; Guidoin et al., 2000). Later the Kennedy Ligament Augmentation Device (LAD) (polypropylene; 3M, St. Paul, MN) were designed to promote tissue ingrowth, but these grafts had limited clinical success because of inflammatory reactions mediated by the non-degradable graft and subsequent failure of the construct (Koski et al., 2000). To regenerate a ligament that will parallel its native stability, function, and longevity, a novel technique based on tissue engineering approaches has been explored. This chapter will review functional tissue engineering approaches for ligament and tendon reconstruction. In addition, biological and functional performance of regenerated ligaments and tendons will be reviewed. Challenges in the current strategies and advanced options in this technology will be also discussed.

17.2

Tissue engineering approaches for ligament and tendon repair

Tissue engineering is an emerging interdisciplinary research field that has the significant potential to improve function of human tissues or organs (Langer and Vacanti, 1993; Vacanti and Langer, 1999). Organ failure and tissue loss, produced as a result of injury or other type of damage, affect millions of people annually and more than $400 billion is spent annually in the United States on patients suffering from organ failure or tissue loss. Tissue engineering potentially offers new medical solutions for hundreds of thousands of patients suffering from organ failure or tissue loss, and equally dramatic decrease in costs of care. Because the development of native tissue is a complex regeneration process, research in tissue engineering requires a collaborative approach with different disciplines including biological sciences, engineering, materials science, medicine, and various relevant biotechnologies. In the late 1980s Langer, Vacanti and co-workers described the potential use of biodegradable polymers combined with tissue-specific cells in culture to regenerate tissues and organs for transplantation (Vacanti et al., 1988; Cima et al., 1991; Langer and Vacanti, 1995). Subsequently, researchers in several disciplines have expanded the scope of this technology and are developing engineered replacements for a variety of

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tissues, including nerve, skin, cardiac valves, myocardium, hepatic tissue, pancreas, and bladder. The foundation of knowledge in this emerging field has broad applicability and may provide solutions to a variety of orthopedic disorders, including fracture non-union, cartilage injury, and tendon and ligament insufficiency. The triad of tissue engineering is based on the three basic components of biologic tissues including cells, extracellular matrix (ECM), and regulatory signals. The development of a functional engineered ligament is predicated on a system that uses (1) reparative cells with the capacity for proliferation and matrix synthesis, (2) a biologic scaffold that serves as a temporary structural template with adequate mechanical properties, and (3) an environment that provides sufficient nutrient transport, and appropriate chemical and mechanical regulatory stimuli. Current research on ligament tissue engineering attempts to replicate the physiologic milieu present during normal ligament development and repair. The application of mechanical environment designed to mimic the normal ligament can enhance the properties of tissue engineered constructs. Ex vivo systems use a bioreactor in which a ligament is developed on a structural scaffold for subsequent host implantation. Exogenous mechanical stimuli are applied by the bioreactor to induce seeded cells to synthesize collagen and ECM. The potential tissue replacement rendered by this system would allow immediate implantation of a functional ligament that would undergo physiologic remodeling in the host knee. The successful implication of tissue engineering requires that the cells that populate the scaffold lay down collagen in an organized fashion analogous to the native ligament, and that this neoligament have the mechanical and biochemical properties required for physiologic loading following implantation.

17.2.1 Cell therapy Cell therapy by implanting cell-seeded scaffolds or injecting cells into the defect site has tremendous potential in ligament and tendon tissue engineering. The ideal source for use in ligament/tendon engineering paradigm should provide cells that are readily available for clinical use, show robust proliferation, and possess the potential to elaborate ECM in an organized fashion. A variety of cell types have been investigated in an attempt to define a suitable cell line for ligament/tendon engineering including dermal fibroblasts, cells isolated from ligaments or tendons, mesenchymal stem cells (MSCs). Early investigation focused on fibroblast cell lines with the idea that these differentiated cells possessed the phenotypic characteristics necessary for organized collagen synthesis and ligament formation. Although fibroblasts from ligament and tendon tissue are similar, the choice of appropriate cell type is important for the successful development of tissue engineered ligaments. Ligaments and tendons have different matrix properties and healing capacities,

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lie in different synovial and vascular environments, and are subjected to different dynamic stress. It has been shown that fibroblasts from these tissues have a different intrinsic property in proliferation and matrix production (Wiig et al., 1991; Nagineni et al., 1992; Kobayashi et al., 2000; Hannafin et al., 2006). Cooper et al. (2006) investigated four different cell types from the ACL, medial collateral ligament (MCL), Achilles tendon (AT), and patellar tendon (PT) on three-dimensional (3D) braided polymer scaffolds to determine optimal cell source for tissue-engineered ligaments and suggested the ACL fibroblast as the most suitable cells. Dunn et al. (1995) compared intra-articular rabbit ACL fibroblasts and extra-articular rabbit PT fibroblasts in vitro and found PT fibroblasts proliferated more rapidly than ACL fibroblasts. Fibroblast proliferation and function also depended on the tissue culture substrate and both cell types synthesized 10-fold greater collagen on the collagen ligament analog than on the tissue culture plate. The recent studies reported dramatic effects of passage of ligament and tendon fibroblasts on the gene expression of matrix constituents. With increasing passage number, Yao et al. (2006) showed increase in the ratio of type III to type I collagen in tenocytes isolated from human Achilles tendon. Almarza et al. (2008) quantified the gene expression of matrix proteins of fibroblasts from the MCL, ACL, and PT and found a significant increase in type I collagen of rat MCL fibroblasts throughout passage, suggesting MCL fibroblasts' high potential for ligament tissue engineering. Lin et al. (1999) compared human ACL and MCL cell proliferation in vitro on a synthetic biodegradable polymer fiber scaffold. Human ACL and MCL fibroblasts showed rapid proliferation that was enhanced with mechanical mixing of the culture media and the addition of transforming growth factor beta1 (TGF- 1 ). Collectively, these early studies established the capacity of fibroblasts originating from various mesenchymal tissues to proliferate, synthesize collagen, and respond to mechanical and biochemical growth factors making them a potential source for ligament engineering. Although adult fibroblasts retain many of the phenotypic qualities necessary for collagen synthesis, they are relatively quiescent and have limited potential for further differentiation (Van Eijk et al., 2004). Accordingly, bone marrow stromal cells (BMSC) are an attractive candidate for ligament engineering, and their value is currently under investigation. BMSCs are derived from a source that maintains some degree of self-renewal and has the potential to differentiate into cells of multiple mesenchymal lineages. Moreover, the synthetic and proliferative systems of these cells are robust, and they have the potential to readily adapt to their local niche. From the perspective of clinical utility, BMSCs are routinely collected from patients for use in bone grafting and can be easily accessed in most patients without significant additional surgery or the risk of immune reaction (Vunjak-Novakovic et al., 2004). These cells can be rapidly expanded in cell culture media and stimulated to differentiate into a fibroblastlike cell lineage (Altman et al., 2001; Vunjak-Novakovic et al., 2004).

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A comparative evaluation of goat BMSCs, ACL fibroblasts, and skin fibroblasts was undertaken to evaluate the optimal cell source for ACL engineering (Van Eijk et al., 2004). The aforementioned cells were seeded onto a degradable suture material and cultured for a maximum of 12 days. Each of the cell types attached, proliferated, and synthesized ECM rich in collagen type I. However, the scaffolds seeded with BMSCs showed the highest DNA content and collagen production. Although further characterization of the role of BMSCs is yet to be established, these cells hold tremendous promise for use in ACL engineering.

17.2.2 Biomaterial scaffolds In ligament and tendon tissue engineering, the success of tissue regeneration relies on the development of suitable scaffolds to direct 3D tissue ingrowth. Optimal scaffold structure is required to enhance nutrient transport within 3D constructs for the survival of transplanted cells and vascularization from the host to provide successful engraftment. Additionally, the scaffold must have sufficient initial strength to withstand mechanical stress, yet it must subsequently degrade over time and yield to the progress of native tissue ingrowth. A variety of scaffold materials have been considered as ligament and tendon scaffolds, including biologic materials such as collagen and silk, as well as synthetic polymers and composite materials (Dunn et al., 1995; Lin et al., 1999; Chen et al., 2003; Bourke et al., 2004; Cooper et al., 2005; Laurencin and Freeman, 2005; Lee et al., 2005). Collagen is a major component of the ECM and is the primary constituent of a normal ligament. Investigators hypothesized that using a collagen-based product would most closely replicate the structural framework of the native ACL. Although the fibrous collagen scaffold supported cell attachment, spreading, and fiber coverage with ECM, the capacity of the construct to support mechanical loading diminished over time. A number of crosslinking attempts (chemical, physical, and biological) have been undertaken to improve the mechanical integrity of these constructs and they improve initial strength, control the rate of degradation, and also influence the response evoked in host cells (Caruso and Dunn, 2005). Although this approach may improve the mechanical limitations of collagen grafts, the predominant chemical crosslinking agents such as glutaraldehyde, formaldehyde, cyanamide, and carbodiimide are further limited by the potential toxic residues and there is no commonly accepted ideal crosslinking treatment (Khor, 1997). Silk has excellent mechanical properties and predictable biodegradation. Like collagen, silk has also been used as a surgical suture material that, when properly processed, is inexpensive and biocompatible. Its high homogeneity in secondary structure provides a remarkable mechanical property and environmental stability (Greenwald et al., 1994). Altman and co-workers have developed a processed silk scaffold that has shown promise as a ligament replacement solution. When

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woven into a `wire-rope' scaffold (bundles of fibers are wound into fibrils that are then woven into cords), the construct shows mechanical properties similar to the native ACL (Altman et al., 2001, 2002a; Vunjak-Novakovic et al., 2004). Moreover, it is hypothesized that the microenvironment created by this construct facilitates mass transport and increases the surface area for cell attachment. In initial studies, Altman and co-workers showed that silk scaffolds seeded with human BMSCs and subjected to cyclic translational and rotational strain induce the synthesis of collagen and other fibroblast markers, with corresponding morphologic changes consistent with a fibroblast-like cell differentiation (Altman et al., 2001; Vunjak-Novakovic et al., 2004). The most commonly investigated synthetic polymers are resorbable polyester polymers that are both biocompatible and provisional in design, including polyglycolic acid (PGA), polylactic acid (PLA), polycaprolactone (PCL), and polycarbonate (Marler et al., 1998). These polymers are gradually reabsorbed, reducing the likelihood of inflammatory reaction. Subsequently, they are replaced by tissue ingrowth, obviating the need for long-term structural integrity. Polymer selection allows control over the rate of degradation, the mechanical properties of the construct, and the cellular response. Moreover, the characteristics of these materials can be altered with specific surface, structure, and formulation modifications, providing tremendous flexibility to the overall scaffold design (Cooper et al., 2005). Additionally, most of these polymers are currently approved for other surgical applications and have established safety profiles. Further modification of the synthetic polymers with ECM proteins has been shown to improve the cell adhesion properties (Lu et al., 2005).

17.2.3 Growth factors Numerous growth factors are employed to control cellular functions and promote tissue regeneration during natural ligament and tendon healing. Research in a rabbit model has indicated that endogenous growth factors such as platelet-derived growth factor (PDGF) and TGF- are present in high concentrations during the acute phase of ligament injury, and these levels return to baseline 2 to 4 weeks after injury (Lee et al., 1998). An increase in mRNA transcripts for growth factors and growth factor receptors is also seen in the subacute phase of injury, with levels of endothelin (ET-1), TGF- , and insulinlike growth factor (IGF) increased 2.5 to 5 times the control level 3 to 14 weeks after injury (Sciore et al., 1998). These data indicate that there is temporal growth factor response following ligament injury. With greater understanding of the pertinent local growth agents and their significance during normal ligament development and repair, researchers will be able to more accurately recapitulate these processes in vitro. Fibroblast growth factor (FGF), TGF, PDGF, epidermal growth factor (EGF), IGF, and growth and differentiation factor (GDF) have all shown the capacity to

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improve cell proliferation or matrix elaboration in ligament engineering constructs (Goh et al., 2003). Murray et al. (2003) reported the effects of selected growth factors on human ACL cell responses in a collagen±glycosaminoglycan (CG) scaffold. The addition of TGF- 1 to the culture medium resulted in increased cell number, increased collagen production and increased expression of -smooth muscle actin within the CG scaffold. PDGF-AB has been shown to lead to increased cell proliferation rates within the scaffold and increased collagen production. A study by Weiler et al. (2004) demonstrated that local delivery of PDGF-BB improved the mechanical properties of tendon graft after ACL reconstruction. bFGF has been shown to induce the proliferation of various cell types, including endothelial cells, fibroblasts, and smooth muscle cells (Gospodarowicz et al., 1986). It also has been shown to promote angiogenesis and to enhance wound healing and tissue repair. Local controlled delivery of bFGF has been shown to increase the mechanical strength of the regenerated ACL using the PLLA braid scaffold combined with the gelatin hydrogel incorporating bFGF (Kimura et al., 2008). GDF was applied on injured tendons and enhanced tendon healing was observed with GDF5 (Wolfman et al., 1997; Rickert et al., 2005). Tashiro et al. (2006) also demonstrated favorable effects of GDF5 on ligament healing in a study using a gap injury model of the medial collateral ligament in rat knee joints. The potential of the production of ligament tissue from BMSCs has been explored. Unlike ligament cells and tenocytes used in ligament and tendon engineering, BMSCs have potential to differentiate into multiple mesenchymal lineages and do not intrinsically produce fibroblasts specific proteins. Studies have demonstrated that biochemical cues can direct differentiation of BMSCs to a fibroblast phenotype. Moreau et al. (2005) have attempted to establish specific media formulations and growth factor combinations that support BMSC differentiation toward a fibroblast phenotype. They found that media supplemented with ascorbate-2-phosphate was potent in promoting BMSC proliferation and they cited three growth factor and media combinations that enhanced fibroblast differentiation: (1) EGF and TGF, (2) bFGF and TGF, and (3) growth factor-free advanced Dulbecco's minimal essential medium (ADMEM).

17.2.4 Mechanical factors Ligaments and tendons are regularly oriented, dense connective tissue and they consist mainly of closely packed collagen fibers interspersed with fibroblasts that lie along the long axis of ligaments. The fibroblasts in the ligament are continuously stimulated by dynamic stress and align along collagen fibers. It is well known that mechanical loading can affect tissue development of the musculoskeletal tissues, such as bones, cartilages, ligaments, and tendons. Woo et al. (2006) demonstrated the beneficial effect of exercise and dynamic loading on ligament strength and the detrimental effects of prolonged immobilization on

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ultimate failure strength. The importance of physiologic strain in maintaining in vivo ligament strength has prompted research into the role of mechanical stimulation in the development of an engineered ligament construct. Wang et al. (2003) studied the effect of fibroblasts orientation on the alignment of cellproduced collagenous matrix using a novel in vitro model in which the orientation of cells could be controlled via microgrooves. In this study, when cells were oriented along the direction of the microgrooves, they produced an aligned collagenous matrix, suggesting that cell orientation affects collagenous matrix alignment. Park et al. (2006) examined the effect of external mechanical loading on the matrix production by the cells. In this study, ligament fibroblasts were cultivated on micropatterned silicone substrates and subjected to cyclic stretching. With stretching on a microgrooved surface, cell proliferation and collagen production were greatly enhanced. Several theories have been proposed to explain the effect of mechanical stimulation on cellular response with regard to cell morphology and matrix production. It is generally believed that mechanotransduction signals are sensed by deformation of the cytoskeleton that is transmitted from the surrounding matrix via transmembrane cellular adhesion molecules, resulting in reorganization of the cytoskeleton and initiating upregulation of ECM synthesis (Flickinger et al., 1992; Gudi et al., 1998; Berry et al., 2003). Application of mechanical stimulation can direct undifferentiated cells such as BMSCs into cells expressing ligament fibroblast markers. Altman et al. (2001, 2002b) observed that human BMSCs subjected to multidimensional mechanical strains in the absence of biochemical growth factors can induce the differentiation of the cells towards a fibroblast-like cell lineage. This was corroborated by up-regulation of ligament fibroblast markers including collagen types I and III, as well as tenascin-C, orientation of ECM, and the absence of production of bone or cartilage-specific cell markers (Altman et al., 2001). More recently, Van Eijk et al. (2004) examined the effect of mechanical stimulation of BMSCs cultured on a porous 3D braided poly(lactic-co-glycolic acid) (PLGA) scaffold. The seeded scaffolds were subjected to static mechanical load, resulting in alignment, proliferation, and differentiation of the cells. The precise mechanisms underlying cell proliferation and differentiation in response to mechanical stimulation are unclear. It is likely that these phenotypic changes are the result of the influence of mechanical signal transduction pathways as well as differing rates of nutrient, metabolite, and oxygen mass transfer induced by these stimuli (Altman et al., 2001). Henshaw et al. (2006) have found increased expression of ACL fibroblast 1 and 1 integrin subunits in response to intrinsic and extrinsic strain, possibly implicating these receptors in the mechanotransduction pathway. Further investigation will be necessary to delineate the role of mechanical stimulation in the generation of ligament, including the cellular pathways underlying mechanotransduction and a quantitative analysis of the optimal stimulus for tissue formation.

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427

Reconstruction of ligaments and tendons

The aforementioned investigations represent the foundation of knowledge of ligament and tendon tissue engineering. Collectively, the data suggest that fibroblasts and mesenchymal stem cells can be cultured on natural and synthetic ligament scaffolds, and that these cells respond to a number of biochemical and mechanical stimuli by proliferation, differentiation, and elaboration of ECM. Despite a lot of potential means to construct optimal tissue-engineered ligaments and tendons, the absence of ligament-specific markers makes it difficult to evaluate the outcomes and determine an optimal tissue engineering approach. Various biomechanical and biological parameters have been suggested to determine the success of the reconstructed ligaments and tendons. Most often used biomechanical assessment is a measure of structural and materials properties of the reconstructed tissues, such as force, stiffness, stress, and modulus, compared with normal ligaments and tendons (Butler et al., 2004). In addition, biological assessment should be taken into account. The major ECM component of ligaments and tendons is collagen (primarily types I and III) and collagen is primarily responsible for tissue strength (Thomopoulos et al., 2003). Quantification of specific expression of collagen types I and III, with the relative ratio of ~7 : 1, has been proposed as a successful indicator for reconstructed ligaments (Amiel et al., 1984; Martin et al., 2001). Another important component of the ligament and tendon ECM is proteoglycan such as decorin, aggrecan, biglycan, fibromodulin, lumican, and versican. They provide viscoelastic properties to the tissues and influence other properties of the tissue, including fibril diameter, and cellular proliferation and migration (Yoon and Halper, 2005; Wang, 2006). Given that decorin is known to inhibit collagen fibrillogenesis, down-regulating the decorin expression by antisense gene therapy would potentially enhance the diameter of newly synthesized collagen fibrils and thus improve the mechanical property of regenerated ligament (Nakamura et al., 2000). The components of ECM of the ligament and tendon appear to respond to mechanical stimulation placed on the cells. It has been shown that the application of mechanical stress, without ligament growth and regulatory factors, induced differentiation of mesenchymal stem cells (MSC) into a ligament cell lineage by up-regulating ligament fibroblast markers, including collagen types I and III and tenascin-C (Vunjak-Novakovic et al., 2004). Functionality of the regenerated tissue can also be distinguished by evaluating organization and remodeling of the deposited ECM. The most important markers in ligament remodeling are the matrix metalloproteinases (MMPs), enzymes secreted from fibroblasts, facilitating digestion of ECM (Bramono et al., 2004). The activity of MMPs are regulated by tissue inhibitor of metalloproteinase-2 (TIMP-2) and 2-macroglobulin (Munshi et al., 2004). The interplay between MMPs and TIMPs controls remodeling of unorganized

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ECM, facilitating organization of collagen sturctures of developing ligament and tendon tissue. Furthermore, cell morphology and the ultrastructure of collagen network (collagen pattern, collagen fibril diameter) should be taken into account. Establishment of success criteria could provide clues for the future design of microenvironment to engineer functional ligaments and tendons. Previous studies demonstrated the efficacy of the application of biochemical or mechanical stimulation in ligament and tendon tissue regeneration. These findings represent the most elemental steps toward the realization of a tissueengineered ligament replacement. The development of native ligaments and tendons is a complex regeneration process driven by a cascade of multiple growth factors acting at the necessary site, concentration, and timing. bFGF is up-regulated in the injured area following ligament injury and this area is infiltrated by fibroblasts. With the progression of healing, TGF- induces matrix deposition and synthesis of a higher proportion of type I collagen arranged along the functional axis of the injured ligament. More recently, sequential administration of bFGF and TGF- over extended culture has been demonstrated to enhance matrix ingrwoth and collagen type I production of BMSCs (Moreau et al., 2006). The precise mechanism of action, temporal demand, and physiologic concentration of these agents will likely be the topics of future investigation, as well as the incorporation of the relevant factors into biodegradable polymers to achieve sustained release in vitro. Recent studies have evaluated combined effects of either biochemical or mechanical stimuli on promoting cellular adaptation or gene expression in the ligament and tendon engineering. The combined effect of locally released bFGF and mechanical strain on cellular morphology and gene expression of BMSCs was investigated under bFGF-coated 3D polymer scaffold under static and cyclically strained conditions (Petrigliano et al., 2007). Cells subjected to both mechanical stimulation and bFGF (500 ng) demonstrated the highest upregulation of stress-resistive (collagen I, III) and stress-responsive proteins (tenascin-C). The concurrent application of biochemical and mechanical stimuli may be important in promoting the adaptation of BMSCs and driving the transcription of genes essential for synthesis of a functional ligament replacement tissue. Most recently, sequential biochemical and mechanical stimulation has been explored to develop optimal ligament tissue (Moreau et al., 2008). Administration of mechanical stimulation at the peak of growth-factor-induced cell activity enhanced matrix ingrowth, cell and ECM alignment, and total collagen type I.

17.4

Future trends

A suitable tissue-engineered ACL replacement may obviate many of the current shortcomings of current autograft and allograft techniques, while allowing patients to promptly return to activity with an immediately functional ACL

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replacement. Current research has found that natural and synthetic scaffolds can sustain cell adhesion, growth, and matrix deposition under appropriate conditions, and that growth factors and mechanical stimuli have a role in modulating cellular response. Although these advancements are promising, many obstacles persist. Ligaments or tendons connect to subchondral bone through a complex fibrocartilage interface, with phenotypically three different zones (ligament, fibrocartilage, and bone) where resident cells and extracellular matrix composition have different morphology and organization (Benjamin et al., 1986; Sagarriga Visconti et al., 1996; Thomopoulos et al., 2003). The interface region is composed of nonmineralized fibrocartilage region within which ovoid chondrocytes are surrounded by type II collagen matrix, and mineralized fibrocartilage region with hypertrophic chondrocytes embedded in calcified matrix. This organized 3D network of cells and ECM permits a gradual transition of mechanical load between ligament and bone, contributing to minimize the formation of stress concentrations. For the success of ligament and tendon tissue engineering, additional design strategy to mimic the native zonal organization may require to meet the mechanical and structural properties of ligament-to-bone interface (Spalazzi et al., 2006). Recent novel technologies such as solid freeform fabrication (SFF) fabrication technique allow the creation of a complex scaffold incorporating a mechanical and structural properties' gradient. Osteochondral composite scaffold has been demonstrated using the TheriForm 3D printing process for articular cartilage repair and supported formation of cartilage tissue in the upper region and had a similar tensile strength of the lower bone region to native cancellous human bone (Sherwood et al., 2002). The development of a composite scaffold mimicking the zonal region presented in native ligament±bone constructs would direct successful ligament development and integration with host bone after implantation. Facilitation of distinct cellular and matrix development will be feasible with co-seeding of cells from ligament and bone tissue. In addition, an ideal scaffold should provide the appropriate biochemical stimuli with spatial and temporal control to promote tissue ingrowth as well as tissue-specific matrix deposition during regeneration. As research continues, it is expected that ligament engineering techniques will lead to the production of a new generation of scaffolds that will mirror the structural and mechanical variation of the native ligament and result in the fabrication of a compatible and durable ligament replacement. The mechanical environment can direct and modulate the cell differentiation and collagen organization. However, the precise local mechanical conditions that promote successful ligament development and repair have yet to be defined. The design of a bioreactor allowing complicated loading conditions similar to knee motion on appropriate development stages and regions of ligaments should help develop functional outcomes for clinical applications.

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Sources of further information and advice

Because of the complex mechanical and structural requirements of native ligaments and tendons, design and fabrication of scaffolds are important research areas for the successful ligament and tendon tissue engineering. O'Shea and Miao (2008) discuss the current scaffold strategies that can be applied to produce a composite structure to promote complex tissue growth such as a transitional zone of fibrocartilage at ligament-to-bone interface. The limited blood supply and altered expression of growth factors after ligament and tendon injuries make it difficult to regenerate tissues. The mechanical and structural functions of scaffolds can be further improved by combining bioactive factors to enhance the quality of healing tissues. Hollister (2006) describes design and fabrication strategies of material/biofactor hybrid that would have computationally optimized 3D structural and biofactor topology.

17.6

References

Almarza A J, Augustine S M and Woo S L Y (2008), `Changes in gene expression of matrix constituents with respect to passage of ligament and tendon fibroblasts'. Ann Biomed Eng 36(12): 1927±1933. Altman G H, Horan R L, Martin I, Farhadi J, Stark P R H, Volloch V, Richmond J C, Vunjak-Novakovic G and Kaplan D L (2001), `Cell differentiation by mechanical stress'. Faseb J 15(14): 270±272. Altman G H, Horan R L, Lu H H, Moreau J, Martin I, Richmond J C and Kaplan D L (2002a), `Silk matrix for tissue engineered anterior cruciate ligaments'. Biomaterials 23(20): 4131±4141. Altman G H, Lu H H, Horan R L, Calabro T, Ryder D, Kaplan D L, Stark P, Martin I, Richmond J C and Vunjak-Novakovic G (2002b), `Advanced bioreactor with controlled application of multi-dimensional strain for tissue engineering'. J Biomech Eng-T Asme 124(6): 742±749. Amiel D, Frank C, Harwood F, Fronek J and Akeson W (1984), `Tendons and ligaments: a morphological and biochemical comparison'. Journal of Orthopedic Research 1(3): 257±265. Bach B R, Levy M E, Bojchuk J, Tradonsky S, Bush-Joseph C A and Khan N H (1998a), `Single-incision endoscopic anterior cruciate ligament reconstruction using patellar tendon autograft ± minimum two-year follow-up evaluation'. Am J Sport Med 26(1): 30±40. Bach B R, Tradonsky S, Bojchuk J, Levy M E, Bush-Joseph C A and Khan N H (1998b), `Arthroscopically assisted anterior cruciate ligament reconstruction using patellar tendon autograft ± five- to nine-year follow-up evaluation'. Am J Sport Med 26(1): 20±29. Barrack R L, Bruckner J D, Kneisl J, Inman W S and Alexander A H (1990), `The outcome of nonoperatively treated complete tears of the anterior cruciate ligament in active young adults'. Clin Orthop Relat R 259: 192±199. Benjamin M, Evans E J and Copp L (1986), `The histology of tendon attachments to bone in man'. J Anat 149: 89±100. Berry C C, Cacou C, Lee D A, Bader D L and Shelton J C (2003), `Dermal fibroblasts

Tissue engineering for ligament and tendon repair

431

respond to mechanical conditioning in a strain profile dependent manner'. Biorheology 40(1±3): 337±345. Bolton C W and Bruchman W C (1985), `The Gore-Tex expanded polytetrafluoroethylene prosthetic ligament ± an in vitro and in vivo evaluation'. Clin Orthop Relat R 196: 202±213. Bourke S L, Kohn J and Dunn M G (2004), `Preliminary development of a novel resorbable synthetic polymer fiber scaffold for anterior cruciate ligament reconstruction'. Tissue Eng 10(1±2): 43±52. Bramono D S, Richmond J C, Weitzel P P, Kaplan D L and Altman G H (2004), `Matrix metalloproteinases and their clinical applications in orthopaedics'. Clin Orthop Relat Res 428: 272±285. Butler D L, Juncosa N and Dressler M R (2004), `Functional efficacy of tendon kepair processes'. Annu Rev Biomed Eng 6: 303±329. Caborn D N M and Johnson B M (1993), `The natural history of the anterior cruciate ligament-deficient knee ± a review'. Clin Sport Med 12(4): 625±636. Caruso A B and Dunn M G (2005), `Changes in mechanical properties and cellularity during long-term culture of collagen fiber ACL reconstruction scaffolds'. J Biomed Mater Res A 73A(4): 388±397. Chen J S, Altman G H, Karageorgiou V, Horan R, Collette A, Volloch V, Colabro T and Kaplan D L (2003), `Human bone marrow stromal cell and ligament fibroblast responses on RGD-modified silk fibers'. J Biomed Mater Res A 67A(2): 559±570. Cima L G, Vacanti J P, Vacanti C, Ingber D, Mooney D and Langer R (1991), `Tissue engineering by cell transplantation using degradable polymer substrates'. J Biomech Eng-T Asme 113(2): 143±151. Cooper J A, Lu H H, Ko F K, Freeman J W and Laurencin C T (2005), `Fiber-based tissue-engineered scaffold for ligament replacement: design considerations and in vitro evaluation'. Biomaterials 26(13): 1523±1532. Cooper J A, Bailey L O, Carter J N, Castiglioni C E, Kofron M D, Ko F K and Laurencin C T (2006), `Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament'. Biomaterials 27(13): 2747±2754. Crawford C, Kainer M, Jernigan D, Banerjee S, Friedman C, Ahmed F and Archibald L K (2005), `Investigation of postoperative allograft-associated infections in patients who underwent musculoskeletal allograft implantation'. Clin Infect Dis 41(2): 195± 200. Dunn M G, Liesch J B, Tiku M L and Zawadsky J P (1995), `Development of fibroblastseeded ligament analogs for ACL reconstruction'. J Biomed Mater Res 29(11): 1363±1371. Fetto J F and Marshall J L (1980), `The natural history and diagnosis of anterior cruciate ligament insufficiency'. Clin Orthop Relat R 147: 29±38. Flickinger K S, Carter W G and Culp L A (1992), `Deficiency in integrin-mediated transmembrane signaling and microfilament stress fiber formation by aging dermal fibroblasts from normal and Downs syndrome patients'. Exp Cell Res 203(2): 466± 475. Frank C B and Jackson D W (1997), `Current concepts review ± the science of reconstruction of the anterior cruciate ligament'. J Bone Joint Surg Am 79A(10): 1556±1576. Goh J C H, Ouyang H W, Teoh S H, Chan C K C and Lee E H (2003), `Tissueengineering approach to the repair and regeneration of tendons and ligaments'. Tissue Eng 9: S31±S44.

432

Regenerative medicine and biomaterials in connective tissue repair

Gospodarowicz D, Neufeld G and Schweigerer L (1986), `Fibroblast growth factor'. Mol Cell Endocrinol 46(3): 187±204. Greenwald D, Shumway S, Albear P and Gottlieb L (1994), `Mechanical comparison of 10 suture materials before and after in vivo incubation'. J Surg Res 56(4): 372± 377. Gudi S R P, Lee A A, Clark C B and Frangos J A (1998), `Equibiaxial strain and strain rate stimulate early activation of G proteins in cardiac fibroblasts'. Am J PhysiolCell Ph 43(5): C1424±C1428. Guidoin M F, Marois Y, Bejui J, Poddevin N, King M W and Guidoin R (2000), `Analysis of retrieved polymer fiber based replacements for the ACL'. Biomaterials 21(23): 2461±2474. Hannafin J A, Attia E A, Henshaw R, Warren R F and Bhargava M A (2006), `Effect of cyclic strain and plating matrix on cell proliferation and integrin expression by ligament fibroblasts'. J Orthop Res 24(2): 149±158. Henshaw D R, Attia E, Bhargava M and Hannafin L A (2006), `Canine ACL fibroblast integrin expression and cell alignment in response to cyclic tensile strain in threedimensional collagen gels'. J Orthop Res 24(3): 481±490. Hollister S J (2006), `Porous scaffold design for tissue engineering (vol 4, pg 518, 2005)'. Nat Mater 5(7): 590±590. Jackson D W, Grood E S, Goldstein J D, Rosen M A, Kurzweil P R, Cummings J F and Simon T M (1993), `A comparison of patellar tendon autograft and allograft used for anterior cruciate ligament reconstruction in the goat model'. Am J Sport Med 21(2): 176±185. Khor E (1997), `Methods for the treatment of collagenous tissues for bioprostheses'. Biomaterials 18(2): 95±105. Kimura Y, Hokugo A, Takamoto T, Tabata Y and Kurosawa H (2008), `Regeneration of anterior cruciate ligament by biodegradable scaffold combined with local controlled release of basic fibroblast growth factor and collagen wrapping'. Tissue Eng Pt CMeth 14(1): 47±57. Kobayashi K, Healey R M, Sah R L, Clark J J, Tu B P, Goomer R S, Akeson W H, Moriya H and Amiel D (2000), `Novel method for the quantitative assessment of cell migration: a study on the motility of rabbit anterior cruciate (ACL) and medial collateral ligament (MCL) cells'. Tissue Eng 6(1): 29±38. Koh J and Dietz J (2005), `Osteoarthritis in other joints (hip, elbow, foot, ankle, toes, wrist) after sports injuries'. Clin Sport Med 24(1): 57±70. Koski J A, Ibarra C and Rodeo S A (2000), `Tissue-engineered ligament ± cells, matrix, and growth factors'. Orthop Clin N Am 31(3): 437±452. Langer R and Vacanti J P (1993), `Tissue engineering'. Science 260(5110): 920±926. Langer R and Vacanti J P (1995), `Artificial organs'. Sci Am 273(3): 130±133. Laurencin C T and Freeman J W (2005), `Ligament tissue engineering: an evolutionary materials science approach'. Biomaterials 26(36): 7530±7536. Lee C H, Shin H J, Cho I H, Kang Y M, Kim I A, Park K D and Shin J W (2005), `Nanofiber alignment and direction of mechanical strain affect the ECM production of human ACL fibroblast'. Biomaterials 26(11): 1261±1270. Lee J, Harwood F L, Akeson W H and Amiel D (1998), `Growth factor expression in healing rabbit medial collateral and anterior cruciate ligaments'. J Appl Physiol 18: 19±25. Lin V S, Lee M C, O'Neal S, McKean J and Sung K L P (1999), `Ligament tissue engineering using synthetic biodegradable fiber scaffolds'. Tissue Eng 5(5): 443± 451.

Tissue engineering for ligament and tendon repair

433

Lu H H, Cooper J A, Manuel S, Freeman J W, Attawia M A, Ko F K and Laurencin C T (2005), `Anterior cruciate ligament regeneration using braided biodegradable scaffolds: in vitro optimization studies'. Biomaterials 26(23): 4805±4816. Marler J J, Upton J, Langer R and Vacanti J P (1998), `Transplantation of cells in matrices for tissue regeneration'. Adv Drug Deliver Rev 33(1±2): 165±182. Martin I, Jakob M, Schafer D, Dick W, Spagnoli G and Heberer M (2001), `Quantitative analysis of gene expression in human articular cartilage from normal and osteoarthritic joints'. Osteoarthr Cartilage 9(2): 112±118. Mody B S, Howard L, Harding M L, Parmar H V and Learmonth D J (1993), `The Abc carbon and polyester prosthetic ligament for ACL-deficient knees ± early results in 31 cases'. J Bone Joint Surg Br 75(5): 818±821. Moreau J, Chen J, Kaplan D and Altman G (2006), `Sequential growth factor stimulation of bone marrow stromal cells in extended culture'. Tissue Eng 12(10): 2905±2912. Moreau J E, Chen J S, Bramono D S, Volloch V, Chernoff H, Vunjak-Novakovic G, Richmond J C, Kaplan D L and Altman G H (2005), `Growth factor induced fibroblast differentiation from human bone marrow stromal cells in vitro'. J Orthop Res 23(1): 164±174. Moreau J E, Bramono D S, Horan R L, Kaplan D L and Altman G H (2008), `Sequential biochemical and mechanical stimulation in the development of tissue-engineered ligaments'. Tissue Eng Part A 14(7): 1161±1172. Munshi H G, Wu Y I, Mukhopadhyay S, Ottaviano A J, Sassano A, Koblinski J E, Platanias L C and Stack M S (2004), `Differential regulation of membrane type 1matrix metalloproteinase activity by ERK 1/2- and p38 MAPK-modulated tissue inhibitor of metalloproteinases 2 expression controls transforming growth factorbeta1-induced pericellular collagenolysis'. J Biol Chem 279(37): 39042±39050. Murray M M, Rice K, Wright R J and Spector M (2003), `The effect of selected growth factors on human anterior cruciate ligament cell interactions with a threedimensional collagen-GAG scaffold'. J Orthop Res 21(2): 238±244. Nagineni C N, Amiel D, Green M H, Berchuck M and Akeson W H (1992), `Characterization of the intrinsic properties of the anterior cruciate and medial collateral ligament cells ± an in vitro cell culture study'. J Orthop Res 10(4): 465± 475. Nakamura N, Hart D A, Boorman R S, Kaneda Y, Shrive N G, Marchuk L L, Shino K, Ochi T and Frank C B (2000), `Decorin antisense gene therapy improves functional healing of early rabbit ligament scar with enhanced collagen fibrillogenesis in vivo'. J Orthop Res 18(4): 517±523. O'Shea T M and Miao X G (2008), `Bilayered scaffolds for osteochondral tissue engineering'. Tissue Eng Pt B-Rev 14(4): 447±464. Owings M F and Kozak L J (1998), `Ambulatory and inpatient procedures in the United States'. Anesth Analg 13(139): 1±119. Park S A, Kim I A, Lee Y J, Shin J W, Kim C R, Kim J K, Yang Y I and Shin J W (2006), `Biological responses of ligament fibroblasts and gene expression profiling on micropatterned silicone substrates subjected to mechanical stimuli'. J Biosci Bioeng 102(5): 402±412. Petrigliano F A, English C S, Barba D, Esmende S, Wu B M and McAllister D R (2007), `The effects of local bFGF release and uniaxial strain on cellular adaptation and gene expression in a 3D environment: implications for ligament tissue engineering'. Tissue Eng 13(11): 2721±2731. Richmond J C, Manseau C J, Patz R and Mcconville O (1992), `Anterior cruciate

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reconstruction using a Dacron ligament prosthesis ± a long-term study'. Am J Sport Med 20(1): 24±28. Rickert M, Wang H L, Wieloch P, Lorenz H, Steek E, Sabo D and Richter W (2005), `Adenovirus-mediated gene transfer of growth and differentiation factor-5 into tenocytes and the healing rat Achilles tendon'. Connect Tissue Res 46(4±5): 175± 183. Sagarriga Visconti C, Kavalkovich K, Wu J and Niyibizi C (1996), `Biochemical analysis of collagens at the ligament-bone interface reveals presence of cartilage-specific collagens'. Arch Biochem Biophys 328(1): 135±142. Sciore P, Boykiw R and Hart D A (1998), `Semiquantitative reverse transcriptionpolymerase chain reaction analysis of mRNA for growth factors and growth factor receptors from normal and healing rabbit medial collateral ligament tissue'. J Orthop Res 16(4): 429±437. Sherwood J K, Riley S L, Palazzolo R, Brown S C, Monkhouse D C, Coates M, Griffith L G, Landeen L K and Ratcliffe A (2002), `A three-dimensional osteochondral composite scaffold for articular cartilage repair'. Biomaterials 23(24): 4739±4751. Spalazzi J P, Doty S B, Moffat K L, Levine W N and Lu H H (2006), `Development of controlled matrix heterogeneity on a triphasic scaffold for orthopedic interface tissue engineering'. Tissue Eng 12(12): 3497±3508. Tashiro T, Hiraoka H, Ikeda Y, Ohnuki T, Suzuki R, Ochi T, Nakamura K and Fukui N (2006), `Effect of GDF-5 on ligament healing'. J Orthop Res 24(1): 71±79. Thomopoulos S, Williams G R, Gimbel J A, Favata M and Soslowsky L J (2003), `Variation of biomechanical, structural, and compositional properties along the tendon to bone insertion site'. J Orthop Res 21(3): 413±419. Vacanti J P and Langer R (1999), `Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation'. Lancet 354: Si32±Si34. Vacanti J P, Morse M A, Saltzman W M, Domb A J, Perezatayde A and Langer R (1988), `Selective cell transplantation using bioabsorbable artificial polymers as matrices'. J Pediatr Surg 23(1): 3±9. Van Eijk F, Saris D B F, Riesle J, Willems W J, Van Blitterswijk C A, Verbout A J and Dhert W J A (2004), `Tissue engineering of ligaments: a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source'. Tissue Eng 10(5±6): 893±903. Vunjak-Novakovic G, Altman G, Horan R and Kaplan D L (2004), `Tissue engineering of ligaments'. Annu Rev Biomed Eng 6: 131±156. Wang J H C (2006), `Mechanobiology of tendon'. J Biomech 39(9): 1563±1582. Wang J H C, Jia F Y, Gilbert T W and Woo S L Y (2003), `Cell orientation determines the alignment of cell-produced collagenous matrix'. J Biomech 36(1): 97±102. Weiler A, Forster C, Hunt P, Falk R, Jung T, Unterhauser F N, Bergmann V, Schmidmaier G and Haas N P (2004), `The influence of locally applied plateletderived growth factor-BB on free tendon graft remodeling after anterior cruciate ligament reconstruction'. Am J Sport Med 32(4): 881±891. Wiig M E, Amiel D, Ivarsson M, Nagineni C N, Wallace C D and Arfors K E (1991), `Type-I procollagen gene-expression in normal and early healing of the medial collateral and anterior cruciate ligaments in rabbits ± an in situ hybridization study'. J Orthop Res 9(3): 374±382. Wolfman N M, Hattersley G, Cox K, Celeste A J, Nelson R, Yamaji N, Dube J L, DiBlasioSmith E, Nove J, Song J J, Wozney J M and Rosen V (1997), `Ectopic

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induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family'. J Clin Invest 100(2): 321±330. Woo S L Y, Abramowitch S D, Kilger R and Liang R (2006), `Biomechanics of knee ligaments: injury, healing, and repair'. J Biomech 39(1): 1±20. Yao L, Bestwick C S, Bestwick L A, Maffulli N and Aspden R M (2006), `Phenotypic drift in human tenocyte culture'. Tissue Eng 12(7): 1843±1849. Yoon J H and Halper J (2005), `Tendon proteoglycans: biochemistry and function'. J Musculoskelet Neuronal Interact 5(1): 22±34.

18

Cell-based therapies for the repair and regeneration of tendons and ligaments R . K . W . S M I T H , The Royal Veterinary College, UK

Abstract: The importance of many orthopaedic diseases is the persistent morbidity associated with poor functional repair. The complex array of cellular responses available with cell therapy, where live cells are implanted into the damaged tissue, offer an improved therapeutic effect in those diseases where current therapeutic strategies have minimal effectiveness. This chapter will discuss the current options for the use of cell therapies to treat tendon and ligament injuries and review both the experimental data and the clinical results for its use in the horse to treat naturally occurring overstrain injury which can be considered a `proving ground' for the treatment of similar human diseases. Key words: tendon, tendinopathy, cell therapy, stem cell, horse.

18.1

Introduction

The importance of many orthopaedic diseases lies in their persistent morbidity associated with poor functional repair. There has therefore been considerable interest in regenerative strategies for injuries where natural repair mechanisms do not deliver functional recovery and for which current therapeutic strategies have minimal effectiveness. At the forefront of these strategies is the use of cellbased therapy where live cells are implanted into the damaged tissue and the complex cellular responses are hoped will mediate an improved therapeutic effect. This chapter will discuss the current options for the use of cell-based therapies to treat tendon and ligament injuries. These injuries have many attributes that make them appropriate for cell-based therapies. Cell therapy is a fledging technology where many questions still need to be answered but this chapter will review the progress in our understanding of the role and effect of cell therapy, both in vitro and in vivo in laboratory animal models. Finally, the experience in the clinical use of bone marrow-derived mesenchymal stromal cells (MSCs) will be described in a species where the natural injury has similarities to the disease in humans and which can be viewed as a `proving ground' for such therapies.

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18.2

437

The rationale behind the use of cells to treat tendon and ligament injuries

Tendons and ligaments vary in their role depending on their location. However, they require two important mechanical properties ± structural strength to withstand the loads placed on them and elasticity. Tendons can be divided into two broad categories ± weight-bearing tendons and positional tendons. The latter requires high stiffness for precise movements while the former functions to store elastic energy during locomotion and hence has greater elasticity. The horse has evolved this function to an extreme with its superficial digital flexor tendon stretching up to 16% at the gallop1 and helping making the horse 120% efficient at the gallop.2 After injury, most tendons and ligaments are capable of mounting a fibrotic response that restores some function to the structure. However, this fibrous repair is functionally deficient compared with normal tendon is a number of ways. Scar tissue has less strength than normal tendon and ligament and so greater amounts are laid down to provide the necessary structural strength, which results in increased structural stiffness.3 In addition, one of the mechanisms whereby reparative cells gain access to the injured area are by the formation of adhesions, especially in intra-thecal portions of the tendon and the presence of these adhesions that can further limit the normal movement and elasticity of the structure. For some tendon injuries, pain and lameness are persistent and debilitating for patients. However, in many, pain is not usually a long-term feature, including the very common injury to the superficial digital flexor tendon in athletic horses. However, the inferior functionality of the repair to this structure results in reduced performance and a substantial risk of re-injury ± more than 50% of racehorses will suffer a re-injury within 2 years of returning to full work in spite of myriad of different treatments.4 There is therefore a primary need to restore functionality after this injury and this has encouraged the development of regenerative strategies. Regenerative medicine traditionally utilises three different components ± a scaffold, an anabolic stimulus and a cell source. Post-injury, tendon does not exhibit a deficiency with cellular infiltration but recent studies5 using transgenic rats have indicated that while there is a population of cells derived from the bone marrow that infiltrates the injury early, these are more likely to be components of the white blood cell series involved in debridement and the cells remaining within the reparative tissue and therefore most likely to be involved with new matrix synthesis were mostly locally derived from the peritendinous tissues. Given that natural repair produces a functionally inferior structure through the synthesis of scar tissue, a beneficial effect of the addition of an exogenous cell source capable of synthesising a matrix more resembling tendon tissue can be envisaged. Equine digital flexor tendon strain injuries provide many of the

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elements required for tendon tissue engineering ± the lesion manifests within the central core of the tissue,6 thus providing a natural enclosure for implantation and, by the time of cell implantation, is filled with granulation tissue which acts in the role of a scaffold. It has the added advantage of being highly vascularised and therefore capable of nutritional support of the implanted cells. The cytokine and mechanical environments, which are potentially important drives for differentiation, are provided by the intra-tendinous location of the cells and by the addition of anabolic factors at the same time as cell implantation.

18.3

Cell choice for tendon and ligament treatment

18.3.1 Differentiated cells Tendon-derived cells A logical source of cells for making new tendon tissue would be the tenocytes themselves.7 However, the removal of sections of tendon to digest out cells leads to formation of a secondary lesion at the donor site, unacceptable for flexor tendons in the horse.8 Furthermore, owing to the different cell characteristics that arise in different tendons and regions of tendon,9 using cells from positional tendons or from other areas of tendon may also not be ideal. Ligament-derived cells Ligament-derived cells have certain advantages over tendon-derived cells as they are often more metabolically active than other fibroblasts10 and hence potentially capable of more effective and rapid matrix synthesis. This is currently being attempted in clinical cases in Australia using autologous cells derived from the ligamentum nuchae (Casey P, personal communication) but no data has been published to date. Other fibroblast sources Fibroblasts can be sourced from many other tissues, including the skin and tendon sheath synovium.7 Dermal fibroblasts have been shown to be capable of functionally bridging a tendon defect in laboratory animal models with similar histological and tensile properties of the tenocyte seeded scaffold,11 although in vitro these cells behave differently from tenocytes.12

18.3.2 Undifferentiated cells Embryonic Embryonic stem cells (ESCs) are derived from the inner cell mass of the embryo and offer great potential as they are truly pluripotential but have the

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disadvantages of being allogenic (although with greater immunological tolerance) and associated with a risk of teratoma formation. Recent studies in the horse have suggested that cell lines can be derived from equine embryos.13±15 Although some lines have not formed terratomata when implanted into immunodeficient mice, this may just reflect that they are not true ESCs. However, even with the development of these cells for clinical use there are considerable ethical issues which hamper their use in the human. These issues may be, but not necessarily, less for equine use. Umbilical cord ± Wharton's jelly While umbilical cord blood is a relatively rich source of haematopoietic stem cells, studies have not consistently shown that umbilical cord blood and peripheral blood are reliable sources of MSCs although cells demonstrating multilineage differentiation capabilities have been recovered from both sources.16±19 Certainly work in the horse has not achieved reliable recovery20,21 (unpublished data). Cells from Wharton's jelly in the umbilical cord might be a superior source and it has been possible to recover cells from foals at the time of parturition20 (unpublished data). These cells potentially span the divide between foetal and post-natal stem cells and therefore have greater potential. Furthermore, they can be easily recovered at birth and stored for future use. However, considerably more work is needed in this area before these cells can be advocated for clinical use. Mesenchymal stem cells MSCs are derived from post-natal tissues and are found in varying but sparse amounts in many tissues. The terminology for these cells is not standardised. Mesenchymal stem cells should be reserved for those cell lines where both selfrenewal and multipotentiality (including function) has been demonstrated. Mesenchymal progenitor cells (MPCs) are probably best used to describe cells recovered from individual tissues that demonstrate the ability to differentiate into specific cell types. Tendon progenitor cells Most tissues have a sub or side population of precursor cells (tissue-specific progenitor cells) that are used to replenish cells due to natural turnover and aid in repair post-injury. Certainly evidence of multipotency has been shown for cells derived from rodent tendon.22 The exact site for these cells within tendon is not known but they are most likely to reside in the endotenon tissue between the collagen fascicles and adjacent to the vasculature. More recently a specific stem cell niche has been shown in tendon.23 However, in adult tendon, it has been

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difficult in our laboratories to demonstrate the presence of a cell sub-population capable of differentiating into multiple cell lines with similar ability to bone marrow-derived cells, which may explain why this component of the repair process is limited and hence natural repair inferior to normal tendon. Bone marrow Bone marrow contains a haematopoietic stem cell population from which the cellular components of blood are derived and an MSC population. The latter cell population, derived from the non-haematopoietic cells in bone marrow, that have, for many years, been shown to be multipotential because, in addition to self-replication, they are able to differentiate into osteoblasts (bone), chondrocytes (cartilage), tenocytes (tendon and ligament), fibroblasts (scar tissue), adipocytes (fat) and myofibroblasts (myotubes).24 However, the actual numbers of MSCs in bone marrow are very small, estimated at between 1 in 10 000 and 1 in 100 000 of the nucleated cells in an aspirated bone marrow sample.25 The isolation of MSCs frequently, but not necessarily, begins with subfractionation on a density gradient solution such as Percoll. The MSC-containing fraction is then transferred to culture flasks where their recovery relies on their ability to adhere to the tissue culture plastic and form colonies of cells when plated at low cell densities. This property is used to separate MSCs from haemopoietic cells that are removed with the media when the culture media is changed. Bone marrow aspirates in recent equine studies have yielded 6:4  3:4  109 /l nucleated cells in bone marrow aspirate26,27 which, is comparative to human bone marrow aspirates.28 The MSCs have a fibroblastic morphology in culture.29 Adipose-derived MSCs can also be derived from adipose tissue where some studies have been shown to be indistinguishable from bone marrow-derived MSCs.30 However, others have shown them to differentiate less capably into specific cell lines.31±33 Defining the mesenchymal stem cell population MSCs are defined as cells that have two fundamental properties ± the ability of self-renewal and the capability to differentiate into a limited number of different cell lines.34 Self-renewal is demonstrated by the ability of the cultured cells to divide and that they retain their multipotentiality after a number of passages. Multipotentiality is usually demonstrated by the ability of the cultured stem cell population to differentiate into at least three separate cell lines, most commonly adipocytes, chondrocytes and osteoblasts.

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Adipogenesis is provoked by culturing in media containing isobutyl-methylxanthine, dexamethasone, insulin and indomethacin.30,35 Positive differentiation is usually identified by histological demonstration of the accumulation of intracellular lipid using Oil Red O stain, or can alternatively be identified more objectively by gene expression of fat-specific genes such as the transcription factor, peroxisome proliferator-activated receptor gamma (PPAR).36,37 Chondrogenic differentiation is usually achieved using pellet culture in media containing transforming growth factor beta 1. The differentiation into chondrocytes is demonstrated by a rounded cellular morphology38,39 and the synthesis of cartilage extracellular matrix proteins such as collagen type II and aggrecan,40 and/or gene expression for cartilage expressed genes such as the chondrogenic transcription factor Sox9.41 Osteogenesis in vitro can be induced by the addition into the media of ascorbic acid, -glycerophosphate and dexamethasone,42 where positive differentiation is usually confirmed by increased expression of alkaline phosphatise (although not specific for osteogenesis), the formation of mineralised nodules demonstrated by staining with von Kossa or alizarin red stains, and/or gene expression of bone-specific genes such as the transcription factor, core-binding factor alpha-1 (CBFa1), and extracellular matrix genes such as osteocalcin and bone sialoprotein.43 However, the recovery of cells from a stem cell source, such as bone marrow or fat, yields a heterogeneous population where not all cells demonstrate such multipotentiality. To characterise these cells further and therefore to enable the sorting of cells into a more homogenous cell population, cell surface markers have been used. This include SH2 (CD105), SH3 (CD73) and Stro 1.25,44,45 These cell surface markers are rarely specific for MSCs but by using a panel of both positive and negative (usually against the haematopoietic stem cells) markers, greater confidence in defining a stem cell population can be achieved. Unfortunately, many of the monoclonal antibodies raised against these cell surface markers in human MSCs show limited cross-reactivity in equine MSCs, preventing them being used to sort cells at this time. There is a real need to develop such tools for both quality control and optimising MSC cultures in their use clinically in the horse (see below).

18.4

Mixed cell populations

The nucleated cell population from bone marrow without culture has been assessed in tendon healing models. These preparations have a markedly reduced proportion of MSCs but so far the results of these studies have suggested that these preparations may be as effective as the culture cells.46 This technique has the advantage of allowing `patient-side' treatments without the need for an interval for culture, thereby optimising timing and cost of treatment.

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Regenerative medicine and biomaterials in connective tissue repair

Allogenic versus autologous sources

An allogenic source would allow an `off-the shelf' product which could then be given at an optimum time in the disease process rather than one governed by culture times. This would also potentially make the product cheaper. In addition, the product could be standardised more easily although maintaining this standard throughout multiple population doublings necessary to supply sufficient cells would be a concern. MSCs are also not completely immunotolerant although they have been shown to suppress the immune response.47 Furthermore, allogenic cells would be a possible source of disease transmission and further regulatory issues would need to be addressed.

18.6

Proposed beneficial actions of stem cells on tendon healing

The goal in the use of stem cells is to engineer new tendon tissue utilising cellular synthetic machinery. This can be achieved either by the stem cells differentiating into tenocytes and synthesising the tendon matrix themselves or via a paracrine or trophic effect to provoke resident cell populations to synthesise new tissue. It is not known whether one or both of these actions occur after stem cell implantation, although it is likely that the latter action is the most important. A third effect may have secondary or primary benefits and that is the suppression of inflammation via demonstrated effects of MSCs on natural killer cells, and T and B cell function.47±51

18.7

Stem cell-induced tenogenesis ± in vitro

Tenocytic differentiation of MSCs can potentially be induced by a number of key factors ± an appropriate mechanical environment, contact with tenocytes and tendon extracellular matrix, and certain soluble growth factors and mediators.

18.7.1 Tenogenesis in 2D and 3D culture systems In vitro equine MSCs cultured in 2D and 3D matrices can be induced to synthesise matrices with some (but not all) the characteristics of tendon extracellular matrix. Equine MSCs can synthesise an abundant and remarkably well-structured matrix when cultured in vitro in a bioreactor within the coagulated supernatant of the bone marrow (unpublished data). However, while several confident determinants of osteogenic, lipidogenic and chondrogenic differentiation are available, demonstration of tenogenic differentiation has been hampered by the lack of a definitive tenocytic or tendon matrix marker. At present tenocytes are described as having fibroblast morphology (similar to

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MSCs), and so cannot be identified from appearance alone. Collagen type I is the primary protein synthesised by tenocytes, but this does not differentiate these cells from fibroblasts capable of producing connective tissues, including scar tissue. The synthesis of the glycoprotein cartilage oligomeric matrix protein (COMP) provides a more discriminating analysis but it too is not specific to tendon although it does have a restricted distribution to tissues primarily designed to withstand load (e.g. cartilage, tendon and fibrocartilage). Scleraxis52 and tenomodulin,53±55 genes encoding for transcription factors, have been shown to be expressed only by partially differentiated mesenchymal progenitor cells that are the precursors of connective tissue and tendon and therefore have been considered good markers for tendon tissue. However, more recent studies have suggested they are not as specific for this tissue as previously thought (unpublished data). The use of a `signature' of a broad range of synthesised extracellular matrix proteins will enable a better characterisation of tenogenesis.

18.7.2 Stem cells cultured on tendon explants/matrices Culturing MSCs on different substrates has been shown to enhance or induce differentiation into different lineages including osteogenic, chondrogenic and neurogenic. MSCs survive well on acellularised tendon matrices and equine MSCs, cultured on fresh acellular equine tendon sections, not only survived, proliferated and invaded the matrix, but also up-regulated COMP gene expression while down-regulating collagen I and III gene expression in comparison with gene expression when cultured on 2D matrices for a short period (unpublished data). Longer contact appears to change the morphology of implanted cells and using this in vitro model, longer culture times (3 weeks) resulted in greater similarities to tenocytes with MSCs lining up with the collagen fascicles.

18.8

Stem cell-induced tenogenesis ± in vivo

18.8.1 Laboratory animal models MSCs have been implanted into surgical defects in tendons in multiple in vivo experiments in laboratory animals with almost universally positive outcomes.56±60 Most of these models used surgically created defects in rabbit or rat tendons and have variously showed regeneration of new tendon-like tissue in defects implanted with MSCs in a biodegradable scaffold (collagen gel, Vicryl knitted mesh or fibrin glue) as assessed by histology or simple biochemical assays. However, not all have shown an improvement in microstructure and, because of the use of allogenic cells, an inflammatory reaction persisted. Furthermore, the implanted cells exhibited fibroblast morphology but were not fully characterised as tenocytes. In more recent studies, MSC implantation was associated with both improved strength and quality of reparative tissue (determined by collagen I/III ratio).61,62

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Thus, MSC seeded constructs implanted in vivo have shown the ability to integrate into the tissue and synthesise tissue-specific extracellular matrix; however, it is unclear which factors are initiating this functional differentiation. There have been limited experimental studies performed in the horse. However, the survival of significant numbers of bone marrow MSCs has been shown after experimental injection.63,64

18.8.2 Clinical experience with naturally occurring equine tendon disease Technique Two techniques are currently available for the treatment of equine tendon and ligament injuries with MSCs. One utilises a cultured cell population derived from bone marrow26 and the other relies on a mixed cell population derived from adipose tissue. Each technique has its strengths and weaknesses. Bone marrow-derived MSCs We have chosen to use bone marrow-derived MSCs as they are the most investigated and characterised post-natally derived stem cell and they appear to perform superiorly to MSCs recovered from other tissues (including tendon) in terms of differentiation into known cell types.65 Bone marrow is recovered from the sternum (or tuber coxae) under standing sedation and transferred to a laboratory in specially designed containers for culture and expansion of MSCs. Cultures from aspirated equine bone marrow have shown, as for other species, ability to differentiate into different lineages.27,39,66 After approximately 3 weeks, the cultured cells are transferred back to the veterinarian (10±50  106 cells, depending on the extent of the lesion) and implanted into the damaged tendon of the same horse under ultrasound guidance. The cells are suspended in bone marrow supernatant for implantation so that no `foreign' material is implanted and to gain potential beneficial effects from the rich mix of growth factors present in the supernatant.67 After implantation, the limb is bandaged and the horse undergoes a week of box rest to allow the cells to `take up residence in the tissue'. Thereafter the horse enters a controlled exercise programme for up to 48 weeks. This procedure has now become routine in equine clinical practice in the UK, Europe and Australia and training courses are run to educate veterinarians on the technology, criteria for treatment and the practicalities of the technique to minimise inappropriate use. Adipose-derived MSCs The currently available technique utilises a mixture of cells derived from the adipose tissue (taken surgically from the tail head) once the cells containing fat

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have been removed. This does not involve a culture step and therefore has the advantage of cheaper cost and speed of preparation (cells are returned to the practitioner within 48 hours). However, the cell mixture is thought to be heterogeneous with regards to cell type, only an estimated 2% being MSCs. The cells shipped back to the veterinarian and are implanted under ultrasound guidance as outlined above. This technique is used only in the USA. Safety aspects Safety has been a major concern in the human field because of the possibility that stem cells, which, by definition, have self-replicative capacity, may be able to provoke neoplasia when implanted into other tissues. Prior to using BMMSCs clinically, six horses with large core lesions in their SDFTs were treated to ensure safety of the technique. The results from this initial trial indicated that the technique did not cause any worsening of the injury. Furthermore, there was no reaction or enlargement of the tendon post-implantation, and no bone or cartilage was formed based on gamma scintigraphy and ultrasonography. Our current clinical experience in more than 700 horses has demonstrated that implantation of autologous MSCs has not resulted in the formation of different normal or abnormal (e.g. tumorous) tissues within the implantation site. One horse has developed ossification in the subcutaneous tissues but not within the tendon itself. Diseases treated ± tendon, ligament, meniscal, cruciate Currently, a wide range of tendon and ligament injuries have been treated in the horse, the most common being tendinopathy of the superficial digital flexor tendon which is the most common overstrain tendon injury in the horse. I proposed that the optimum time to implant the cells is after the initial inflammatory phase but before fibrous tissue formation. It was hypothesised that the presence of mature fibrous tissue within the tendon would (a) make implantation more difficult and (b) reduce the benefits of the stem cell therapy due to its persistence both of which have been supported by clinical experience of delayed implantation of bone marrow-derived MSCs and outcome ± successes had an average interval between injury and implantation of 44 days while, horses suffering re-injury, this was 83 days (p < 0:004). Current recommendations are that bone marrow is aspirated within 1 month of injury and, for the same reason, known recurrent injuries are not considered ideal cases because significant fibrosis would already be present. The time of implantation may be further optimised by pre-injury storage of cells. Lesions to tendons and ligaments that damage the surface of the structure, such as following percutaneous injury or in intra-thecal tendon and ligament tears, have not been considered candidates for stem cell therapy because of the

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inability to retain the cells within the lesion after implantation. In fact, for lesions present within a tendon sheath, the implantation is performed after tenoscopic evaluation to ensure that there are no previously unrecognised surface defects through which the cells could leak. This may be an unnecessary worry, as intra-synovial MSCs appear to engraft better into the soft tissues of the joint and induce the formation of new tissue.68 Therefore MSCs may be particularly useful for treating injuries to structures such as tendon, meniscal and intra-articular ligament tears. Outcome Bone marrow-derived MSCs During the initial safety trial, core lesions were found to fill in quickly when a hypoechoic lesion was still visible at the time of implantation. The longitudinal pattern, however, remained inferior to normal tendon but improved with exercise. Since this initial trial, in excess of 700 horses have been treated with this technique. At the most recent evaluation of clinical outcome (September 2007), 172 racehorses had been treated with >1 year follow-up. For National Hunt racehorses (n ˆ 145), the re-injury rate was 18% (23% when injuries to untreated contralateral limb were included). When only those horses which had entered full training and had been followed up for 2 years after treatment were included, the re-injury rate rose to 24% (35% with contralateral re-injuries). This re-injury rate was significantly better than for the same type of horse treated conventionally and followed for the same period of time (56% re-injury rate for National Hunt horses;4 p < 0:05). In further support for this improvement in outcome, re-injury rates for sports horses (all disciplines combined; n ˆ 109 with more than 1 year follow-up) was improved by a similar degree (13% compared with 23±43% reported for different sports horse disciplines.4 While this data shows encouraging signs of efficacy, it is not able to prove definitively improved healing over conventionally treated animals because no contemporary control animals were included. It is frequently not possible to obtain a control population for clinical cases treated in a referral institute within the equine industry. In addition, equine superficial digital flexor tendonitis is a highly variable condition where many factors influence the prognosis. Proof of efficacy will come from controlled experimental studies which are currently being performed and longer follow-up of carefully characterised clinical cases. Three cases which have died through unrelated causes have been analysed histologically and showed excellent healing with minimal inflammatory cells, and crimped organised collagen fibres. In contrast, a contralateral untreated suspensory ligament injury in one of these horses, which was clinically silent at the time of implantation, showed persistent inflammatory cells and poorly organised collagen fibres (unpublished data).

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Adipose-derived MSCs A large number of horses have been treated in the USA with this technique although no outcome data has been published to date. A pilot study using two groups of four horses in which lesions were created in the SDFT with collagenases has been performed to assess the efficacy of over phosphate buffered saline-injected control tendons. This showed a significant improvement in tissue organisation as assessed by histological score in the adipose-derived MSC treated tendons (Nixon AJ, personal communication).

18.9

Conclusions

There are some encouraging aspects to this technology, although definitive proof of efficacy is still lacking. This is essential before full confidence in the technology can be achieved. Furthermore there have been no direct comparisons between the two techniques currently available for use commercially. It must be remembered that there are still considerable gaps in our knowledge although the technology is developing rapidly. Although cell-based therapies are likely to be another instrument for tackling orthopaedic disease in the future, it is also likely that we will need to be selective in choosing the right clinical cases. It is also hoped that experience gained from treating clinical cases in horses will provide sufficient supportive data to encourage the translation of this technology into the human field where largely randomised control trials will lead to better evidencebased medicine.

18.10 Sources of further information and advice Richardson LE, Dudhia J, Clegg PD, Smith RKW (2007) Stem cells in veterinary medicine ± attempts at regenerating equine tendon after injury. Trends in Biotechnology 9: 409±16. Smith RK, Webbon PM. (2005) Harnessing the stem cell for the treatment of tendon injuries: heralding a new dawn? Br J Sports Med 39: 582±4. Taylor SE, Smith RK, Clegg PD. (2007) Mesenchymal stem cell therapy in equine musculoskeletal disease: scientific fact or clinical fiction? Equine Vet J 39: 172±80.

18.11 References 1. Stephens PR, Nunamaker DM, Butterweck DM. Application of a Hall-effect transducer for measurement of tendon strains in horses. Am J Vet Res 1989; 50(7): 1089±95. 2. Minetti AE, Ardig OL, Reinach E, Saibene F. The relationship between mechanical work and energy expenditure of locomotion in horses. J Exp Biol 1999; 202(Pt 17): 2329±38. 3. Crevier-Denoix N, Collobert C, Pourcelot P, et al. Mechanical properties of pathological equine superficial digital flexor tendons. Equine Vet J 1997; 23: 23±6.

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4. Dyson SJ. Medical management of superficial digital flexor tendonitis: a comparative study in 219 horses (1992±2000). Equine Vet J 2004; 36(5): 415±19. 5. Kajikawa Y, Morihara T, Watanabe N, et al. GFP chimeric models exhibited a biphasic pattern of mesenchymal cell invasion in tendon healing. J Cell Physiol 2007; 210(3): 684±91. 6. Webbon PM. A post mortem study of equine digital flexor tendons. Equine Vet J 1977; 9(2): 61±7. 7. Kryger GS, Chong AK, Costa M, Pham H, Bates SJ, Chang J. A comparison of tenocytes and mesenchymal stem cells for use in flexor tendon tissue engineering. J Hand Surgery 2007; 32(5): 597±605. 8. Webbon PM. Preliminary study of tendon biopsy in the horse. Equine Vet J 1986; 18(5): 383±7. 9. Goodman SA, May SA, Heinegard D, Smith RK. Tenocyte response to cyclical strain and transforming growth factor beta is dependent upon age and site of origin. Biorheology 2004; 41(5): 613±28. 10. Amiel D, Frank C, Harwood F, Fronek J, Akeson W. Tendons and ligaments: a morphological and biochemical comparison. J Orthop Res 1984; 1(3): 257±65. 11. Liu W, Chen B, Deng D, Xu F, Cui L, Cao Y. Repair of tendon defect with dermal fibroblast engineered tendon in a porcine model. Tissue Engineering 2006; 12(4): 775±88. 12. Evans CE, Trail IA. Fibroblast-like cells from tendons differ from skin fibroblasts in their ability to form three-dimensional structures in vitro. J Hand Surgery 1998; 23(5): 633±41. 13. Saito S, Sawai K, Minamihashi A, Ugai H, Murata T, Yokoyama KK. Derivation, maintenance, and induction of the differentiation in vitro of equine embryonic stem cells. Methods Mol Biol 2006; 329: 59±79. 14. Fortier LA. Stem cells: classifications, controversies, and clinical applications. Vet Surg 2005; 34(5): 415±23. 15. Li X, Zhou SG, Imreh MP, Ahrlund-Richter L, Allen WR. Horse embryonic stem cell lines from the proliferation of inner cell mass cells. Stem Cells Devel 2006; 15(4): 523±31. 16. Reed SA, Johnson SE. Equine umbilical cord blood contains a population of stem cells that express Oct4 and differentiate into mesodermal and endodermal cell types. J Cell Physiol 2008; 215(2): 329±36. 17. Wexler SA, Donaldson C, Denning-Kendall P, Rice C, Bradley B, Hows JM. Adult bone marrow is a rich source of human mesenchymal 'stem' cells but umbilical cord and mobilized adult blood are not. Br J Haematol 2003; 121(2): 368±74. 18. Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 2004; 103(5): 1669±75. 19. Koerner J, Nesic D, Romero JD, Brehm W, Mainil-Varlet P, Grogan SP. Equine peripheral blood-derived progenitors in comparison to bone marrow-derived mesenchymal stem cells. Stem Cells 2006; 24(6): 1613±19. 20. Hoynowski SM, Fry MM, Gardner BM, et al. Characterization and differentiation of equine umbilical cord-derived matrix cells. Biochem Biophys Res Commun 2007; 362(2): 347±53. 21. Koch TG, Heerkens T, Thomsen PD, Betts DH. Isolation of mesenchymal stem cells from equine umbilical cord blood. BMC Biotechnol 2007; 7: 26. 22. Salingcarnboriboon R, Yoshitake H, Tsuji K, et al. Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property. Exp Cell Res 2003; 287(2): 289±300.

Cell-based therapies in repair and regeneration

449

23. Bi Y, Ehirchiou D, Kilts TM, et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nature Medicine 2007; 13(10): 1219±27. 24. Caplan AI. Mesenchymal stem cells. J Orthop Res 1991; 9(5): 641±50. 25. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284(5411): 143±7. 26. Smith RK, Korda M, Blunn GW, Goodship AE. Isolation and implantation of autologous equine mesenchymal stem cells from bone marrow into the superficial digital flexor tendon as a potential novel treatment. Equine Vet J 2003; 35(1): 99± 102. 27. Vidal MA, Kilroy GE, Johnson JR, Lopez MJ, Moore RM, Gimble JM. Cell growth characteristics and differentiation frequency of adherent equine bone marrowderived mesenchymal stromal cells: adipogenic and osteogenic capacity. Vet Surg 2006; 35(7): 601±10. 28. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284: 143±7. 29. Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 2004; 36(4): 568±84. 30. De Ugarte DA, Morizono K, Elbarbary A, et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells, Tissues, Organs 2003; 174(3): 101±9. 31. Im GI, Shin YW, Lee KB. Do adipose tissue-derived mesenchymal stem cells have the same osteogenic and chondrogenic potential as bone marrow-derived cells? Osteoarthritis Cartilage/OARS, Osteoarthritis Res Soc 2005; 13(10): 845±53. 32. Park J, Gelse K, Frank S, von der Mark K, Aigner T, Schneider H. Transgeneactivated mesenchymal cells for articular cartilage repair: a comparison of primary bone marrow-, perichondrium/periosteum- and fat-derived cells. J Gene Med 2006; 8(1): 112±25. 33. Kisiday JD, Kopesky PW, Evans CH, Grodzinsky AJ, McIlwraith CW, Frisbie DD. Evaluation of adult equine bone marrow- and adipose-derived progenitor cell chondrogenesis in hydrogel cultures. J Orthop Res 2008; 26(3): 322±31. 34. Lee EH, Hui JH. The potential of stem cells in orthopaedic surgery. J Bone Joint Surg Br 2006; 88(7): 841±51. 35. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002; 13(12): 4279±95. 36. Gimble J, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy 2003; 5(5): 362±9. 37. Neubauer M, Fischbach C, Bauer-Kreisel P, et al. Basic fibroblast growth factor enhances PPARgamma ligand-induced adipogenesis of mesenchymal stem cells. FEBS Lett 2004; 577(1±2): 277±83. 38. Fortier LA, Nixon AJ, Williams J, Cable CS. Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells. Am J Vet Res 1998; 59(9): 1182±7. 39. Worster AA, Brower-Toland BD, Fortier LA, Bent SJ, Williams J, Nixon AJ. Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-beta1 in monolayer and insulin-like growth factor-I in a three-dimensional matrix. J Orthop Res 2001; 19(4): 738±49. 40. Yoo JU, Barthel TS, Nishimura K, et al. The chondrogenic potential of human bonemarrow-derived mesenchymal progenitor cells. J Bone Joint Surg 1998; 80(12): 1745±57.

450

Regenerative medicine and biomaterials in connective tissue repair

41. Asou Y, Nifuji A, Tsuji K, et al. Coordinated expression of scleraxis and Sox9 genes during embryonic development of tendons and cartilage. J Orthop Res 2002; 20(4): 827±33. 42. Herbertson A, Aubin JE. Dexamethasone alters the subpopulation make-up of rat bone marrow stromal cell cultures. J Bone Miner Res 1995; 10(2): 285±94. 43. Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997; 64(2): 295±312. 44. Barry FP, Boynton RE, Haynesworth S, Murphy JM, Zaia J. The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105). Biochem Biophys Res Commun 1999; 265(1): 134±9. 45. Gronthos S, Graves SE, Ohta S, Simmons PJ. The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors. Blood 1994; 84(12): 4164±73. 46. Crovace A, Lacitignola L, De Siena R, Rossi G, Francioso E. Cell therapy for tendon repair in horses: an experimental study. Vet Res Commun 2007; 31 Suppl 1: 281±3. 47. Ryan JM, Barry FP, Murphy JM, Mahon BP. Mesenchymal stem cells avoid allogeneic rejection. J Inflammation 2005; 2: 8. 48. English K, Barry FP, Field-Corbett CP, Mahon BP. IFN-gamma and TNF-alpha differentially regulate immunomodulation by murine mesenchymal stem cells. Immunol Lett 2007; 110(2): 91±100. 49. Corcione A, Benvenuto F, Ferretti E, et al. Human mesenchymal stem cells modulate B-cell functions. Blood 2006; 107(1): 367±72. 50. Klyushnenkova E, Mosca JD, Zernetkina V, et al. T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression. J Biomed Sci 2005; 12(1): 47±57. 51. Sotiropoulou PA, Perez SA, Gritzapis AD, Baxevanis CN, Papamichail M. Interactions between human mesenchymal stem cells and natural killer cells. Stem Cells 2006; 24(1): 74±85. 52. Schweitzer R, Chyung JH, Murtaugh LC, et al. Analysis of the tendon cell fate using Scleraxis, a specific marker for tendons and ligaments. Development 2001; 128(19): 3855±66. 53. Docheva D, Hunziker EB, Fassler R, Brandau O. Tenomodulin is necessary for tenocyte proliferation and tendon maturation. Mol Cell Biol 2005; 25(2): 699±705. 54. Oshima Y, Shukunami C, Honda J, et al. Expression and localization of tenomodulin, a transmembrane type chondromodulin-I-related angiogenesis inhibitor, in mouse eyes. Investigative Ophthalmol Visual Sci 2003; 44(5): 1814±23. 55. Shukunami C, Oshima Y, Hiraki Y. Molecular cloning of tenomodulin, a novel chondromodulin-I related gene. Biochem Biophys Res Commun 2001; 280(5): 1323± 7. 56. Chong AK, Ang AD, Goh JC, et al. Bone marrow-derived mesenchymal stem cells influence early tendon-healing in a rabbit Achilles tendon model. J Bone Joint Surg 2007; 89(1): 74±81. 57. Awad HA, Boivin GP, Dressler MR, Smith FN, Young RG, Butler DL. Repair of patellar tendon injuries using a cell-collagen composite. J Orthop Res 2003; 21(3): 420±31. 58. Awad HA, Butler DL, Boivin GP, et al. Autologous mesenchymal stem cellmediated repair of tendon. Tissue Engineering 1999; 5(3): 267±77. 59. Butler DL, Juncosa-Melvin N, Boivin GP, et al. Functional tissue engineering for tendon repair: a multidisciplinary strategy using mesenchymal stem cells, bioscaffolds, and mechanical stimulation. J Orthop Res 2008; 26(1): 1±9.

Cell-based therapies in repair and regeneration

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60. Young RG, Butler DL, Weber W, Caplan AI, Gordon SL, Fink DJ. Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair. J Orthop Res 1998; 16(4): 406±13. 61. Hankemeier S, van Griensven M, Ezechieli M, et al. Tissue engineering of tendons and ligaments by human bone marrow stromal cells in a liquid fibrin matrix in immunodeficient rats: results of a histologic study. Arch Orthop Trauma Surgery 2007; 127(9): 815±21. 62. Hankemeier S vGM, Hurschler C, Westdoerp J, Jagodzinski M, Bosch U, Krettek C, Zeichen J. Tissue engineering of ligaments by human bone marrow stromal cells in a liquid fibrin matrix: histological, biomechanical, and molecular biological results of a study with immunodeficient rats. Proceedings of the 51st Orthopaedic Research Society Meeting 2005; 30: 157. 63. Carstanjen B, Desbois C, Hekmati M, Behr L. Successful engraftment of cultured autologous mesenchymal stem cells in a surgically repaired soft palate defect in an adult horse. Canadian J Vet Res/Rev Canadienne Recherche Vet 2006; 70(2): 143±7. 64. Guest DJ, Smith MR, Allen WR. Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: preliminary study. Equine Vet J 2008; 40(2): 178±81. 65. Strassburg S. SR, Goodship AE, Hardingham TE, Clegg PD. Adult and late foetal equine tendon contain cell populations with weak progenitor properties in comparison to bone marrow derived mesenchymal stem cells. Proceedings of the 52nd Orthopaedic Research Society 2006; 31: 1113. 66. Arnhold SJ, Goletz I, Klein H, et al. Isolation and characterization of bone marrowderived equine mesenchymal stem cells. Am J Vet Res 2007; 68(10): 1095±105. 67. Smith JJ, Ross MW, Smith RK. Anabolic effects of acellular bone marrow, platelet rich plasma, and serum on equine suspensory ligament fibroblasts in vitro. Vet Comp Orthop Traumatol 2006; 19(1): 43±7. 68. Murphy JM, Fink DJ, Hunziker EB, Barry FP. Stem cell therapy in a caprine model of osteoarthritis. Arthritis Rheumatism 2003; 48(12): 3464±74.

19

Scaffolds for tendon and ligament tissue engineering J . C . H . G O H and S . S A H O O , National University of Singapore, Singapore

Abstract: An optimal scaffold that biomimics the mechanical and functional characteristics of tendons and ligaments is essential for successful tissue engineering of these dense connective tissues. In this chapter, we review the requirements and criteria that such scaffolds should meet, and the various biomaterials and fabrication techniques used in developing such scaffolds. Several synthetic and natural biomaterials, as well as their composites, have been fabricated into fibre-based, gel-based or hybrid scaffolds in an attempt to best achieve the mix of mechanical and functional properties. A new breed of biofunctional scaffolds incorporating growth factors, genes, and active functional groups are currently been developed. Lastly, tissue engineering strategies to regenerate the tendon/ligament±bone interface are also reviewed. Key words: connective tissues, fibrous scaffolds, biomimetic scaffolds, silk, collagen, polyesters.

19.1

Criteria and requirements for tendon/ligament tissue engineering scaffolds

Successful regeneration of tendons and ligaments using a tissue engineering (TE) approach would require the engineered tissue to possess similar mechanical and functional characteristics as the dense connective tissues (Langer and Vacanti 1993; Louie et al. 1998; Lin et al. 1999; Woo et al. 1999; Koob 2002; Butler et al. 2003, 2004a,b; Goh et al. 2003, 2004). The scaffold plays a central role in providing these characteristics to the engineered tissue as it functions as the template upon which seeded cells grow and lay down the new tissue over a certain period of time, during which the evolving construct should possess sufficient mechanical properties to support the healing tissue. A TE scaffold is essentially an engineered replacement of the native extracellular matrix (ECM), and the ideal scaffold would be the one that could biomimic the mechanical and functional characteristics of the desired tissue. A scaffold that is biocompatible, biodegradable and porous, and that possesses a geometry that imparts required mechanical properties and sufficient surface area for cell attachment, growth and proliferation, a suitable degradation rate, and biofunctionality to influence cell

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fate (Mikos and Temenoff 2000; Chen et al. 2001; Hutmacher 2001) is thus desirable. Difficulty in fabricating such a scaffold has been a limitation facing tendon/ligament TE research (Calve et al. 2004; Caplan 2004; Goh et al. 2004; Vunjak-Novakovic et al. 2004). In the following section, the various biomaterials that have been used in tendon and ligament TE applications are summarized.

19.2

Biomaterials for tendon and ligament tissue engineering

Several synthetic polymeric systems as well as natural biomaterials have been used for fabrication of scaffolds for tendon and ligament TE (Table 19.1). These include biodegradable polyesters, poly(-hydroxy acids), polyanhydrides, poly(orthoesters), polyurethanes and polycarbonates, biopolymers such as collagen, chitin and silk, and composites incorporating both synthetics and natural materials.

19.2.1 Synthetic biomaterials Owing to their ease of fabrication, reproducibility, and the versatility of the processing techniques involved, synthetic polymers have been commonly used as the biomaterials for scaffolds. Unlike autografts, synthetic polymeric scaffolds carry no risk of disease transmission and can also be sterilized easier than grafts made from natural materials without sacrificing the mechanical properties of the scaffold (Laurencin and Freeman 2005). Fibrous tissues such as tendons and ligaments require fibrous scaffolds for optimal tissue engineering, and synthetic polymers can be processed using various textile technologies to fabricate scaffolds in the form of woven or non-woven fabrics. Synthetic poly(-hydroxy acids) like polyglycolic acid, polylactic acid and their copolymer, poly-lactic-co-glycolic acid (PLGA) have been effectively used to fabricate mechanically strong and biodegradable porous scaffolds for tendon Table 19.1 Comparison of mechanical properties of several types of biomaterial fibres with tendon and ligament tissues (Wren et al. 2001; Altman et al. 2003; Lu et al. 2005; Dressler et al. 2006; Woo et al. 2006) Material

UTS (MPa)

Modulus (GPa)

Strain at break (%)

Bombyx mori silk Collagen X-linked Poly-L-lactic acid (PLLA) Patellar tendon Anterior cruciate ligament

740 47±72 28±50 50±150 13±46

10 0.4±0.8 1.2±3.0 0.1±0.6 0.1±0.3

20 12±16 2±6 15±30 10±40

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and ligament TE (Ouyang et al. 2003; Ge et al. 2005). Properties such as design flexibility, controllable porosity and degradation rate, complete in vivo bioresorption, the existence of several PLGA-based products approved by the US Food and Drug Administration (FDA) for human use (Athanasiou et al. 1996), make PLGA an attractive choice. However, rapid hydrolytic biodegradation of PLGA often causes complete loss of mechanical strength and integrity of PLGA-based scaffolds within weeks; such scaffolds would thus fail to support an injured tendon or ligament throughout its healing period that generally extends to months (Durselen et al. 2001; Wu and Ding 2004). This is one of the reasons behind excellent in vitro results but poor in vivo performance of grafts made of synthetic biomaterials (Bellincampi et al., 1998; Guidoin et al., 2000). An alternative biomaterial that does not degrade as rapidly as PLGA would be better suitable for tendon/ligament TE. Among polyesters, polycaprolactone (PCL) and poly- L -lactic acid (PLLA) have the advantage of slow biodegradability and have been used to fabricate scaffolds for ligaments (Cooper et al. 2007; Petrigliano et al. 2007); however, their excessive hydrophobicity results in poor cell attachment and proliferation on such scaffolds (Ouyang et al. 2002; Sahoo et al. 2007). PLLA reconstructed anterior cruciate ligament (ACL) in a sheep model retained only 12.3% of the ultimate tensile strength after 48 weeks (Laitinen et al. 1993). Attempts to improve PLLA biocompatibility by combining with collagen also failed to improve overall mechanical strength of the graft substitute because of poor integration between the PLLA and collagen (Dunn et al. 1997). While novel synthetic polymers such as poly(desamino tyrosyl-tyrosine ethyl ester carbonate) or poly(DTE carbonate) have been developed to overcome the problems of insufficient initial mechanical properties and rapid biodegradation, none of these possesses any significant improvements over the conventional polyesters (Bourke et al. 2004).

19.2.2 Natural biomaterials Several natural materials, such as collagen and silk, possess the advantage of being inherently fibrous, and their better mechanical properties and slow rate of biodegradation could ensure a gradual transfer of load from the scaffold to the healing ligament/tendon. Collagen-based scaffolds evoked immense initial interest as collagen is the principal component of tendon/ligament ECM (Dunn et al. 1995; Bellincampi et al. 1998). However, derived collagen, even after physical or chemical crosslinking, fails to reproduce the mechanical properties of natural collagenous structures, and is also limited by its allogenicity. Recently, the focus has shifted to the use of Bombyx mori silk, which also possesses a fibrous structure with excellent mechanical properties and slow biodegradation (Altman et al. 2002, 2003; Vepari and Kaplan 2007).

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Collagenous scaffolds are back in attention recently in the form of decellularized ECM-derived scaffolds, because of improved decellularization techniques. Such scaffolds have been derived from tendons/ligaments (Tischer et al. 2007; Whitlock et al. 2007) and other collagenous tissues including bovine pericardium (Integra Life Science), human dermal collagen (Alloderm), porcine small intestinal submucosa (Badylak et al., 1995, 1999; Dejardin et al., 1999; Musahl et al., 2004; Liang et al., 2006) and human umbilical vein (Abousleiman et al. 2008). The preserved natural three-dimensional ultrastructural hierarchy of ECM collagen, as well as the presence of other structural and functional proteins including elastin, growth factors and proteoglycans is expected to make these scaffolds mechanically and functionally viable, and thus an attractive choice for TE applications.

19.2.3 Composites and novel materials As glycosaminoglycans (GAGs) are a key component of tendon/ligament ECM and play an important role in improving biocompatibility, cell-adhesion properties and stimulate various in vitro tissue regenerative processes, novel composite materials have been developed incorporating GAGs into other synthetic or natural biomaterials. Hybrid polymers combining chitosan with hyaluronan or alginate have been used to develop biocompatible fibrous scaffolds with improved mechanical properties (Majima et al. 2005, 2007). Modified hyaluronic acid-based scaffolds showed improved bone marrow stromal cell (BMSC) growth and viability, as well as enhanced expression of collagen type I and other fibroblast markers over short-term culture.

19.3

Scaffold architecture

Since tendons and ligaments are fibrous tissues, TE approaches for their regeneration require fibre-based scaffolds. Such scaffolds could be in the form of parallel bundles (Altman et al. 2002), or fabricated into braided, knitted, woven or non-woven fabrics depending on the textile technology used.

19.3.1 Fibre-based scaffolds Braided and woven scaffolds Traditionally, braided and woven structures derived from synthetic polyester fibres have been used for ligament/tendon TE. While such scaffolds possess high initial mechanical properties and show good results in in vitro conditions (Karamuk et al. 1999; Laurencin et al. 1999; Van Eijk et al. 2004; Cooper et al. 2005, 2006; Lu et al. 2005; Freeman et al. 2007), there are only a few studies demonstrating their in vivo efficacy. In one study, braided PLLA scaffolds have

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shown favourable results 3 months post-implantation in a rabbit model (Cooper et al. 2007), but the long-term outcome of such scaffolds is still unknown. Silk derived from silkworms has also been used to fabricate fibrous scaffolds in the form of twisted cables or braids (Altman et al. 2002; Fan et al. 2008a,b). Such scaffolds have been shown to support attachment and proliferation of ligament cells as well as stem cells, and have been evaluated extensively via in vitro studies (Fan et al. 2008a,b). By controlling the number of yarns used, as well as the design of the construct (such as twisted, cabled or wire-rope, braided design), the mechanical properties of the scaffolds can be adjusted according to requirements (Horan et al. 2006). While cabled yarns possess the structural flexibility and mechanical properties desired for tendon/ligament TE applications, braided scaffolds were found to be unfavourable for cyclical mechanical loading and tissue ingrowth. Knitted scaffolds In comparison with braided scaffolds that often lack sufficient porosity and encounter problems of nutrient transmission and cell infiltration, knitted scaffolds have high porosity and preserved internal connective spaces under mechanical tension (Ouyang et al. 2003, 2005; Ge et al. 2005). These spaces allow enough cells to be seeded initially and permit ECM to form and deposit therein during the repair process, helping in functional integration of the engineered tissue into the surrounding tissues. Such knitted scaffolds have been coated with thin films of biomaterials such as PCL and collagen, with random or aligned layers of nanofibres, or with macroporous sponges of collagen or silk to produce hybrid scaffolds, These various coating techniques could modulate the mechanical properties and facilitate cell attachment and proliferation in the hybrid scaffolds (Sahoo et al. 2006b, 2007). PLGA and silk-based knitted scaffolds have been shown to be effective in in vivo repair and regeneration of tendons and ligaments in several injury models. Knitted PLGA scaffolds seeded with BMSCs using a fibrin gel were found to effectively repair rabbit patellar tendon defects over 12 weeks, while knitted silk scaffolds reinforced with collagen or silk sponges (Fig. 19.1) have been observed to heal rabbit medial collateral ligament (MCL) defects over 12 weeks and ACL defects over 24 weeks in separate studies (Ouyang et al. 2003; X. Chen et al. 2008; Fan et al. 2008c).

19.3.2 Gel-based scaffolds Water is a major component in all tissues, with the 3D fibrous network of proteins in the ECM surrounded by a hydrogel of GAGs. Thus several hydrogels, derived from collagens, fibrin and hyaluronic acid, have been used as scaffolds for engineering tendons and ligaments. However, gel-based systems

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19.1 Knitted silk microfibrous scaffolds reinforced with microporous silk sponge; BMSC-seeded composite scaffolds used to reconstruct and regenerate rabbit ACL (indicated by arrow) (Fan et al. 2008c).

lack mechanical properties required to replace or regenerate an injured tendon/ ligament, and are usually used only as a method of cell delivery. They have also been used, alone or in combination with cells, to repair partial tendon/ligament defects (Juncosa-Melvin et al. 2006a; Hankemeier et al. 2007), and combined with fibrous or spongy scaffolds to constitute hybrids scaffolds (Ouyang et al. 2003; Juncosa-Melvin et al. 2006b,c; Takezawa et al. 2007). A plastic compression technique has also been used to rapidly process collagen gels into dense and strong matrices that could be better suitable for TE applications (Brown et al. 2005).

19.3.3 Composite/hybrid scaffolds In an attempt to combine the mechanical advantages of a microscaled structure and the biomimetic nanoscaled features of gels, sponges and nanofibres, several hybrid scaffolds have been developed. Composite fibre and gel based scaffolds Fibrous scaffolds are often combined with gel systems such as fibrin or collagen gel as a media for cell delivery (Ouyang et al. 2003; Juncosa-Melvin et al. 2006b,c; Takezawa et al. 2007). However, the gel component is usually poorly integrated with the fibrous component, rendering the composite inherently unstable in mechanically dynamic environments like the knee joint. Porosity limitation of 3D gels also hinders nutrient transmission and cell proliferation within them (Louie et al. 1998; Jockenhoevel et al. 2001).

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Composite fibre and sponge-based scaffolds Microporous sponges have also been used in the place of gels to reinforce fibrous scaffolds. Silk, collagen and gelatin sponges have been incorporated into silk-based knitted microfibrous scaffolds using freeze-drying technique to form hybrid scaffolds that could regenerate injured ligaments in rabbit models (X. Chen et al. 2008; Fan et al. 2008b,c). After 6 months of implantation of BMSCseeded silk fibre-sponge scaffolds into the ACL site, the regenerated ligament possessed adequate mechanical properties and was observed to be uniformly populated with fibroblast-like cells within a collagenous matrix (Fig. 19.2) (Liu et al. 2008). Hybrid nano-microfibrous scaffolds Collagen type I fibrils in ECM are typically nanofibrous with diameters ranging between 50 and 500 nm. To be truly biomimetic, scaffolds would need to be made up of building blocks that possess dimensions similar to the natural ECM proteins; with this objective, microfibrous scaffolds have been coated with nanoscaled fibres to form nano-microscaffolds that showed improved cell proliferation and differentiation (Fig. 19.3). Nanofibres can be produced using the facile and versatile technique of electrospinning, and such fibres can form a porous nanostructure that closely mimics the natural ECM nanostructure (Fig. 19.4). Such hybrid scaffold systems combine the advantages of mechanical integrity of macrofibres and the large biomimetic surface of nanofibres (Sahoo et al. 2006b). Biphasic/triphasic scaffolds: interfacial tissue engineering Most of the current TE approaches aim at regenerating the tendon/ligament midsubstance. Such grafts require mechanical fixation with the adjacent bones and

19.2 Hybrid silk sponge reinforced fibrous scaffold(s) and the reconstructed ACL showing uniform population of cells within a collagenous matrix (Liu et al. 2008).

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19.3 Nano-microscaffold, formed by coating nanofibres on a microfibrous scaffold, demonstrated favourable BMSC proliferation on the scaffold surface (Sahoo et al. 2006b).

often fail to preserve or re-establish the anatomic soft tissue-to-bone enthesis post-surgery, thus compromising graft stability and long-term functional outcome. Consequently, there exists a significant need for engineering and regenerating the tendon/ligament±bone interface to facilitate functional graft-tobone integration. Towards this end, complex scaffolds have been developed by incorporating different phases, suitable for engineering tendon/ligament, cartilage and bone, into different zones of the scaffold. As an example, the ligament phase has been made up of a PLGA fibrous mesh, the interfacial phase of PLGA microspheres and the bone phase of sintered composite PLGA and bioactive glass microspheres (Spalazzi et al. 2006, 2008). Such scaffolds have been differentially seeded with multiple cell lines appropriate for regenerating the different target tissues (bone, fibrocartilage or ligament) (Fig. 19.5).

19.4 Mimicry of native ECM structure by an electrospun polymeric nanofibre matrix seeded with BMSCs.

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19.5 The distinct yet continuous regions at the ligament±bone interface can be mimicked by a tri-phasic scaffold design with separate zones for ligament, cartilage and bone, which could be differentially seeded with mature or precursor cells to regenerate the interface.

19.4

Functional scaffolds

The role of the ECM is not only to provide a template for resident cells to grow, but also to interact with and provide them with necessary stimuli to maintain and direct their function. In order for a TE scaffold to truly biomimic the natural ECM, the scaffold should not only mimic the ECM in structure, but also in function. Functional mimicry of the ECM has been attempted by incorporating biological or chemical signals into the scaffold, either in the form of genes, proteins or their active functional groups.

19.4.1 Growth factors Since growth factors lose their bioactivity rapidly by diffusional loss and/or enzymatic inactivation/degradation (Whalen et al. 1989; Mark Saltzman and Baldwin 1998), a sustained local delivery of bioactive growth factor at the healing site would be desirable for optimal effects in TE applications. Scaffolds

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can mimic ECM function by acting as a depot for sustained release of growth factors. Several putative growth and differentiation factors such as basic fibroblast growth factor (bFGF), transforming growth factor- (TGF- ), epidermal growth factor (EGF), platelet derived growth factor (PDGF), insulin-like growth factor (IGF), and growth and differentiation factor-5 (GDF-5) have been shown to be beneficial in tendon and ligament repair (Goh et al. 2003; Moreau et al. 2005a,b; Hoffmann and Gross 2007; Jenner et al. 2007; Yamada et al. 2008). These growth factors are usually expressed in a spatio-temporal manner, e.g. bFGF levels at the injury site peak within the first week (Chang et al. 1998; Berglund et al. 2006; Kobayashi et al. 2006; WurglerHauri et al. 2007), while TGF- 1 and PDGF-BB are over-expressed in the later phases of tendon healing (Lee et al. 1998; Ngo et al. 2001). Hence, bFGF has been incorporated into electrospun PLGA nanofibres to allow continued release of the growth factor over 1±2 weeks. Modifications of the electrospinning technique could produce either a random dispersion or an axial core-like distribution of the growth factor within the nanofibres; such scaffolds favoured BMSC attachment, subsequent proliferation and fibroblastic differentiation (Sahoo et al. 2006a, 2009) (Fig. 19.6). Such nanofibres when coated over a knitted scaffold could produce a nano-microscaffold capable of continued release of the incorporated growth factor, stimulating proliferation and differentiation of the seeded BMSCs. More commonly, growth factors have been delivered into scaffolds using hydrogels as the media (Whitaker et al. 2001). bFGF has been noncovalently bound and delivered via a collagen gel onto porous 3D PCL scaffolds

19.6 Different distribution patterns of growth factor in electrospun nanofibres to achieve different sustained release patterns.

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(Petrigliano et al. 2007). Bone morphogenetic protein-2 has been covalently tethered onto hyaluronic acid and delivered via a poly(ethylene glycol)-based photopolymerizable hydrogel to improve tendon±bone insertion in rabbits (C.H. Chen et al. 2008).

19.4.2 Functional groups Silk fibres coated with arginine-glycine-aspartic acid (RGD) peptide sequences have been shown to increase cell attachment, proliferation and ECM production by seeded BMSCs (Chen et al. 2003). Similarly, RGD-coated chitosan also demonstrated increased fibroblast proliferation (Masuko et al. 2005).

19.4.3 Gene therapy Gene transfer, wherein genetic material is delivered into target cells to alter their functions, can also be used prolong the delivery of high concentrations of growth factors at the repair site. Bone morphogenetic protein-12 (BMP12) and PDGF-B transfected mesenchymal stem cells (MSCs) have been shown to benefit healing of tendon/ligament defects when delivered by direct injection into injury site or via treated allografts (Lou et al. 2001; Li et al. 2007). Scaffold-based gene delivery has been shown using lacZ gene-marked allogenic MSCs in a fibrin glue embedded into 3-D fibrous scaffolds (Goh et al. 2003). Gene silencing through RNA-interference and short-interfering RNA (siRNA) is another promising technique whereby expression of target genes can be decreased, providing a valuable tool for promoting and directing the growth of functional tissues for TE applications (Cheema et al. 2007). Increased collagen type V and decorin levels in healing tendons and ligaments are known to adversely affect collagen type I fibrillogenesis. Antisense gene therapy that down-regulates decorin and collagen type V expression have resulted in formation of thicker collagen fibrils and significantly improved healing of tendon/ligament defects (Nakamura et al. 2000; Shimomura et al. 2003).

19.5

Future trends

Tendons and ligaments are highly ordered, complex tissues with mechanical properties that are crucial for normal joint kinematics. The current options for the therapy of tendon and ligament injuries do not provide satisfactory long-term results and new therapeutic modalities are required. For complete functional restoration of injuries to these tissues, tissue engineered constructs should possess a blend of biological activity and mechanical stability. Development of an optimal scaffold with appropriate biologic architecture and mechanical strength is the key to overcome this major future challenge.

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Mechanical stimulation, growth factors and hormones may be needed for an optimal control of the differentiation process during regeneration. A major deterrent to tendon/ligament TE research is the lack of knowledge about the precise local conditions that promote successful tendon/ligament development and repair. Future studies would need to define the temporal role of growth factors and exogenous stimulation in prompting organized collagen deposition. Engineering the interface between tendon/ligament and bone is another key issue that needs to be addressed in order to generate a complete bone±ligament± bone substitute that could replace current auto- or allograft transplants.

19.6

References

Abousleiman, R. I., Y. Reyes, et al. (2008). `The human umbilical vein: a novel scaffold for musculoskeletal soft tissue regeneration'. Artif Organs 32(9): 735±42. Altman, G. H., R. L. Horan, et al. (2002). `Silk matrix for tissue engineered anterior cruciate ligaments'. Biomaterials 23(20): 4131±41. Altman, G. H., F. Diaz, et al. (2003). `Silk-based biomaterials'. Biomaterials 24(3): 401± 16. Athanasiou, K. A., G. G. Niederauer, et al. (1996). `Sterilization, toxicity, biocompatibility and clinical applications of polylactic acid/polyglycolic acid copolymers'. Biomaterials 17(2): 93±102. Badylak, S. F., R. Tullius, et al. (1995). `The use of xenogeneic small intestinal submucosa as a biomaterial for Achilles tendon repair in a dog model'. J Biomed Mater Res 29(8): 977±85. Badylak, S., S. Arnoczky, et al. (1999). `Naturally occurring extracellular matrix as a scaffold for musculoskeletal repair'. Clin Orthop Relat Res 367 Suppl: S333±43. Bellincampi, L. D., R. F. Closkey, et al. (1998). `Viability of fibroblast-seeded ligament analogs after autogenous implantation'. J Orthop Res 16(4): 414±20. Berglund, M., C. Reno, et al. (2006). `Patterns of mRNA expression for matrix molecules and growth factors in flexor tendon injury: differences in the regulation between tendon and tendon sheath'. J Hand Surg [Am] 31(8): 1279±87. Bourke, S. L., J. Kohn, et al. (2004). `Preliminary development of a novel resorbable synthetic polymer fiber scaffold for anterior cruciate ligament reconstruction'. Tissue Eng 10(1±2): 43±52. Brown, R. A., M. Wiseman, et al. (2005). `Ultrarapid engineering of biomimetic materials and tissues: fabrication of nano- and microstructures by plastic compression'. Advanced Functional Materials 15(11): 1762±70. Butler, D. L., M. R. Dressler, et al. (2003). Functional tissue engineering: assessment of function in tendon and ligament repair. In Functional Tissue Engineering. F. Guilak, D. L. Butler, S. A. Goldstein and D. Mooney. New York, Springer-Verlag: 213±26. Butler, D. L., N. Juncosa, et al. (2004a). `Functional efficacy of tendon repair processes'. Annu Rev Biomed Eng 6: 303±29. Butler, D. L., J. T. Shearn, et al. (2004b). `Functional tissue engineering parameters toward designing repair and replacement strategies'. Clin Orthop Relat Res 427 Suppl: S190±9. Calve, S., R. G. Dennis, et al. (2004). `Engineering of functional tendon'. Tissue Eng 10(5±6): 755±61.

464

Regenerative medicine and biomaterials in connective tissue repair

Caplan, A. I. (2004). Design parameters for functional tissue engineering. In Functional Tissue Engineering. F. Guilak, D. L. Butler, S. A. Goldstein and D. Mooney, Springer: 129±38. Chang, J., D. Most, et al. (1998). `Molecular studies in flexor tendon wound healing: the role of basic fibroblast growth factor gene expression'. J Hand Surg [Am] 23(6): 1052±8. Cheema, S. K., E. Chen, et al. (2007). `Regulation and guidance of cell behavior for tissue regeneration via the siRNA mechanism'. Wound Repair Regen 15(3): 286± 95. Chen, C. H., H. W. Liu, et al. (2008). `Photoencapsulation of bone morphogenetic protein-2 and periosteal progenitor cells improve tendon graft healing in a bone tunnel'. Am J Sports Med 36(3): 461±73. Chen, G., T. Ushida, et al. (2001). `Development of biodegradable porous scaffolds for tissue engineering'. Materials Science and Engineering: C 17(1): 63±69. Chen, J., G. H. Altman, et al. (2003). `Human bone marrow stromal cell and ligament fibroblast responses on RGD-modified silk fibers'. J Biomed Mater Res A 67(2): 559±70. Chen, X., Y. Y. Qi, et al. (2008). `Ligament regeneration using a knitted silk scaffold combined with collagen matrix'. Biomaterials 29(27): 3683±92. Cooper, J. A., H. H. Lu, et al. (2005). `Fiber-based tissue-engineered scaffold for ligament replacement: design considerations and in vitro evaluation'. Biomaterials 26(13): 1523±32. Cooper, J. A., Jr., L. O. Bailey, et al. (2006). `Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament'. Biomaterials 27(13): 2747±54. Cooper, J. A., Jr., J. S. Sahota, et al. (2007). `Biomimetic tissue-engineered anterior cruciate ligament replacement'. Proc Natl Acad Sci USA 104(9): 3049±54. Dejardin, L. M., S. P. Arnoczky, et al. (1999). `Use of small intestinal submucosal implants for regeneration of large fascial defects: an experimental study in dogs'. J Biomed Mater Res 46(2): 203±11. Dressler, M. R., D. L. Butler, et al. (2006). `Age-related changes in the biomechanics of healing patellar tendon'. J Biomech 39(12): 2205±12. Dunn, M. G., J. B. Liesch, et al. (1995). `Development of fibroblast-seeded ligament analogs for ACL reconstruction'. J Biomed Mater Res 29(11): 1363±71. Dunn, M. G., L. D. Bellincampi, et al. (1997). `Preliminary development of a collagenPLA composite for ACL reconstruction'. Journal of Applied Polymer Science 63(11): 1423±1428. Durselen, L., M. Dauner, et al. (2001). `Resorbable polymer fibers for ligament augmentation'. J Biomed Mater Res 58(6): 666±72. Fan, H., H. Liu, et al. (2008a). `Enhanced differentiation of mesenchymal stem cells cocultured with ligament fibroblasts on gelatin/silk fibroin hybrid scaffold'. Biomaterials 29(8): 1017±27. Fan, H., H. Liu, et al. (2008b). `Development of a silk cable-reinforced gelatin/silk fibroin hybrid scaffold for ligament tissue engineering'. Cell Transplant 17(12): 1389±401. Fan, H., H. Liu, et al. (2008c). `In vivo study of anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold'. Biomaterials 29(23): 3324±37. Freeman, J. W., M. D. Woods, et al. (2007). `Tissue engineering of the anterior cruciate ligament using a braid-twist scaffold design'. J Biomech 40(9): 2029±36. Ge, Z., J. C. Goh, et al. (2005). `Characterization of knitted polymeric scaffolds for

Scaffolds for tendon and ligament tissue engineering

465

potential use in ligament tissue engineering'. J Biomater Sci Polym Ed 16(9): 1179± 92. Goh, J. C., H. W. Ouyang, et al. (2003). `Tissue-engineering approach to the repair and regeneration of tendons and ligaments'. Tissue Eng 9 Suppl 1: S31±44. Goh, J. C., H. W. Ouyang, et al. (2004). `Tissue engineering techniques in tendon and ligament replacement'. Med J Malaysia 59 Suppl B: 47±8. Guidoin, M. F., Y. Marois, et al. (2000). `Analysis of retrieved polymer fiber based replacements for the ACL'. Biomaterials 21(23): 2461±74. Hankemeier, S., M. van Griensven, et al. (2007). `Tissue engineering of tendons and ligaments by human bone marrow stromal cells in a liquid fibrin matrix in immunodeficient rats: results of a histologic study'. Arch Orthop Trauma Surg 127(9): 815±21. Hoffmann, A. and G. Gross (2007). `Tendon and ligament engineering in the adult organism: mesenchymal stem cells and gene-therapeutic approaches'. Int Orthop 31(6): 791±7. Horan, R. L., A. L. Collette, et al. (2006). `Yarn design for functional tissue engineering'. J Biomech 39(12): 2232±40. Hutmacher, D. W. (2001). `Scaffold design and fabrication technologies for engineering tissues ± state of the art and future perspectives'. J Biomater Sci Polym Ed 12(1): 107±24. Jenner, J. M., F. van Eijk, et al. (2007). `Effect of transforming growth factor-beta and growth differentiation factor-5 on proliferation and matrix production by human bone marrow stromal cells cultured on braided poly lactic-co-glycolic acid scaffolds for ligament tissue engineering'. Tissue Eng 13(7): 1573±82. Jockenhoevel, S., G. Zund, et al. (2001). `Fibrin gel ± advantages of a new scaffold in cardiovascular tissue engineering'. Eur J Cardiothorac Surg 19(4): 424±30. Juncosa-Melvin, N., G. P. Boivin, et al. (2006a). `Effects of cell-to-collagen ratio in stem cell-seeded constructs for Achilles tendon repair'. Tissue Eng 12(4): 681±9. Juncosa-Melvin, N., G. P. Boivin, et al. (2006b). `The effect of autologous mesenchymal stem cells on the biomechanics and histology of gel±collagen sponge constructs used for rabbit patellar tendon repair'. Tissue Eng 12(2): 369±79. Juncosa-Melvin, N., J. T. Shearn, et al. (2006c). `Effects of mechanical stimulation on the biomechanics and histology of stem cell±collagen sponge constructs for rabbit patellar tendon repair'. Tissue Eng 12(8): 2291±300. Karamuk, E., J. Mayer, et al. (1999). Embroidery technology for medical textiles. Medical Textiles, 2nd International Conference, Bolton. Kobayashi, M., E. Itoi, et al. (2006). `Expression of growth factors in the early phase of supraspinatus tendon healing in rabbits'. J Shoulder Elbow Surg 15(3): 371±7. Koob, T. J. (2002). `Biomimetic approaches to tendon repair'. Comp Biochem Physiol A Mol Integr Physiol 133(4): 1171±92. Laitinen, O., T. Pohjonen, et al. (1993). `Mechanical properties of biodegradable poly-Llactide ligament augmentation device in experimental anterior cruciate ligament reconstruction'. Arch Orthop Trauma Surg 112(6): 270±4. Langer, R. and J. P. Vacanti (1993). `Tissue engineering'. Science 260(5110): 920±6. Laurencin, C. T. and J. W. Freeman (2005). `Ligament tissue engineering: an evolutionary materials science approach'. Biomaterials 26(36): 7530±6. Laurencin, C. T., A. M. A. Ambrosio, et al. (1999). `Tissue Engineering: Orthopedic Applications'. Annual Rev Biomed Eng 1(1): 19±46. Lee, J., F. L. Harwood, et al. (1998). `Growth factor expression in healing rabbit medial collateral and anterior cruciate ligaments'. Iowa Orthop J 18: 19±25.

466

Regenerative medicine and biomaterials in connective tissue repair

Li, F., H. Jia, et al. (2007). `ACL reconstruction in a rabbit model using irradiated Achilles allograft seeded with mesenchymal stem cells or PDGF-B gene-transfected mesenchymal stem cells'. Knee Surg Sports Traumatol Arthrosc 15(10): 1219±27. Liang, R., S. L. Woo, et al. (2006). `Long-term effects of porcine small intestine submucosa on the healing of medial collateral ligament: a functional tissue engineering study'. J Orthop Res 24(4): 811±19. Lin, V. S., M. C. Lee, et al. (1999). `Ligament tissue engineering using synthetic biodegradable fiber scaffolds'. Tissue Eng 5(5): 443±52. Liu, H., H. Fan, et al. (2008). `A comparison of rabbit mesenchymal stem cells and anterior cruciate ligament fibroblasts responses on combined silk scaffolds'. Biomaterials 29(10): 1443±53. Lou, J., Y. Tu, et al. (2001). `BMP-12 gene transfer augmentation of lacerated tendon repair'. J Orthop Res 19(6): 1199±202. Louie, L., I. V. Yannas, et al. (1998). Tissue engineered tendon. In Frontiers in Tissue Engineering. C. W. J. Patrick, A. G. Mikos and L. V. McIntire, Pergamon, Elsevier Science Ltd.: 412±442. Lu, H. H., J. A. Cooper, Jr, et al. (2005). `Anterior cruciate ligament regeneration using braided biodegradable scaffolds: in vitro optimization studies'. Biomaterials 26(23): 4805±16. Majima, T., T. Funakosi, et al. (2005). `Alginate and chitosan polyion complex hybrid fibers for scaffolds in ligament and tendon tissue engineering'. J Orthop Sci 10(3): 302±7. Majima, T., T. Irie, et al. (2007). `Chitosan-based hyaluronan hybrid polymer fibre scaffold for ligament and tendon tissue engineering'. Proc Inst Mech Eng [H] 221(5): 537±46. Mark Saltzman, W. and S. P. Baldwin (1998). `Materials for protein delivery in tissue engineering'. Adv Drug Deliv Rev 33(1±2): 71±86. Masuko, T., N. Iwasaki, et al. (2005). `Chitosan±RGDSGGC conjugate as a scaffold material for musculoskeletal tissue engineering'. Biomaterials 26(26): 5339±47. Mikos, A. G. and J. S. Temenoff (2000). `Formation of highly porous biodegradable scaffolds for tissue engineering'. Electronic Journal of Biotechnology 3(2): 114. Moreau, J. E., J. Chen, et al. (2005a). `Growth factor induced fibroblast differentiation from human bone marrow stromal cells in vitro'. J Orthop Res 23(1): 164±74. Moreau, J. E., J. Chen, et al. (2005b). `Sequential growth factor application in bone marrow stromal cell ligament engineering'. Tissue Eng 11(11±12): 1887±97. Musahl, V., S. D. Abramowitch, et al. (2004). `The use of porcine small intestinal submucosa to enhance the healing of the medial collateral ligament ± a functional tissue engineering study in rabbits'. J Orthop Res 22(1): 214±20. Nakamura, N., D. A. Hart, et al. (2000). `Decorin antisense gene therapy improves functional healing of early rabbit ligament scar with enhanced collagen fibrillogenesis in vivo'. J Orthop Res 18(4): 517±23. Ngo, M., H. Pham, et al. (2001). `Differential expression of transforming growth factorbeta receptors in a rabbit zone II flexor tendon wound healing model'. Plast Reconstr Surg 108(5): 1260±7. Ouyang, H. W., J. C. H. Goh, et al. (2002). `Characterization of anterior cruciate ligament cells and bone marrow stromal cells on various biodegradable polymeric films'. Mat Sci Eng: C 20(1±2): 63±69. Ouyang, H. W., J. C. Goh, et al. (2003). `Knitted poly-lactide-co-glycolide scaffold loaded with bone marrow stromal cells in repair and regeneration of rabbit Achilles tendon'. Tissue Eng 9(3): 431±9.

Scaffolds for tendon and ligament tissue engineering

467

Ouyang, H. W., S. L. Toh, et al. (2005). `Assembly of bone marrow stromal cell sheets with knitted poly (L-lactide) scaffold for engineering ligament analogs'. J Biomed Mater Res B Appl Biomater 75(2): 264±71. Petrigliano, F. A., C. S. English, et al. (2007). `The effects of local bFGF release and uniaxial strain on cellular adaptation and gene expression in a 3D environment: implications for ligament tissue engineering'. Tissue Eng 13(11): 2721±31. Sahoo, S., J. C. H. Goh, et al. (2006a). `FGF-2 releasing nanofibrous scaffolds for tendon tissue engineering'. Tissue Eng 12(4): 1053. Sahoo, S., H. Ouyang, et al. (2006b). `Characterization of a novel polymeric scaffold for potential application in tendon/ligament tissue engineering'. Tissue Eng 12(1): 91±9. Sahoo, S., J. C. H. Goh, et al. (2007). `Development of hybrid polymer scaffolds for potential applications in ligament and tendon tissue engineering'. Biomedical Materials 2(3): 169±173. Sahoo, S., L. T. Ang, et al. (2009). `Growth factor delivery through electrospun nanofibres in scaffolds for tissue engineering applications'. J Biomed Mater Res A, in press. Shimomura, T., F. Jia, et al. (2003). `Antisense oligonucleotides reduce synthesis of procollagen alpha1 (V) chain in human patellar tendon fibroblasts: potential application in healing ligaments and tendons'. Connect Tissue Res 44(3±4): 167± 72. Spalazzi, J. P., S. B. Doty, et al. (2006). `Development of controlled matrix heterogeneity on a triphasic scaffold for orthopedic interface tissue engineering'. Tissue Eng 12(12): 3497±508. Spalazzi, J. P., E. Dagher, et al. (2008). `In vivo evaluation of a multiphased scaffold designed for orthopaedic interface tissue engineering and soft tissue-to-bone integration'. J Biomed Mater Res A 86(1): 1±12. Takezawa, T., K. Ozaki, et al. (2007). `Reconstruction of a hard connective tissue utilizing a pressed silk sheet and type-I collagen as the scaffold for fibroblasts'. Tissue Eng 13(6): 1357±66. Tischer, T., S. Vogt, et al. (2007). `Tissue engineering of the anterior cruciate ligament: a new method using acellularized tendon allografts and autologous fibroblasts'. Arch Orthop Trauma Surg 127(9): 735±41. Van Eijk, F., D. B. Saris, et al. (2004). `Tissue engineering of ligaments: a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source'. Tissue Eng 10(5±6): 893±903. Vepari, C. and D. L. Kaplan (2007). `Silk as a biomaterial'. Progress in Polymer Science 32(8±9): 991±1007. Vunjak-Novakovic, G., G. Altman, et al. (2004). `Tissue engineering of ligaments'. Annu Rev Biomed Eng 6: 131±56. Whalen, G. F., Y. Shing, et al. (1989). `The fate of intravenously administered bFGF and the effect of heparin'. Growth Factors 1(2): 157±64. Whitaker, M. J., R. A. Quirk, et al. (2001). `Growth factor release from tissue engineering scaffolds'. J Pharm Pharmacol 53(11): 1427±37. Whitlock, P. W., T. L. Smith, et al. (2007). `A naturally derived, cytocompatible, and architecturally optimized scaffold for tendon and ligament regeneration'. Biomaterials 28(29): 4321±9. Woo, S. L., K. Hildebrand, et al. (1999). `Tissue engineering of ligament and tendon healing'. Clin Orthop Relat Res (367 Suppl): S312±23. Woo, S. L., S. D. Abramowitch, et al. (2006). `Biomechanics of knee ligaments: injury, healing, and repair'. J Biomech 39(1): 1±20.

468

Regenerative medicine and biomaterials in connective tissue repair

Wren, T. A., S. A. Yerby, et al. (2001). `Mechanical properties of the human achilles tendon'. Clin Biomech 16(3): 245±51. Wu, L. and J. Ding (2004). `In vitro degradation of three-dimensional porous poly(D,Llactide-co-glycolide) scaffolds for tissue engineering'. Biomaterials 25(27): 5821± 30. Wurgler-Hauri, C. C., L. M. Dourte, et al. (2007). `Temporal expression of 8 growth factors in tendon-to-bone healing in a rat supraspinatus model'. J Shoulder Elbow Surg 16(5 Suppl): S198±203. Yamada, M., K. Akeda, et al. (2008). `Effect of osteogenic protein-1 on the matrix metabolism of bovine tendon cells'. J Orthop Res 26(1): 42±8.

Index

A260/280 absorbance ratio, 259 -dystrobrevin, 368 2-macroglobulin, 427 A/O ratio, 399 -smooth muscle actin, 43, 56 7 1 integrin, 368 ABO genes, 399 abrader, 206 abrasion arthroplasty, 214 Achilles tendon, 422 ACI see autologous chondrocyte implantation acromionizer, 206 ACT see autologous chondrocyte transplantation ADAM, 311±12 ADAMTS-1, 312 ADAMTS-4, 146, 311 ADAMTS-5, 146, 148, 261, 311 adenovirus, 284 adenovirus associated viruses, 284 adipocytes, 440 adipogenesis, 441 advanced Dulbecco's minimal essential medium, 425 AFM see atomic force microscopy agarose, 303 agarose gel, 114 aggrecan, 9, 114, 125, 146, 157, 272, 282, 286, 306, 361, 400, 427, 441 aggrecan 1, 260 aggrecanase, 143, 146 Alcian-blue-staining, 277, 397 alginate, 118, 307 alizarin red stains, 441

alkaline phosphatase, 318 American Orthopaedic Society for Sports Medicine, 330 anabolic steroids, 405 animal models, for screening new clinical techniques of cartilage repair, 179±81 articular cartilage thickness in rabbit, dog, sheep, goat, horse and human, 186 current status, 194±6 early equine models, 181±5 articular defects in carpus, 181±4 articular defects in femoropatellar and femorotibial joints, 184±5 articular defects in tarsus, 185 repair tissue in full-thickness articular cartilage defects in equine radial carpal bones, 184 equine femoropatellar and femorotibial joints repair, 185±94 advantages of equine models, 193±4 articular cartilage repair models in equine trochlea, 191±3 cartilage defects on medial femoral condyle and its validation, 186, 189, 191 collection sites for osteochondral blocks to measure articular cartilage, 187 creation of defects on medial trochlear ridge of the femur, 193 non-calcified and calcified cartilage and subchondral bone plate thickness, 188±9

470

Index

overall arthroscopic grade and treatment group in ACT study, 195 tissue harvest site diagram, 194 histologic comparison microfractured articular defects, 191 repair tissue at 18 months, 196 microfractured full-thickness cartilage defects at 12 months, 190 review of models in non-equine species, 179±81 goat, 180±1 pig, 181 rabbits, 179±80 sheep, 180 annexin II, 400 anterior cruciate ligament, 210, 235, 317, 333, 419, 422, 454 aponeurosis, 386 arthroscopic mosaicplasty, 206±19 defect preparation, 206 donor site filling by different biodegradable plugs, 215 graft harvest, 207±8 graft implantation, 208±9 medial femoral condyle resurfacement, 205 perpendicular access to the damaged area by spinal needle, 206 portal selection, 206 articular cartilage, 4±7, 19±23, 254 arthroscopic evaluation, 340±2 ICRS score, 342 Oswestry arthroscopy score, 341±2 biomechanical properties, 272 cell sheet technology for repair, 255 cell therapies for repair, 266±91 cartilage macromolecular network and biomechanical properties, 271±2 chemical compounds to enhance matrix production, 277±9 chondrocyte, 267±70 exogenous growth factors to promote chondrogeneic phenotype, 281±3 gene therapy to deliver chondrogeneic factors, 283±4 hydrostatic pressure control of chondrocyte phenotype and chondrogenesis, 284±5

low oxygen tension in cartilage repair, 285±9 mesenchymal and stem cells, 273±7 phenotypic changes, 273 strategies to maintain chondrogeneic phenotype, 279±81 chondrocytes, 5±6 dual function of immature cartilage during postnatal growth, 89±95 anisotropy index, 93 electron micrographs of chondrocytes, 95 epiphyseal growth, 91 light micrographs of medial femoral condyle, 92 mineralised longitudinal septa, 94 extracellular matrix, 6±7 schematic representation, 7 general structure and function, 84±8 electron micrograph of adult human articular cartilage, 88 electron micrograph of chondrocytes, 87 interterritorial matrix compartment, 90 light micrograph of femoral condyle, 86 matrix organisation, 89 histology, 342±4 articular pathologic scoring systems, 343 cartilage repair histology, 343±4 inter-species differences, 98±101 mechanical testing, 13 MRI, 338±40 patient-based outcome measures, 333±8 Cincinnati knee rating scale, 334±5 IKDC, 336±7 KOOS, 337 Lysholm, 333±4 Marx activity rating scale, 337±8 SF-36, 335±6 Tegner, 334 physiological mechanism, 95±8 decreased height in decreased bone growth rate, 97 epiphyseal bone growth rate, 97 immature and mature tissue, 96

Index length-gain in epiphyseal bone vs articular cartilage layer, 98 process centred outcome measures, 338±44 repair outcome measures, 330±44 schematic representation of zones and regions, 5 structure, 83±101 articular cartilage injury, 138 articular epiphyseal complex, 89 articular stability, 220 Artscan 1000 computerised indentometric device, 213 ascorbate-2-phosphate, 425 ascorbic acid, 255 aspiration, 214 atelocollagen, 253, 262±3 atomic force microscopy, 111±13, 114 autologous chondrocyte implantation, 125, 140, 193, 218±19, 266 biopsy of repair after chondrocyte implantation with periosteum, 242 cartilage defect on femoral condyle opening wedge osteotomy with plate fixation, 237 sutured collagen membrane, 237 cartilage tissue repair, 228±46 autologous chondrocytes, 230±2 cartilage repair imaging evaluation, 242±3 chondrocyte implantation and osteoarthritis, 244±5 chondrogeneic cell implantation, 228 chondrogeneic cells comparison, 228±30 clinical follow-up results, 239, 241±2 conclusions and future trends, 245±6 randomised controlled studies, 243±4 human chondrocytes in monolayer culture, 236 human clinical use and studies, 233±9 biomechanical malalignment, 238 cartilage harvest, 233±5 chondrocyte implantation, 236±7 coexisting knee pathology, 237 harvest sites, 235 indications, 233 ligamentous insufficiency, 238

471

meniscal deficiencies, 238 osteochondral defects, 238 postoperative rehabilitation, 238±9 surgical technique, 233 in vitro cell expansion, 235±6 hyalograft scaffold cultured chondrocytes, 240 implanted Hyalograft scaffold, 241 trans-arthroscopic implantation of chondrocytes, 241 with cultured chondrocytes prepared for implantation, 240 other joints besides knee joint, 239 autologous chondrocyte transplantation, 142, 185, 233, 303 autologous osteochondral mosaicplasty arthroscopic mosaicplasty, 206±19 defect preparation, 206 graft harvest, 207±8 graft implantation, 208±9 portal selection, 206 for cartilage tissue repair, 201±20 clinical results, 213±17 from author's institution, 213±16 from other sources, 216±17 mosaicplasty resurfacing technique development, 202±4 pitfalls and complications, 212±13 patellofemoral complaints, 213 septic or thromboembolic complications, 212 postoperative management and rehabilitation, 209±10 preoperative planning, 204 role in treatment compared with other procedures, 217±19 ACI, 218±19 microfracture, 217 osteochondral allograft transplantation, 218 osteochondral autograft transplantation, 217±18 surgical instruments and choice of surgical technique, 204±5 autoradiography, 148, 229 B-mode ultrasonography, 384 -tricalcium phosphate, 310 Bandi scoring system, 213, 216

472

Index

basic fibroblast growth factor, 425, 428, 461 BD Falcon, 255 benign hypermobility syndrome, 139 betglycan, 272 biceps brachii, 401 biglycan, 272, 361, 427 biomaterials scaffolds for tissue engineering for ligament and tendon repair, 423±4 tendon and ligament tissue engineering, 453±5 composites and novel materials, 455 natural biomaterials, 454±5 synthetic biomaterials, 453±4 vs tendon and ligament tissues mechanical properties, 453 biomechanical conditioning chondrocytes, 117±28 biomechanical loading systems, 120 cell source, 125±8 mechanical loading regimens, 120±5 scaffold materials, 128 compressive cell strain system, 121 GAG synthesis and [3H]-thymidine incorporation by bovine chondrocytes, 122 SO4 and [3H]-thymidine incorporation by freshly isolated bovine chondrocytes, 127 by full depth bovine chondrocytes, 129 by human monolayer-expanded chondrocytes, 127 intermittent loading regimens effect, 124 biomimetic scaffolds, 308 Bionano interface technology, 251 blastema, 228 blastocyst, 306 Blood Transfusion Centre, 399 BMP see bone morphogenetic protein BMP-2, 308, 462 BMP-12, 462 Bombyx mori silk, 454 Bonar score, 402 bone marrow mesenchymal stem cells, 303 bone marrow stromal cell, 276, 422, 455

bone metaplasia, 396 bone morphogenetic protein, 282±3, 284, 306 bone sialoprotein, 441 Breinan study 2001, 232 brevican, 400 brevis muscles, 365 brushing, 88 c-jun-terminal kinase, 147 cadherin-11, 361 calcification, 396 calcitonin gene-related peptide, 409 calcium phosphate, 310 calcium pyrophosphate dehydrate crystal deposition disease, 164 cancer progression, 43±4 carbodiimide, 423 carbon rods, 215 cartilage articular see articular cartilage grading/scoring systems cartilage degeneration, 168±70 cartilage repair, 170±3 ICRS visual histological assessment score for assessing repair quality, 173 injury see cartilage injury major pathology and pathobiology, 157±65 crystal deposition disease, 163±5 OA pathology and pathobiology, 157±62 osteochondrosis dissecans, 163 post-traumatic lesions, 162 measuring biomechanical properties of cells, 106±30 osteoarthritis and other diseases, 155±74 future trends, 174 normal joint, 156±7 in vivo repair, 165±8 osteophyte development stages and their structural organisation, 167 staging according to Gelse et al., 166 repair cell sheet technologies, 251±63 histology, 343±4

Index using animal models to screen new clinical techniques, 178±96 tissue repair autologous chondrocyte implantation, 228±46 autologous osteochondral mosaicplasty, 201±20 cartilage histopathology assessment system, 343 cartilage injury animal models, 143±6 direct injury to articular surface, 143±5 indirect cartilage injury, 145±6 clinical in vivo, 138±43 aetiology of JSI, 139 chronic injury, 142±3 natural history of JSI, 139±42 surgical models of osteoarthritis, 145 tissue response, 137±49 in vitro, 146±8 explantation, 146±7 impact loading, 147±8 low velocity injurious load, 148 re-cutting, 147 cartilage oligomeric matrix protein, 443 CC chemokine receptor 2, 47±8 CD29, 50 CD34, 48 CD44, 50 CD45, 48 CD73, 50 CD90, 50 CD105, 50 CD106, 50 CD166, 50 cell adhesion molecules, 400 cell proliferation, 268±70 cell sheet technologies allograft study, 254±8 articular chondrocytes from Japanese white rabbits, 254 cell proliferations on temperatureresponsive surface, 254±5 cell sheets harvesting, 255±6 chondrocyte sheets transplantation, 256 articular cartilage repair, 255 cartilage repair, 251±63

473

cell detachment mechanism, 252 challenge for cartilage repair, 253±8 chondrocyte sheets properties, 258±62 gene expression analysis, 260±2 human articular chondrocytes, 258 primer design and RT-PCR, 259±60 RNA isolation and cDNA synthesis, 259 fibronectin expression, 260 future trends in cartilage repair, 262 histological findings after transplantation of layered chondrocytes sheets, 257 allografted chondrocyte sheet and injured sites, 256, 258 layered chondrocyte sheets transplantation, 257 list of primers used in real-time PCR, 259 present clinical applications overview, 252±3 regulations regarding regenerative medicine in Japan, 262±3 relative expressions of mRNA of key genes, 261 cell surface proteins, 50 cell therapy articular cartilage repair, 266±91 differentiated cells ligament-derived cells, 438 other fibroblast sources, 438 tendon-derived cells, 438 for tendons and ligaments repair and reconstruction, 436±47 allogeneic vs autologous sources, 442 cell choice, 438±41 mixed cell populations, 441 rationale behind use of cells, 437±8 stem cell-induced tenogenesis ± in vitro, 442±3 stem cell-induced tenogenesis ± in vivo, 443±7 stem cells proposed beneficial actions on tendon healing, 442 tissue engineering for ligament and tendon repair, 421±3 undifferentiated cells, 438±41 adipose-derived, 440

474

Index

bone marrow, 440 defining the MSC population, 440±1 embryonic, 438±9 mesenchymal stem cells, 439 tendon progenitor cells, 439±40 umbilical cord, 439 cell transplantation therapy, 253 cells ECM structure basic aspects, 354±62 structure and function, 356±60 fibroblast, 356±7 other cells, 358±60 stem cells, 357±8 Charnley's total hip arthroplasty, 331 chitosan, 303, 307 chondral injury, 149 see also cartilage injury chondrocalcinosis, 163, 164 chondrocyte-agarose model, 114, 121 chondrocyte clusters, 161 chondrocytes, 5±6, 99±100, 157, 159, 267±70, 282, 307, 317, 440 adult cartilage, 270 autologous, 230±2 autologous implantation, 125, 140, 193, 218±19, 266 autologous transplantation, 185, 233, 303 biomechanical conditioning, 117±28 biomechanical loading systems, 120 cell source, 125±8 mechanical loading regimens, 120±5 scaffold materials, 128 biomechanics measurement, 107±16 AFM, 111±13 cytoindentation and cytocompression, 113 isolated chondrocytes compression in 3D scaffolds, 114±16 micropipette aspiration, 109±11 in situ within cartilage explants, 107±8 cartilage cell therapy, 273±4 cell proliferation and hypertrophy, 268±70 cartilage hypertrophy and regulation, 269±70 growth in cartilage, 268±9

chemical compounds to enhance matrix production, 277±9 chondrocyte sheets transplantation, 256 chondrogeneic markers expression in hBM cells under hypoxia treatment, 288 chondrogenesis in human bone marrow MSCs, 278 cultured in Hyalograft scaffold, 240 electron micrographs, 87, 95 embryonic origin and chondrocyte differentiation, 267±8 exogenous growth factors to promote chondrogeneic phenotype, 281±3 BMP, 282±3 FGF, 281 IGF-1, 283 TGF, 282 gene therapy to deliver chondrogeneic factors, 283±4 HIF-1, HIF-1-DN proteins overexpression on binding activity and chondrogeneic markers expression, 290 hydrostatic pressure control of phenotype and chondrogenesis, 284±5 hypertrophic cartilage mineralisation, 270 implantation, 236±7 osteoarthritis, 244±5 intracellular biomechanics, 116±17 low oxygen tension in cartilage repair, 285±9 hypertrophy inhibition by hypoxia, 289 hypoxia-induced chondrogeneic differentiation of stem cells, 287±9 use of hypoxia to favour differentiated chondrocytes, 285±7 measuring biomechanical properties, 106±30 compression in 3D scaffolds, 115 compressive stiffness values, 112 future trends, 129±30 micropipette aspiration, 108±9 protocols used to assess conditioning in model systems, 119

Index phenotypic changes, 273 strategies to maintain chondrogeneic phenotype, 279±81 testing of response to biomechanical stimuli, 118 trans-arthroscopic implantation in Hyalograft scaffold, 241 chondrogenesis, 267 control by hydrostatic pressure, 284±5 in human bone marrow MSCs, 278 ChondroGide, 236 chondroid cells, 358±9 chondroid metaplasia, 359 chondroitin sulfate, 302 chondron, 107, 111, 130 chondroscopy, 341 chrondrogeneic differentiation, 441 Cincinnati knee rating scale, 213, 243, 334±5 ciprofloxacin, 395, 405 Clostridium hystoliticum collagenase, 274 COL1A1, 399 COL2A1, 286, 289 COL3A1, 399 COL10a1, 269, 289 COL5A1 genes, 399, 400 collagen, 85, 157, 271±2, 285, 303, 310, 423, 454 adaptation regulation to mechanical loading, 377±80 biomechanical properties of articular cartilage, 272 proteoglycans, 271±2 structure and formation of ECM, 360±1 in vivo turnover, 376±7 collagen fibres, 9 collagen fibrils, 91±2 collagen gel, 443 collagen type I, 125, 126, 261, 376, 377, 379, 397, 399, 426, 427, 428, 443 collagen type II, 114, 124, 125, 126, 128, 254, 260, 282, 284, 285, 286, 306, 441 collagen type III, 379, 397, 399, 426, 427, 428 collagen type V, 399, 462 collagen type VI, 368 collagen type X, 282 collagenase, 276, 311, 397

475

Collagenase P, 255 collagen±glycosaminoglycan scaffold, 425 composite/hybrid scaffolds, 457±9 biphasic/triphasic scaffolds, 458±9 composite fibre and gel based scaffolds, 457 composite fibre and sponge-based scaffolds, 458 hybrid nano-microfibrous scaffolds, 458 hybrid silk sponge reinforced scaffold, 458 ligament±bone interface mimicked by tri-phasic scaffold design, 460 nano-microscaffold, 459 native ECM structure mimicry, 459 computed tomography, 204 confocal microscopy, 114 connective tissue growth factor, 379 connexin 32, 357 connexin 43, 357 continuous passive motion, 179 controlled passive motion, 209 core-binding factor alpha-1, 441 corticosteroids, 405 creep, 365 crimp, 354 cryotherapy, 214 crystal deposition disease, 163±5 gout, 163±4 pseudogout, 164±5 urate crystal deposition, 164 crystallopathies, 155 CXCL13, 314 cyanamide, 423 CYP27A1, 402 cytochalasin treatment, 361 cytocompression, 113 cytoindentation, 113, 114 cytokines, 313, 395 cytoskeleton, 117 3D agarose constructs, 118 3D alginated bead culture, 280 3D fast spin-echo, 141 D-tubocurarine, 355 decorin, 272, 361, 362, 427, 462 degenerative arthritis, 220

476

Index

degenerative theory, 403±4 dermatan sulphate proteoglycan 3, 148 desmin, 45 destabilisation of medial meniscus, 146 detritus-rich synovitis, 161 dexamethasone, 278±9, 441 dGEMRIC, 242 digital image correlation, 117 discoidin domain receptor-2, 271 Dispoposplasty System, 204 Dulbecco's Modified Eagle's Medium, 126, 274 Dulbecco's Modified Eagle's Medium/ F12, 254 dynamic mechanical stimulation, 291 dystrophin-associated protein complex, 368 ECM see extracellular matrix EDTA see ethylenediaminetetraacetic acid elastin, 361, 396 electromagnetic therapy, 410 electrophoresis, 259 electrophoretic mobility shift assay, 289 electrospinning, 310 embryonic stem cells, 305±6, 438±9 endothelin, 424 enthesis, 362±5 fibrocartilaginous, 363 fibrous, 363 epiligament, 354 epitenon, 354 epithelial-to-mesenchymal transition, 44, 46 equilibrium modulus, 110 equine tendon disease, 444±7 diseases treated, 445±6 outcome, 446±7 adipose-derived MSCs, 447 bone marrow-derived MSCs, 446 safety aspects, 445 technique, 444±5 adipose-derived MSCs, 444±5 bone marrow-derived MSCs, 444 erythromycin, 274 ethylenediaminetetraacetic acid, 275 extracellular matrix, 6±7, 40, 85±7, 254, 272, 394, 452

cell structure basic aspects, 354±62 matrix and contracting skeletal muscle, 380±1 schematic representation, 7 structure and formation, 360±2 collagens, 360±1 other molecules, 361±2 extracellularly regulated kinase, 147 factor VIIa, 137 fetal calf serum, 126 fascicles, 354 fast spin-echo sequence, 339 fat-suppressed proton density-weighted T2 fast spin-echo, 242 fat suppressed spoiled gradient-echo, 141 femoral condyle, 99 Fetal Bovine Serum, 255 FGF see fibroblast growth factor fibre-based scaffold, 455±6 braided and woven scaffolds, 455±6 knitted scaffolds, 456 knitted silk microfibrous scaffolds reinforced with microporous silk sponge, 457 fibril diameter, 355 fibril-fibroblast mechanotransduction, 387 fibrin glue, 128, 185, 443 fibrinoid degeneration, 396 fibripositors, 360 fibroblast growth factor, 126, 281, 424 fibroblast growth factor-2, 51 fibroblasts, 44±5, 232, 307, 317, 440 structure and function, 356±7 fibrocartilage, 172, 253, 273, 302 cells structure and function, 358±9 four zones of tissue at fibrocartilaginous enthesis classical appearance, 358 at human Achilles tendon enthesis, 359 fibrocartilaginous, 396 fibrocyte, 46±9 myofibroblast precursor cells, 47 fibromodulin, 272, 362, 427 fibronectin, 148, 252, 260, 368, 400, 403±4 fibronectin I, 260 fibronexus, 41 fibrosis, 52±3

Index fibrous OA synoviopathy, 161 fibularis, 365 Flexercell, 120 fluorescent dye PKH26, 231 fluoroquinolones, 395, 405 formaldehyde, 423 formaldehyde gel, 259 FRZB, 147 functional adaptation, 3 functional groups, as functional scaffolds, 462 fungizone, 274 GAG see glycosaminoglycan galactosylhydroxy-lysyl-glucotransferase, 376 gel-based scaffold, 456±7 gelatinases, 311, 397 gelatine, 303 gene therapy, as functional scaffolds, 462 Gentamicine, 276 gliding processes, 387 glucosamine, 302 glutaraldehyde, 280, 423 glyceraldehyde-3-phosphate dehydrogenase, 260 glycoproteins, 361 glycosaminoglycan, 124, 282, 285, 303, 307, 455 Goldner Trichome, 309 golgi-to-plasmalemmal carriers, 360 Gore-tex, 420 gout, 163±4 grading, 168±9 graft-versus-host-disease, 305 green fluorescent protein, 231, 381 growth and differentiation factor, 424, 425 growth and differentiation factor-5, 425, 461 growth factors, 463 different distribution patterns in electrospun nanofibres, 461 as functional scaffolds, 460±2 tissue engineering for ligament and tendon repair, 424±5 growth hormone, 378 guanine-thymine dinucleotide repeat polymorphism, 400

477

haematopoietic stem cells, 439, 440 Hannover ankle evaluations, 213 Hdac4, 289 hexosamine, 183, 185 HIF-1, 286±7 HIF-1, 288 Histological Histochemical Grading System, 343 hormones, 463 Hospital for Special Surgery, 213 HYAFF 11, 236, 307, 314 hyaline cartilage, 159, 301 hyaline degeneration, 396 Hyalograft-C, 235±6, 237 Hyalograft scaffold cultured chondrocytes, 240 implanted, 241 trans-arthroscopic implantation of chondrocytes, 241 with cultured chondrocytes prepared for implantation, 240 hyaluronic acid, 307 hydrogels, 307±8, 461±2 hydrostatic pressure, 284±5 hydroxyapatite, 215, 310 hydroxyproline, 377 Hypaque-Ficoll density gradient, 275 hyperplastic synoviopathy, 161 hyperthermia, and tendon rupture, 406 hypertrophy, 268±70 cartilage hypertrophy and regulation, 269±70 growth in cartilage, 268±9 inhibition by hypoxia, 289 loading adaptation of human tendon, 382±3 hypoxia, 291 hypoxia-responsive elements, 287 hypoxic degeneration, 396 ICRS see International Cartilage Repair Society IGF-1 see insulin-like growth factor 1 IKDC see International Knee Documentation Committee subjective knee form immature mammalian articular cartilage see articular epiphyseal complex Indian hedgehog promoter, 269

478

Index

indomethacin, 441 infarcted heart see myocardial infarction inflammatory OA synoviopathy, 161 insulin, 441 insulin-like growth factor, 424, 461 insulin-like growth factor 1, 281, 283, 284, 286, 378 integrins, 128, 260, 400 interferon- , 61 interleukin-1, 61, 273 interleukin-1 , 146, 395 International Cartilage Repair Society, 172±3, 213, 338 ICRS score, 342 ICRS visual histological assessment score for assessing repair quality, 173 International Knee Documentation Committee subjective knee form, 336±7 International Society for Cellular Therapy, 17 isobutyl-methyl-xanthine, 441 joint surface damage, 138 joint surface injury, 138 aetiology, 139 natural history, 139±42 joint tissues biomechanical properties, 4±15 creep measurement, 13 degenerative changes in matrix, 16 isotropic elastic parameters of cartilage, 14 mechanical testing of articular cartilage, 13 destruction typing, 168 normal joint, 156±7 staging of destruction according to Otte, 170 JSI see joint surface injury K-CX Decalcifying Solution, 256 K-wire, 206, 208 Kager's fat pad, 363 kinking, 88 knee chondral lesions, 139 knee injury and osteoarthritis outcome score, 337

KOOS see knee injury and osteoarthritis outcome score L-C-proline, 376 L-N-proline, 376 L-Sox5, 283, 284 lacZ gene-marked allogeneic MSCs, 462 latent TGF1 binding protein, 57 Leeds-Keio, 420 lentivirus, 284 leukaemia inhibitor factor, 313 leukocyte-specific protein-1, 48 levofloxacin, 405 Ligament Augmentation Device, 420 ligamental tear, 220 ligaments, 10±11 cell and ECM structure basic aspects, 354±62 cells structure and function, 356±60 ECM structure and formation, 360±2 collagen fibril organisation, 10 description, 351±3 diverse functions, 353 specialised regions, 362±8 entheses, 362±5 myotendinous junctions, 366±8 wrap-around regions, 365±6 and tendon repair by tissue engineering, 419±29 and tendon tissue engineering scaffolds, 452±63 biomaterials, 453±5 criteria and requirements, 452±3 functional scaffolds, 460±2 future trends, 462±3 scaffold architecture, 455±9 and tendons cell-based therapies for repair and regeneration, 436±47 structure, 357±68 ligamentum nuchae, 438 link protein, 272 lipoid degeneration, 396 load bearing, 100 longus muscles, 365 lower critical solution temperature, 251 LTBP-1 see latent TGF1 binding protein lumican, 362, 427 Lysholm scale, 213, 333±4

Index lysyl hydroxylase, 376 MAC, 236 MACI, 219, 235, 236 MACT, 219 magnetic resonance imaging, 27, 139, 141, 158, 191, 204, 233, 382, 406 cartilage repair tissue system, 340 process centred outcome measures of articular cartilage repair, 338±40 magnetic twist cytometry, 130 malalignment, 142 Mankin system, 169, 343 Marx activity rating scale, 337±8 matrix metalloproteinase, 144, 308, 311, 427 four main groups, 397±8 in tendinopathy, 397±8 mechanical stimulation, 463 mechanical stress, 43, 55±6 mechanical theory, 404 mechano growth factor, 379 Medial collateral ligament, 422 medical outcome study 36 item short form, 335±6 membrane type MMPs, 398 meniscal tear, 220 meniscus, 8±10, 25 knee joint, 9 mesenchymal progenitor cells, 16±26, 439 articular cartilage, 19±23 joint homeostasis, 17, 19 stem cell response to stimuli, 22 meniscus, 25 mesenchymal stromal cells, 16, 17 adult mesenchymal stem/stromal cell, 18 multipotent progenitor cells, 20±1 other tissues, 25±6 synovial fluid, 24 synovium, 23±4 tendons and ligaments, 24±5 mesenchymal stem cells, 49±54, 273±7, 307, 427, 439, 462 benefits of MSC-to-myofibroblast differentiation for regenerative medicine, 54 defining the population, 440±1 effective use, 60±2

479

MSC-to-myofibroblast differentiation, 50±1 regenerative potential, 49±50 threat of MSC-to-myofibroblast differentiation for regenerative medicine, 52±3 use in cartilage cell therapy, 274±7 challenge in using MSC for cartilage repair, 276±7 chondrogeneic phenotype assessment, 277 choosing appropriate MSC, 275±6 mesenchymal stromal cells, 232 metalloproteinases, 143 microdialysis technique, 376 microfracture, 142 microfracture technique, 17, 140, 214, 217, 253, 302±3 microfractured articular defects, 191 microfractured full-thickness cartilage defects at 12 months, 190 micropipette aspiration, 109±11 microtrauma, 162, 395 Millipore filter, 229 mineralisation, 91 mini-arthrotomy mosaicplasty 100% filling rate by cutting the graft into each other, 207 grafts implantation into medial femoral condyle, 203 harvest site on medial femoral condyle, 207 mitogen activated protein kinase, 147 MMP see matrix metalloproteinase MMP3, 144, 148, 261, 395 MMP9, 144, 146, 398 MMP13, 144, 261 modified O'Driscoll score, 344 Moloney Murine Leukaemia Virus, 284 reverse transcriptase, 259 monocytes, 137 mosaicplasty, 140, 266 clinical results, 213±17 control arthroscopy on medial femoral condyle, 214 development, 202±4 mini-arthrotomy mosaicplasty 100% filling rate by cutting the graft into each other, 207

480

Index

grafts implantation into medial femoral condyle, 203 harvest site on medial femoral condyle, 207 open mosaicplasty trochlear defect resurfacement, 205 rehabilitation protocol, 210±12 role in treatment compared to other procedures, 217±19 technique, 201 see also specific mosaicplasty MosaicPlasty Complete System, 204 Movin score, 401±2 MRI see magnetic resonance imaging mRNA, 395 MSC see mesenchymal stem cells mucoid degeneration, 359, 396 myocardial infarction, 53 myocyte enhancer factor-2, 269 myoepithelium, 44 myofibroblast, 440 cell differentiation, 55±60 factors modulating differentiation, 59±60 mechanical stress, 55±6 TGFb1 pro-fibrotic cytokine, 57±9 connective tissue repair and regeneration, 39±62 definition, 44±6 effective use, 60±2 future trends, 62 and MSC phenotype, 49±54 origin, 46±9 fibrocyte, 46±9 precursor cells, 46 tissue construction, 41±4 differentiation spectrum, 42 myotendinous junctions, 366±8 domains, 367 thenar muscle in the human hand, 367 transmembrane receptors, 368 myxoid degeneration, 396 N-isopropylacrylamide monomer solution, 251 nalidixic acid, 405 nectin, 368 neurocan, 400 nitric oxide, 123, 126, 130, 408±9

nitric oxide synthase, 409 Nkx3/Bapx1, 269 non-steroidal anti-inflammatory drug, 258 norfloxacin, 405 Notch1, 22 NOX, 60 Ntelo see synthetic N-telopeptide nucleofection methods, 284 O'Driscoll score, 173 ofloxacin, 405 Oil Red O stain, 441 OOCHAS see cartilage histopathology assessment system open mosaicplasty, 209±10 trochlear defect resurfacement, 205 optimal cutting temperature, 280 Orthopaedic Research Society International, 343 OsScore, 343±4 osteoarthritic cartilage, 111 osteoarthritis, 137, 138, 140, 146, 301 chondrocyte implantation, 244±5 grading according to Mankin et al., 169 according to Pritzker et al., 171 hip osteoarthritis and femoral condyle, 158 joint main structures schematic representation, 156 Mankin et al. grading system vs Otte staging, 170 normal vs osteoarthritic cartilage fibrillation and matrix loss, 160 and other cartilage diseases, 155±74 pathology and pathobiology, 157±62 cartilage cells, 159±61 cartilage matrix, 159 subchondral bone, 162 synovial membrane, joint capsule and inflammation, 161±2 role of cytokines, 313 surgical models, 145 osteoblasts, 282, 307, 317, 440 osteocalcin, 269, 318, 441 osteochondral allograft transplantation, 218 osteochondral autograft transplantation, 217±18

Index osteochondritis dissecans, 155, 163, 206 osteophytes, 165±7 mature osteophytes, 166±7 osteotomy, 142 Oswestry arthroscopy score, 341±2 oxygen tension, 291 p38, 147 pain, in tendinopathy, 406 paracrine communication, 315, 317±18 parathyroid-hormone-related protein, 269 patellar tendinopathy, 385 patellar tendon, 353, 422 pcDNA3-HIF1, 289 pcDNA3-UT-Flag-Sox9, 289 PDGF-B, 462 pefloxacin, 405 PEG dimethacrylate, 128 penicillin, 274 Percoll, 440 pericellular matrix, 107 periosteal transplantation, 266 peripheral quantitative computed tomography, 242 perlecan, 9±10 peroxisome proliferator-activated receptor gamma, 441 Perspex box, 120 Perspex indenter, 120 Pharmaceutical Law, 262±3 phosphate buffered saline, 109, 274 PINP, 380 plastic compression technique, 457 platelet-derived growth factor, 424, 461 platelet-derived growth factor-AB, 425 plug-in method, 245 poly-L-lactic acid, 262, 279, 309, 454, 455 poly-lactic-co-glycolic acid, 262, 279, 303, 306, 307, 453 poly N-isopropylacrylamide, 251 poly(-hydroxy acids), 453 polyacrylamide gel electrophoresis, 192 polyanhydrides, 453 polycaprolactone, 215, 307, 309, 310, 424, 454 polycarbonate, 424 poly(desamino tyrosyl-tyrosine ethyl ester carbonate), 454

481

polyester, 455 poly(ethylene glycol), 128, 303, 306, 307 polyethylene terephthalate, 420 polyethylene terephthalate-polypropylene, 420 polyglycolic acid, 279, 307, 424, 453 polyglycolic±polylactic copolymer scaffold, 308±9 polyglycolide, 262 polyglyconate, 215 polylactate, 215 polylactic acid, 307, 424, 453 polymerase chain reaction, 259 poly(methyl methacrylate), 307 poly(orthoesters), 453 polypropylene, 420 polytetrafluoroethylene, 420 poly(vinyl alcohol), 307 polyvinylidene difluoride membrane, 256 porous polyurethane scaffold, 125 posterior cruciate ligament, 331 postnatal development, 89±95 precursor cells, 46, 93, 95 Pridie drilling, 140, 214 procollagen, 360 proline, 376 prolyl-4-hydroxylase, 376 pronase E, 254, 274 prostaglandin E2, 130, 406 prostaglandins, 285, 395 proteoglycan, 86, 114, 124, 128, 157, 254, 271±2, 284, 361 proto-myofibroblast, 41, 45 pseudocolour maps, 117 pseudogout, 164±5 purulent arthritis, 155 pyrophosphate crystals, 163 radial zone, 84±5 range of motion, 209 real-time transcriptase polymerase chain reaction, 234 regenerative medicine components, 437 relaxation modulus, 110 Restore, 236 resurfacing, 220, 245 retinoic acid, 273 reverse transcriptase polymerase chain reaction, 277

482

Index

rigid punch model, 110 RNase-free water, 260 RNeasy Mini kit, 259 Runx2, 289 Runx2/Cbfa, 269 Safranin O staining, 182, 256, 277, 315, 344 sandwich technique, 238 scaffolds architecture, 455±9 composite/hybrid scaffolds, 457±9 fibre-based scaffold, 455±6 gel-based scaffold, 456±7 functional scaffolds, 460±2 functional groups, 462 gene therapy, 462 growth factors, 460±2 musculoskeletal tissue engineering, 301±19 tendon and ligament tissue engineering, 452±63 see also specific scaffold scanning acoustic microscopy, 130 scleraxis, 443 SF-36 see medical outcomes study 36 item short form Sharpey's fibres, 364 Shetland ponies, 182 Sigmacote, 109 silk, 423±4, 454, 456 SLRP see small leucine-rich repeat proteoglycan SMA fusion peptide, 62 small leucine-rich repeat proteoglycan, 9±10 SmartCycler II software program, 259, 260 SocieÂte FrancËaise d'Arthroscopie, 341 sodium ascorbate, 278±9 sodium dodecyl sulphate±polyacrylamide gel electrophoresis, 148 sodium urate crystals, 164 solid freeform fabrication, 429 Sox5, 289 Sox6, 283, 284 Sox9, 282, 283, 284, 286, 289, 441 specialist single cell cytoindentation, 113

staging, 168±9 Stanmore, 243 stem cell niche, 19, 358 stem cells adult stem cells, 304±5 embryonic stem cells, 305±6 induced tenogenesis ± in vitro stem cells cultured on tendon explants/matrices, 443 tenogenesis in 2D and 3D culture systems, 442±3 induced tenogenesis ± in vivo, 443±7 laboratory animal models, 443±4 naturally occurring equine tendon disease, 444±7 influence on tissues and synovial fluid, 27 proposed beneficial actions on tendon healing, 442 role in biomechanical adaptation of tendon, 381±2 structure and function, 357±8 Stro-1, 50 stromelysins, 397±8 Stryker, 420 subchondral bone, 7±8, 162 subchondral bone plate, 168 substance P, 406 Suk's criteria for quality measures, 332 sulphate, 128 superficial digital flexor tendon, 437 superficial zonal proteins, 234 superficial zone, 84 supraspinatus tendon, 401 SYBR Green PCR Master Mix, 259 syncoilin, 368 syndecan, 272 synovial fluid, 11±12, 24 synovial hyperplasia, 161 synovial joints structure and regenerative capacity, 1±28 anatomy, 2 biomechanical properties, 4±15 function, 2±4 future trends, 26±8 mesenchymal progenitor cells, 16±26 synovial membrane, 11±12, 23±4

Index synovio-entheseal complex, 363 synovitis, 146 synovium see synovial membrane synthetic N-telopeptide, 314 T2 mapping, 242 talin, 368 Tegner scale, 334 temperature recovery system, 256 tenascin-C, 362, 368, 380, 399±400, 426, 427, 428 tendinopathy, 394±8 genetics, 398±400 metalloproteinases, 397±8 pain, 406 tendon healing, after acute injuries, 407±10 healing limitations, 409±10 healing modulators, 408±9 remodelling responses, 408 tendon rupture, 400±6 degenerative theory, 403±4 drug-related tendon rupture, 405 and hyperthermia, 406 mechanical theory, 404 tendon±aponeurosis complex, 384 tendons, 10±11 biochemical adaptation to loading, 376±82 collagen adaptation regulation to mechanical loading, 377±80 matrix and contracting skeletal muscle, 380±1 role of stem cells, 381±2 in vivo collagen turnover, 376±7 biomechanics, 375±88 calf tendon fascicles separated by endotenon, 354 showing crimp, 355 categories, 437 cell and ECM structure basic aspects, 354±62 cells structure and function, 356±60 ECM structure and formation, 360±2 cell-based therapies for repair and regeneration, 436±47

483

classical appearance of four zones of tissue at fibrocartilaginous enthesis, 358 collagen fibril organisation, 10 description, 351±3 diverse functions, 353 fibrocartilage at human Achilles tendon enthesis, 359 healing see tendon healing human tendon biomechanics, 382±8 aponeurosis shear in human muscle± tendon complex, 384±5 loading adaptation by hypertrophy, 382±3 mechanical properties of individual human tendon fascicles, 385±8 regional differences in morphology, 383±4 injury and repair mechanics, 394±410 genetics, 398±400 healing after acute injuries, 407±10 pain in tendinopathy, 406 tendinopathy, 394±8 tendon rupture, 400±6 myotendinous junction of thenar muscle in human hand, 367 repair by tissue engineering, 419±29 rupture see tendon rupture specialised regions, 362±8 entheses, 362±5 myotendinous junctions, 366±8 wrap-around regions, 365±6 structure, 357±68 tendons extending over muscle bellies surface, 352 tissue engineering scaffolds, 452±63 biomaterials, 453±5 criteria and requirements, 452±3 functional scaffolds, 460±2 future trends, 462±3 scaffold architecture, 455±9 various types of degeneration, 396 tenoblasts, 395 tenocytes, 356, 394, 395, 438, 440 tendinopathic tendons, 396±7 tenomodulin, 362, 443 tensile modulus, 15 teratoma, 439 tetranectin, 368

484

Index

TGF see transforming growth factor TGF- , 126, 284, 306, 428, 461 TGF- 1, 43, 51, 57±9, 379, 422, 424, 425, 441 activation, 58 TGF- 3, 286 TheriForm 3D printing process, 429 three-dimensional gradient-echo, 242 three-dimensional spoiled T1 gradientecho sequence, 339 thrombospondin-1, 368 thymidene, 128 tidemark, 85, 168, 364 TIMP see tissue inhibitors of metalloproteinase TIMP-1, 144, 260, 261 TIMP-2, 427 tissue engineering approaches for ligament and tendon repair, 420±6 biomaterial scaffolds, 423±4 cell therapy, 421±3 growth factors, 424±5 mechanical factors, 425±6 components cells, 301 scaffolds, 301 signalling molecules, 301 fragment-loaded scaffolds vs scaffolds seeded with cells, 316 future trends, 428±9 histological samples with Goldner Trichome, 309 ligament and tendon reconstruction, 427±8 repair, 419±29 matrix stimulation and cell±cell communications in tissue regeneration, 314±18 cell±scaffold interactions, 314±15 paracrine communication through organised scaffolds, 315, 317±18 scaffolds for musculoskeletal engineering, 301±19 composite scaffolds to repair tissue, 308±10 future trends and perspectives, 318±19 natural and synthetic materials,

307±8 scaffolds for tendon and ligament, 452±63 biomaterials, 453±5 criteria and requirements, 452±3 functional scaffolds, 460±2 future trends, 462±3 scaffold architecture, 455±9 tissue remodelling, 310±13 triad, 421 utilised cell types, 302±6 adult stem cells, 304±5 autologous primary cells, 302±4 embryonic stem cells, 305±6 tissue inhibitors of metalloproteinase, 311, 312±13, 398 tissue remodelling, 95, 310±13 cytokines in osteoarthritis, 313 metalloproteinases and their inhibitors, 310±13 tissue resorption, 95 toluidine blue, 256, 277 traction tendons, 366 transforming growth factor, 282, 284, 424 transitional zone, 84 translational regenerative medicine, 246 Transwell system, 306 trauma, 162 trypsin±EDTA, 276 trypsinisation, 275, 276 tuber coxae, 444 tumour necrosis factor , 61, 313 typing, 168 ultrasound, 27, 406, 410 umbilical cord cells, 232 UpCell, 255 urate crystals, 163 utrophin, 368 vascular endothelial growth factor, 305 versican, 361, 400, 427 Vicryl knitted mesh, 443 vimentin, 44 visual analogue scale, 341 von Kossa, 441 Western Ontario and McMasters Universities, 337

Index Wharton's jelly, 439 Wnt-16, 147 Wnt signalling pathway, 147 Wolff's Law, 107, 129 wrap-around regions, 365±6

Young's model, 111 Young's modulus, 15, 110, 111, 310 Zwick Testing Machines Ltd, 120 zymogens, 137

485