Ted S. Stashak & Christine L. Theoret

EQUINE WOUND

Second Edition

MANAGEMENT

EQUINE WOUND MANAGEMENT

Second Edition

EQUINE WOUND MANAGEMENT

Second Edition

Ted S. Stashak, DVM, MS, Diplomate ACVS, Professor Emeritus Equine SurgeryDepartment of Clinical Sciences

College of Veterinary Medicine and Biomedical SciencesColorado State University

Fort Collins, Colorado

Christine Theoret, DMV, PhD, Diplomate ACVSDirector, Comparative Tissue Healing Laboratory

Associate ProfessorDépartement de biomédecine vétérinaire

Faculté de médecine vétérinaireUniversité de Montréal

Saint-Hyacinthe, Québec

A John Wiley & Sons, Inc., Publication

First edition fi rst published 1991Second edition fi rst published 2008© 1991 Lea & Febiger© 2008 Wiley-Blackwell

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Library of Congress Cataloguing-in-Publication Data

Equine wound management / [edited by] Ted S. Stashak, Christine Theoret. – 2nd ed. p. ; cm. Rev. ed. of: Equine wound management / Ted S. Stashak. 1991. Includes bibliographical references and index. ISBN-13: 978-0-8138-1223-6 (alk. paper) ISBN-10: 0-8138-1223-2 (alk. paper) 1. Horses–Wounds and injuries–Treatment. 2. Horses–Surgery. I. Stashak, Ted S. II. Theoret, Christine. III. Stashak, Ted S. Equine wound management. [DNLM: 1. Horses–injuries. 2. Horses–surgery. 3. Wounds and Injuries–therapy. 4. Wounds and Injuries–veterinary. SF 951 E647 2008] SF951.S77 2008 636.1′08971–dc22 2008016810

A catalogue record for this book is available from the U.S. Library of Congress.

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DisclaimerThe contents of this work are intended to further general scientifi c research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting a specifi c method, diagnosis, or treatment by practitioners for any particular patient. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifi cally disclaim all warranties, including without limitation any implied warranties of fi tness for a particular purpose. In view of ongoing research, equipment modifi cations, changes in governmental regulations, and the constant fl ow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. Readers should consult with a specialist where appropriate. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom.

1 2008

Dedications

To my wife, Gloria, and my children, Angela, Stephanie, and Ryan, for their love and untiring support.To my parents, Theodore and Ann, for emphasizing the value of an education.

Ted S. Stashak

To my parents, Susan and André, for encouraging excellence. Pour Alain qui me procure cet équilibre ô si bénéfi que et pour Mozelle, Marek et Francesca qui me comble de leur allégresse!Gràcies, en especial, a la Pilar i en Jordi per la seva visió positiva i generosa contribució en aquest llibre.

Christine Theoret

v

Table of Contents

Preface ixAcknowledgments xiContributors xiii

Chapter 1 Wound Healing 31.1 Physiology of Wound Healing 51.2 Differences in Wound Healing

between Horses and Ponies 291.3 Wound Repair: Problems in

the Horse and Innovative Solutions 47

Chapter 2 Factors That Infl uence Wound Infection and Healing 692.1 Selected Factors That

Negatively Impact Healing 712.2 Management Practices That

Infl uence Wound Infection and Healing 85

Chapter 3 Topical Wound Treatments 1073.1 Update on Wound Dressings:

Indications and Best Use 1093.2 Topical Wound Treatments

and Wound Care Products 1373.3 The Extracellular Matrix as

a Biologic Scaffold for Wound Healing in Veterinary Medicine 161

Chapter 4 Approaches to Wound Closure 1754.1 Selection of Approaches to

Wound Closure 1774.2 Selection of Suture Materials,

Suture Patterns, and Drains for Wound Closure 193

4.3 New and Innovative Approaches to Wound Closure 225

Chapter 5 Principles and Techniques For Reconstructive Surgery 239

Chapter 6 Management of Wounds of the Head 273

Chapter 7 Management of Wounds of the Neck and Body 333

Chapter 8 Wounds of the Distal Extremities 3738.1 Management of Wounds of

the Distal Extremities 3758.2 Degloving Injuries 4278.3 Treatment of Exuberant

Granulation Tissue 445

Chapter 9 Diagnosis and Management of Wounds Involving Synovial Structures 463

Chapter 10 Tendon and Paratendon Lacerations 489

Chapter 11 Free Skin Grafting 509

Chapter 12 Management of Severely Infected Wounds 543

Chapter 13 Burn Injuries 569

Chapter 14 Sarcoid Transformation at Wound Sites 585

Chapter 15 Lasers: Effects on Healing and Clinical Applications 609

Chapter 16 Bandaging and Casting Techniques for Wound Management 623

Index 659

vii

Preface

The second edition of this textbook was long overdue. Indeed, since the fi rst edition appeared in 1991, a number of ground-breaking studies have modernized the art of wound management in both human and vet-erinary patients. New topical medications, interactive dressings, and surgical procedures are now available, enabling veterinarians to treat serious injuries once deemed incurable. Moreover, research has shown, unequivo-cally, that many aspects of the horse’s healing response are unique, such that a textbook dedicated to the art and science of wound management in this species would be a most valuable tool for equine practitioners. Sub-sequent to the recent publication of two journal volumes on this topic (Clinical Techniques in Equine Practice, June 2004, TS Stashak, guest editor; Veterinary Clinics of North America, April 2005, CL Theoret, guest editor), it seemed most opportune to provide readers with a single, comprehensive source of theoretical and practical information, enhanced by an abundance of helpful tables, line drawings, and color fi gures. Thus, the purpose of this book is to provide an authoritative, state-of-the-art text on equine wounds and their management.

The fi rst chapter provides an update on the physiology of cutaneous wound healing, with a special focus on the newly discovered mediators that govern the mechanisms underlying repair. Horses have a distinctive response to trauma; therefore, the second section of the chapter endeavors to describe the major differences from the relatively normal healing profi le of ponies. The chapter concludes with an enlightening discussion of innovative solutions to the specifi c problems encountered when dealing with a traumatic wound in a horse.

Chapter 2 addresses selected factors that can exert a negative impact on the physiologic mechanisms that contribute to repair. This is followed by a review of wound management practices that infl uence infection and healing. The chapter emphasizes the importance of thoroughly assessing the wound and the patient as well as various measures such as hemostasis, cleansing, debriding, and disinfecting the wound in the fi rst few hours following injury. The subject is approached in the order in which a case involving a wound would be evaluated and managed clinically. Because infection is a major cause of delayed healing, this chapter emphasizes manage-ment practices that reduce its incidence.

The third chapter is devoted to topical wound treatments. It includes an updated list of dressings and wound-care products. The authors have prepared a number of valuable tables in an effort to guide the practitioner through the maze of commercially available products; they outline indications and suggest the best use for each. This chapter is enhanced by a new and separate section focusing on the use of biologic scaffolds engineered from the extracellular matrix as a potential therapeutic option for treating soft tissue injuries in veterinary medicine.

The fourth chapter leads the reader through the decision-making process preceding the closure of a trau-matic wound. Factors which may preclude this approach are considered, as are the selection of suture materials and patterns as well as the use of drains and alternative, innovative approaches to wound closure in the event that this method is deemed appropriate.

Chapter 5 begins with a review of the physical and biomechanical properties of skin which will help the practitioner develop an appropriate surgical plan. That is followed by a detailed description of practical recon-structive techniques that can be used in conjunction with primary or delayed wound closure.

The next series of chapters (6, 7, and 8) is devoted to the management of wounds in various regions of the body: the head, the neck and body, and the distal extremities, with a focus on how to treat degloving injuries and how to prevent or manage the species-specifi c problem of exuberant granulation tissue.

Chapters 9 and 10 focus on the fact that a fair proportion of traumatic wounds in horses compromise deep, underlying structures. They present detailed anatomical reviews that should enable the practitioner to identify involvement of synovial structures or tendons/ligaments and promptly instigate the recommended therapy to improve the overall prognosis.

ix

x PREFACE

Chapter 11 addresses skin grafting, which should be considered for a wound that cannot heal by epitheli-alization and contraction or be closed using conventional or reconstructive suturing techniques. This chapter describes the principles and various techniques of free skin grafting, the advantages and disadvantages of each technique, and the effects of grafts on the wound.

Chapters 12 through 14 focus on the management of wounds suffering from serious debilitating conditions: severe infection, burn injury, and tumor development. Chapter 12 emphasizes the proper selection of antibiotic agents and reviews methods for optimizing their delivery and effi cacy at the site of infection. Chapter 13 reviews the pathophysiology of burn injury and associated pulmonary damage and advises the reader on the appropri-ate treatment for immediate disorders as well as long-term wound care. While burns are uncommon in horses, management of such injuries can be expensive and time consuming. Moreover, cardiovascular involvement, smoke inhalation, and corneal ulceration can compromise the outcome. Finally, Chapter 14 addresses the trans-formation of a wound into a sarcoid tumor, which suspends the healing process, often indefi nitely. It provides the reader with tips on recognizing and solving this unusual problem.

Chapter 15, which addresses laser surgery, was added to this edition of the textbook in response to the wider usage of this modality in veterinary medicine following the introduction in the 1990s of a more affordable waveguide-delivered CO2 laser. The author discusses various clinical applications of laser surgery pertaining to wound management: debridement, removal of exuberant granulation tissue, graft bed preparation.

Chapter 16 is a well-illustrated chapter intended to guide the practitioner in the art of applying bandages, splints, and casts to support wound healing in different regions of the body.

We thank the contributing authors for their willingness to bring all of their valuable experience to this text-book. We are indebted to these people who generously contributed their clinical insight and current research data.

Acknowledgments

First, I wish to thank Ray Kersey, veterinary consulting editor for Wiley-Blackwell, for his persistence in approaching me regarding a long overdue revision of the fi rst edition of Equine Wound Management. I also want to recognize Erin Gardner, commissioning editor, for her attention to detail and follow-up on the plans discussed at our initial meeting that fi nally led to a contract being signed. Antonia Seymour, publishing director, profes-sional; Nancy Simmerman, editorial assistant; and all of the editorial staff at Wiley-Blackwell have been most patient and helpful with the delays in this revision; for this I am most thankful.

Once committed to the revision, and after realizing the magnitude of the family commitments facing me following my retirement from Colorado State University, I realized that I needed to identify a co-editor. Subse-quent to my experience as guest editor for a wound management journal issue, the choice became clear, and I am so pleased that Dr. Christine Theoret agreed to join me in this endeavor. I am also very grateful to her for her willingness to take the lead during times when I was faced with family medical issues that had to take pre-cedence over my editorial responsibilities.

I thank Jenger Smith, a professional photographer who has been involved with several of my textbooks, for continuing to provide the best possible color images for this publication. I am also grateful to Dave Carlson, the medical illustrator for the fi rst edition, for agreeing to provide many new excellent illustrations. I am deeply indebted to Dennis Sylvain, librarian at the Veterinary Teaching Hospital at Colorado State University, for his willingness to do the numerous literature searches required to present the most current information. Truly, without his help it would have been most diffi cult for me to complete this revision.

I am most grateful to our colleagues and referring practitioners for allowing us the courtesy of using some of their case material as examples. I also acknowledge our residents, barn nurses, and students for the care and treatment of many of the cases presented in the text.

Finally, I would like to acknowledge our clients whose interest in providing the best care for their horses allowed us the opportunity to explore new avenues of treatment which we have shared with the readers of this textbook.

Ted S. Stashak with Christine Theoret

xi

Contributors

Ted S. Stashak, DVM, MS, Diplomate ACVSProfessor Emeritus, Equine SurgeryColorado State UniversitySanta Rosa, CA 95409Tel.: (707) 539-1941Fax: (240) 559-1941, then enter PIN number 8576Email: tstashak@sbcglobal.net

Christine L. Theoret, DMV, PhD, Diplomate ACVSDirector, Comparative Tissue Healing LaboratoryAssociate Professor, Equine Surgical AnatomyFaculté de médecine vétérinaireUniversité de MontréalC.P. 5000 St-HyacintheQuébec, CanadaJ2S 7C6Tel: (450) 773-8521, extension 8517Fax: (450) 778-8109Email: christine.theoret@umontreal.ca

Stephen F. Badylak, PhD, DVM, MDMcGowan Institute for Regenerative Medicine100 Technology Drive, Suite 200University of PittsburghPittsburgh, PA 15219Tel.: (412) 235-5144Fax: (412) 235-5224Email: badylaks@upmc.edu

Cooper Williams, DVM720 Houcksville RoadHampstead, MD 21074Tel.: (410) 239-2323Email: cooperwilliamsvmd@comcast.net

Julie Myers-Irvin, PhDUniversity of PittsburghOffi ce of Research, Health Sciences401 Scaife HallPittsburgh, PA 15261Tel.: (412) 383-5213Email: jmyers_irvin@hs.pitt.edu

Spencer Barber, DVM, Diplomate ACVSProfessor, Equine SurgeryWestern College of Veterinary MedicineUniversity of Saskatchewan52 Campus Dr.Saskatoon, SKCanada S7N 5B4Tel.: (306) 966-7063Fax: (306) 966-7159Email: spence.barber@usask.ca

Gary M. Baxter, VMD, MS, Diplomate ACVSProfessor, Equine SurgeryColorado State University300 West DrakeFt. Collins, CO 80523Tel: (970) 297-4471 or 297-0382Fax: (970) 297-1275Email: gary.baxter@colostate.edu

Christophe Céleste, DrVet, MS, Diplomate ACVS and ECVS

Clinician, Equine Emergency and SurgeryFaculté de médecine vétérinaireUniversité de MontréalC.P. 5000 St-HyacintheQuébec, CanadaJ2S 7C6Tel: (450) 778-8100Email: christophe.celeste@umontreal.ca

Ellis Farstvedt, DVM, MS, Diplomate ACVSCSR Equine, Copper Spring Ranch1373 South Pine Butte RoadBozeman, MT 59718Tel.: (406) 522-4044 or (602) 531-7588 (cell)Email: ellis@csrequine.com

Jorge Gomez, DVM, MS, Diplomate ACVSHagyard Equine Medical Institute4250 Ironworks PikeLexington, KY 40511Tel.: (859) 233-0026Email: jgomez@hagyard.com

xiii

R. Reid Hanson, DVM, Diplomate ACVS and ACVECC

Professor, Equine SurgeryCollege of Veterinary MedicineJ.T. Vaughan Hall1500 Wire RoadAuburn UniversityAuburn, Alabama 36849Tel.: (334) 844-4490Fax: (334) 844-4368Email: hansorr@auburn.edu

Henry Jann, DVM, MS, Diplomate ACVSAssociate Professor, Equine SurgeryCollege of Veterinary MedicineOklahoma State UniversityStillwater, OK 74078-2042Tel: (405) 744-8596Fax: (405) 744-6262Email: jann@okstate.edu

Derek C. Knottenbelt, OBE, BVM&S, DVMS, Diplomate ECEIM, MRCVS

Professor, Equine Internal MedicineUniversity of LiverpoolNestonWirral, UKCH64 7TEEmail: knotty@liv.ac.uk

James A. Orsini, DVM, Diplomate ACVSAssociate Professor, Equine SurgeryDirector, International Laminitis Institute at PennVet

New Bolton CenterSchool of Veterinary MedicineUniversity of PennsylvaniaKennett Square, PA 19348Tel: (610) 925-6402Fax: (610) 925-8120Email: orsini@vet.upenn.edu

Yvonne A. Elce, DVM, Diplomate ACVSClinical Assistant Professor, Equine SurgeryCollege of Veterinary MedicineThe Ohio State University601 Vernon L. Tharp StreetColumbus OH 43210Tel: (614) 292-9683Email: elce.1@osu.edu

Beth M. Ross, DVM, Diplomate ACVS126 Montana Dr.Chadds Ford, PA 19317Tel: (484) 770-8135Email: bmrossdvm@hotmail.com

Jim Schumacher, DVM, MS, Diplomate ACVS, MRCVS

Professor, Equine SurgeryCollege of Veterinary MedicineUniversity of TennesseeKnoxville, TN 37901-1071Tel: (865) 755-8239Fax: (865) 974-5773Email: jschumac@utk.edu

Kenneth E. Sullins, DVM, MS, Diplomate ACVSProfessor, Equine SurgeryVa-Md Regional College of Veterinary MedicineLeesburg, VA 20177Tel: (703) 771-6827Fax: (703) 771-6877 or 771-6810Email: sullins@vt.edu

Jacintha M. Wilmink, DVM, PhD, Diplomate RNVAWoumarec (wound management and reconstruction

in horses)Hamsterlaan 46705 CT WageningenThe NetherlandsTel: **31 (0)317 414462Fax: **31 (0)317 414462Email: jwilmink@tiscali.nl

David A. Wilson, DVM, MS, Diplomate ACVSProfessor, Equine SurgerySection Head, Equine Medicine and SurgeryActing Hospital DirectorDepartment of Veterinary Medicine and SurgeryUniversity of Missouri900 E Campus Dr.Columbia, MO 65211Tel: (573) 882-3513Fax: (573) 884-0173Email: wilsonda@missouri.edu

xiv CONTRIBUTORS

EQUINE WOUND MANAGEMENT

Second Edition

Wound Healing11.1 Physiology of Wound Healing Christine L. Theoret

1.2 Differences in Wound Healing between Horses and Ponies Jacintha M. Wilmink

1.3 Wound Repair: Problems in the Horse and Innovative Solutions Christine L. Theoret

Physiology of Wound Healing

Christine L. Theoret, DMV, PhD, Diplomate ACVS

IntroductionPhases of Wound Repair Acute Infl ammation Cellular Proliferation Matrix Synthesis and RemodelingMediators of Wound Repair Cytokines Growth Factors

Other Tissues Involved in Traumatic Wounds Tendons and Ligaments Periosteum HoofConclusionReferences

Introduction

A vital trait of living organisms, continually subjected to insults from the environment, is their capacity for self repair. Whether the injury is surgical or accidental, it will generate an attempt by the host to restore tissue continuity. Two processes are involved in healing: regeneration and repair. Regeneration entails the replacement of damaged tissue with normal cells of the type lost and is only possible in tissues with a sustained population of cells capable of mitosis, such as epithelium, bone, and liver. Repair is a “stop-gap” reaction designed to re-establish the continuity of interrupted tissues with undifferentiated scar tissue (see Figure 4.12 in Chapter 4). Repair is therefore the “second best” method of healing, producing a result which is less biologically useful than the tissue it replaced and possibly adversely affecting adjacent normal tissues.

Traumatic wounds occur commonly in horses. The objective of repair is re-establishment of an epithelial cover and recovery of tissue integrity, strength, and function. Partial-thickness cutaneous wounds, e.g., abrasions and erosions, heal primarily by migration and proliferation of epidermal cells from the remaining underlying epithelium as well as the adnexal structures (hair follicles, sweat and sebaceous glands), with little participation of infl ammatory or mesenchymal cells. In contrast, repair of full-thickness cutaneous wounds hinges principally on three coordinated phases: acute infl ammation, cellular proliferation, and fi nally, matrix synthesis and remod-eling with scar formation (Figure 1.1). These processes rely on the complex interaction between cells, their sur-rounding matrix, and the mediators that govern their numerous activities.

Veterinarians can positively infl uence wound repair by understanding its mechanisms, which will ensure selection of appropriate wound management techniques. Hippocrates once said, “Healing is a matter of time, but it is sometimes a matter of opportunity.”

Over the past decade, research aimed at unveiling and possibly augmenting the reparative mechanisms of the body has yielded advances in the fi eld of cytokines and the ability to readily synthesize most molecules associated with wound repair.2 This chapter aims to provide an update on the physiological, cellular, biochemi-cal, and molecular aspects of wound repair.

5

1.1

This chapter is reprinted, in a modifi ed form, from Equine Surgery, 3rd edition, Theoret CL, Wound repair, pp. 44–62, Copyright (2005),1 with permission from Elsevier.

6 EQUINE WOUND MANAGEMENT

Phases of Wound Repair

Acute Infl ammation

Infl ammation prepares the wound for the subsequent reparative phases. It purges the body of alien sub-stances and disposes of dead tissue, while the participating cellular populations liberate mediators to amplify and sustain the events to follow. Infl ammation encompasses vascular and cellular responses whose intensity is strongly correlated to the severity of trauma.

Vascular Responses

The injured endothelial cell membrane releases phospholipids that are transformed into arachidonic acid and its metabolites, which mediate vascular tone and permeability as well as platelet aggregation. The fi rst response of the damaged blood vessel is vasoconstriction, lasting 5–10 minutes, after which vasodilation, pri-marily of the small venules, ensues and facilitates diapedesis of cells, fl uids, and proteins across the vessel wall into the extravascular space. Coagulated blood and aggregated platelets together form a clot (provisional matrix) within the wound that, despite providing limited strength, seals off the defect and prevents further bleeding. The clot also functions as a scaffold through the presence of a large number of binding sites on blood proteins that are recognized by special surface receptors (integrins) found on migrating infl ammatory and mesenchymal cells.

Activated platelets are among the earliest promoters of infl ammation, via the release of potent chemoat-tractants and mitogens from their storage granules. These serve as signals to initiate and amplify the reparative phases of healing and are detailed later in this chapter. Over time, the surface clot desiccates to form a scab that protects the wound from infection. This scab is in turn lysed by plasmin, a serine proteinase, and sloughs along with dead infl ammatory cells and bacteria as healing proceeds underneath. The provisional extracellular matrix (ECM) will be replaced by granulation tissue in the next phase of repair.

Cellular Responses

Leukocytes are recruited from the circulating blood pool to the site of injury by the numerous vasoactive mediators and chemoattractants supplied by the coagulation and activated complement pathways, by platelets, by mast cells, and by injured or activated mesenchymal cells.3 These signals initiate the processes of rolling, activation, tight adhesion, and fi nally transmigration of infl ammatory cells through the microvascular endothe-lium. Chemoattractants additionally stimulate the release of enzymes by the activated neutrophils, which facili-tate their penetration through vascular basement membranes upon migration. Neutrophil diapedesis is further facilitated by increased capillary permeability following the release of a spectrum of vasodilatory agents. Cel-

Figure 1.1. Temporal profi le of various processes and gain in tensile strength occurring during normal cutaneous wound repair. Reprinted from Clinical Techniques in Equine Practice, 3, Theoret CL, Update on wound repair, pp. 110–122, Copyright (2004), with permission from Elsevier.

WOUND HEALING 7

lular infl ux begins within minutes and neutrophil numbers progressively increase to reach a peak 1 to 2 days after injury. Neutrophils act as a fi rst line of defense in contaminated wounds by destroying debris and bacteria through phagocytosis and subsequent enzymatic and oxygen-radical mechanisms. The principal deg-radative proteinases released by the neutrophils to rid the site of denatured ECM components are neutrophil-specifi c interstitial collagenase, neutrophil elastase, and cathepsin G. Neutrophil migration and phagocytosis cease when contaminating particles are cleared from the site of injury. Most cells then become entrapped within the clot, which is sloughed during later phases of repair. The neutrophils remaining within viable tissue die in a few days and are phagocytosed by the tissue macrophages or modifi ed wound fi broblasts. This marks the termination of the early infl ammatory phase of repair. Although the neutrophils help create a favorable wound environment and serve as a source of pro-infl ammatory cytokines, they are not essential to repair in uninfected wounds.4

The rapid increase in macrophage numbers under infl ammatory conditions is predominantly caused by the emigration of monocytes from the vasculature, followed by differentiation into macrophages to assist resident tissue macrophages at the wound site for a period of days to weeks. In this manner, the responsive and adapt-able pluripotent monocytes can morph into macrophages whose functional properties are determined by the conditions they encounter at the site of mobilization. Similar to neutrophils, the macrophages are phagocytes and thus carry out debridement and microbial killing. Unlike neutrophils, wound macrophages play a key role in the reparative phases of healing. Indeed, adherence to the ECM (which consists of a cross-linked supporting framework of collagen fi brils and elastin fi bers, saturated with proteoglycans and other glycoproteins) stimu-lates monocytes to transform into a phenotype with the ability to continually synthesize and express the various cytokines necessary for their survival as well as for the initiation and propagation of new tissue formation in wounds (Figure 1.2).

A classic series of experiments in the 1970s determined that wounds depleted of both circulating blood monocytes and tissue macrophages exhibit not only severe retardation of tissue debridement but also a marked delay in fi broblast proliferation and subsequent wound fi brosis.5 Although it has long been considered that the infl ammatory response is instrumental in supplying mediators that orchestrate the cell and tissue movements

Inflammatory phase (day 3)

Epidermis

Dermis

Fat

Blood vessel

VEGF

FGF-2

NeutrophilMacrophage

Fibroblast

IGF

KGF PDGFTGF-β1TGF-β2TGF-β3

TGF-β1

FGF-2

TGF-α

Fibrin clotFibrin clot

MacrophageMacrophage

NeutrophilNeutrophil

Platelet plugPlatelet plug

TGF-β1PDGF ABTGF-β1PDGF AB

PDGF BBPDGF BB

VEGFVEGFFGFFGF

TGF-αTGF-αTGF-β1TGF-β1

Figure 1.2. Illustration of a full thickness cutaneous wound showing the cellular and molecular components present 3 days after injury. FGF = basic fi broblast growth factor; IGF = insulin-like growth factor; KGF = keratinocyte growth factor; PDGF = platelet-derived growth factor; TGF = transforming growth factor; VEGF = vascular endothelial growth factor. Modifi ed from Singer and Clark.3

8 EQUINE WOUND MANAGEMENT

necessary for repair, it was recently shown that mice genetically lacking macrophages and functioning neutro-phils are able to repair skin wounds within a similar time frame to wild-type siblings, and that repair appears scar free, possibly in response to an altered local cytokine and growth factor profi le.6 Conversely, clues from the liver suggest that, although macrophages direct fi brosis, they might also be the best equipped cells to resolve it.7

Upon arrival at the site of infl ammation, macrophages participate in bacterial killing via mechanisms that parallel those of the neutrophil. Three inducible secreted neutral proteinases have been identifi ed in macro-phages: elastase, collagenase and plasminogen activator (PA). These proteinases aid in degradation of damaged tissue and debris, which must be cleared before repair can proceed. Despite the new data gleaned from the study on macrophageless mice, acute infl ammation is still considered crucial to the normal outcome of wound repair, particularly in horses where wounds are frequently exposed to infective agents.

Macrophages are regarded as the major infl ammatory cell not only responsible for debridement, but also for recruitment of other infl ammatory and mesenchymal cells, and subsequent induction of angiogenesis, fi bro-plasia and epithelialization. Paradoxically, prolonged infl ammation will retard healing and encourage the development of chronic proliferation of fi broblastic granulation. This is thought to contribute to the pathogenesis of a number of diseases characterized by disproportionate scarring, such as pulmonary fi brosis, hepatic cirrhosis, glomerulonephritis, and dermal keloids in humans. Extensive scarring or fi brosis of any organ may cause cata-strophic loss of function of that organ. In the horse, a comparable condition is the development of exuberant granulation tissue in full-thickness cutaneous wounds (see Treatment of Exuberant Granulation Tissue in Chapter 8).

Infl ammation is a sequence of events: production of mediators; rolling, tethering, and adhesion of neutro-phils to vascular endothelium with subsequent migration through endothelium and basement membranes; altered vascular permeability with passage of fl uid into tissues; neutrophil phagocytosis of invading organisms, and release of biologically active materials; emigration of monocytes from the local vasculature; and maturation of monocytes into infl ammatory macrophages with subsequent removal of the components of infl ammation. Resolution of infl ammation should therefore address each one of these events and halt or potentially reverse it. However, despite the importance of the processes by which infl ammation normally resolves, little research has been done in this area.

Apoptosis, or programmed cell death, is the universal pathway for the elimination of unneeded cells and tissues in a phagocytic process that does not elicit additional infl ammation.8 This mechanism prevails during all phases of wound repair because each relies on rapid increases in specifi c cell populations that either prepare the wound for repair (infl ammatory cells) or deposit new matrices and mature the wound (mesenchymal cells), but then must be eliminated prior to progression to the next phase. Indeed, a mature wound is typically acellular.

In conclusion, it appears that the termination of infl ammation is a complex and closely regulated sequence of events. There are several steps at which the resolution process could go astray, leading to suppuration, chronic infl ammation (Figure 1.3), and/or excessive fi brosis.

Cellular Proliferation

Fibroplasia

The proliferative phase of repair comes about as infl ammation subsides, and is characterized by the appear-ance of red, fl eshy granulation tissue, which ultimately fi lls the defect. Although the earliest part of this phase is very active on a cellular basis, this does not immediately translate into a gain in wound strength. Indeed, during the fi rst 3 to 5 days following injury, mesenchymal cells such as fi broblasts, endothelial, and epithelial cells are rapidly invading the wound in preparation for matrix synthesis and maturation or for coverage; however, these latter reinforcing mechanisms lag somewhat. Granulation tissue is formed by three elements that move into the wound space simultaneously: macrophages debride and produce cytokines and growth factors, which stimulate angiogenesis and fi broplasia; fi broblasts proliferate and synthesize new ECM compo-nents; and new blood vessels carry oxygen and nutrients necessary for the metabolism and growth of mesen-chymal cells, and confer to the granulation tissue its characteristic red, granular appearance (Figure 1.4).3

This stroma, rich in fi bronectin and hyaluronan, replaces the fi brin-containing clot to provide a physical barrier to infection, and importantly, it provides a surface across which mesenchymal cells can migrate. A number

Figure 1.3. This metatarsal wound failed to heal for 7 months as a result of chronic low-grade infl ammation due to exposure as well as superfi cial and deep infection. The wound in fact became larger rather than smaller, illustrating suspension of the healing process. Courtesy of Dr. D. Knottenbelt.

Epithelialization and Angiogenesis (day 5)

Epidermis

Dermis

Fat

Fibroblast

Collagen

Blood vessel

Fibrin clotFibrin clot

Granulation tissueGranulation tissue

u-PAMMP-1,2,3,13 u-PAMMP-1,2,3,13

t-PAMMP-1,2,3,13 t-PAMMP-1,2,3,13 u-PA

MMP-1,2,3 u-PAMMP-1,2,3

Figure 1.4. Illustration of a full-thickness cutaneous wound 5 days after injury showing angiogenesis, fi broplasia, and epithelializa-tion. uPA = urokinase-type plasminogen activator; tPA = tissue-type plasminogen activator; MMP = matrix metalloproteinase. Modifi ed from Singer and Clark.3

9

10 EQUINE WOUND MANAGEMENT

of matrix molecules as well as chemoattractants, cytokines, and growth factors released by infl ammatory cells are believed to stimulate fi broblasts from adjacent uninjured dermis and subcutaneous tissue to proliferate and express integrin receptors to assist migration into the wound space. Integrins are transmembrane proteins that act as the major cell-surface receptors for ECM molecules and thus mediate interactions and transduction of signals between cells and their environment. They are particularly critical to the migratory movements exhibited by wound healing cells such as epithelial and endothelial cells, as well as fi broblasts. Migration immediately precedes advancing capillary endothelial buds but follows macrophages, which have cleared a path by phago-cytozing debris. Fibroblasts themselves also possess an active proteolytic system to aid migration into the cross-linked fi brin blood clot: proteinases include PA, various collagenases, gelatinase, and stromelysin.9

Once fi broblasts have arrived within the wound space, they proliferate and then switch their function to protein synthesis and commence the gradual replacement of provisional matrix by a collagen-rich one, probably under the infl uence of various cytokines and growth factors. As the wound matures, there is a marked increase in the ratio of type I (mature) to type III (immature) collagen; proteoglycans also become abundant within the mature matrix. The greatest rate of connective tissue accumulation within the wound occurs 7 to 14 days after injury, which translates into the period of most rapid gain in tensile strength (Figure 1.1).

Thereafter, collagen content levels off as fi broblasts down-regulate their synthetic mechanisms; this corre-sponds to a much slower gain in wound strength, which occurs as the wound remodels. The fi broblast-rich granulation tissue is subsequently replaced by a relatively avascular and acellular scar as the capillary content regresses and fi broblasts either undergo apoptosis10 or acquire smooth-muscle characteristics and transform into myofi broblasts that participate in wound contraction. The latter phenomena are regulated by the physiological needs and/or the microenvironmental stimuli present at the wound site. It appears that if the signal to down-regulate fi broblast activity is delayed beyond a specifi c time point, apoptosis is permanently impaired, which ultimately leads to an imbalance between collagen synthesis and degradation11 and the formation of excessive scar tissue.

Angiogenesis

Besides initiating the infl ammatory response through interaction with leukocytes, microvascular endothelial cells play a key role in the proliferative phase of repair. The formation of new capillary blood vessels from pre-existing ones (angiogenesis) is necessary to sustain the granulation tissue newly formed within the wound bed. Angiogenesis, in response to tissue injury and hypoxia, is a complex and dynamic process mediated by diverse soluble factors from both serum and the surrounding ECM environment, in particular angiogenic inducers including growth factors, chemokines and angiogenic enzymes, endothelial-specifi c receptors, and adhesion molecules such as integrins,12 many of which are released during the infl ammatory phase of repair.

Construction of a vascular network requires sequential steps that include augmented microvascular perme-ability, the release of proteinases from activated endothelial cells with subsequent local degradation of the basement membrane surrounding the existing vessel, migration and sprouting of endothelial cells into the interstitial space, endothelial cell proliferation and formation of granulation tissue and differentiation into mature blood vessels, eventually followed by regression and involution of the newly formed vasculature as tissue remodels.13 Angiogenesis is dependent not only on the cells and cytokines present, but also on the pro-duction and organization of ECM components which act as both scaffold support through which endothelial cells may migrate and as reservoir and modulator for growth factors. Thus, endothelial cells at the tip of capil-laries begin their migration into the wound in response to angiogenic stimuli and absence of neighboring cells on the second day following injury.

Cytoplasmic pseudopodia extend through fragmented basement membranes; subsequently, the entire endo-thelial cell migrates into the perivascular space. Cells remaining in the parent vessel near the tip of the angio-genic sprouts begin to proliferate, providing a continuous source of microvascular endothelial cells for angiogenesis. When a new capillary sprout fi rst develops it is solid; after it fuses with a neighboring sprout to form an arcade, it becomes canalized and erythrocytes pass into and through it.

Lumen formation probably involves the joining of plasma membranes of individual and/or adjacent cells, as well as extensive intracellular vacuolization followed by fusion of the vacuoles to form “ring cells,” which ultimately fuse to form seamless capillaries. Capillaries then become stable as endothelial cells interact with the new basement membrane within 24 hours of new vessel formation. Once the reconstitution of stroma is complete, there is no longer the need for a rich vascular supply. Angiogenic stimuli are down-regulated or the local con-

WOUND HEALING 11

centration of inhibitors increases and most of the recently formed capillary network quickly involutes through the activity of matrix metalloproteinases (MMPs),14 in particular MMP-1 and MMP-10,15 and apoptosis of endo-thelial cells. The wound color becomes paler as the rich capillary bed disappears from the granulation tissue.

Epithelialization

All body surfaces are covered by epithelium, which acts as a discriminating barrier to the environment. As such, epithelium provides the primary defense against hostile surroundings and is a major factor in maintaining internal homeostasis by limiting fl uid and electrolyte loss. The outer multilayered stratifi ed squamous epithe-lium (the epidermis) interfaces with the musculoskeletal framework by means of a connective tissue layer (the dermis) and a fi bro-fatty layer (the subcutis). Epidermis is attached to the dermis at the level of the basement membrane, a thin, glycoprotein-rich layer composed primarily of laminin and type IV collagen. This attachment is mediated by hemidesmosomes, which physically attach the basal cells of the epidermis to the underlying dermis, as well as by vertically oriented type VII collagen anchoring fi brils, which bind the cytoskeleton.16

It is critical to survival that an extensive full-thickness wound be covered without delay. In addition to the aforementioned hemostatic activities, which establish a temporary barrier, centripetal movement of the residual epithelium below the clot participates in wound closure. Although epithelial migration commences 24 to 48 hours following wounding, the characteristic pink rim of new epithelium (Figure 1.5) is not macroscopically visible until 4 to 6 days later, although this is variable since the rate of wound closure depends on the animal species as well as the wound site, substrate, and size.

For example, epithelialization is accelerated in a partial-thickness wound because migrating cells will arise not only from the residual epithelium at the wound periphery but also from remaining epidermal appendages. Furthermore, the basement membrane is intact in this type of injury, precluding a lengthy regeneration. On the other hand, during second intention healing of a full-thickness wound, epithelialization must await the forma-tion of a bed of granulation tissue to proceed. Wounds in the fl ank region of a horse epithelialize at a rate of 0.2 mm/day, compared with a rate as slow as 0.09 mm/day for wounds in the distal region of limbs.17

In preparation for migration, basal epidermal cells at the wound margin undergo phenotypic alterations that favor mobility and phagocytic activity. Additionally, various degradative enzymes necessary for the prote-olysis of ECM components are up-regulated within cells at the leading edge, facilitating ingestion of the clot and debris found along the migratory route. The migratory route is determined by the array of integrin recep-tors expressed on the surface of migrating epithelial cells, for various ECM proteins. Indeed, a fundamental reason why migrating epidermis dissects the fi brin eschar from wounds is that they cannot interact with the fi brinogen and its derivatives found within the clot since they lack the appropriate integrin.18 Once the wound surface is covered by epithelial cells that contact one another, further migration is inhibited by the expression within the ECM of laminin, a major cell adhesion factor for epithelial cells, from the margin of the wound inward.

Figure 1.5. Large full-thickness metatarsal wound that healed partially by second intention and was subsequently grafted suc-cessfully. The wound showed excellent epithelialization from the healing margin of the wound. Note the island of epithelium cor-responding to a graft. The healthy epithelial tissue is character-ized by an area of hyperemia adjacent to it. Courtesy of Dr. D. Knottenbelt.

12 EQUINE WOUND MANAGEMENT

Although initial cell migration does not require an increase in cellular multiplication, epidermal cells at the wound margin do begin to proliferate 1 to 2 days after injury to replenish the migratory front. This corresponds histologically to epithelial hyperplasia (Figure 1.6), as cellular mitosis increases 17-fold within 48 to 72 hours. The new cells leapfrog over those at the wound margin and adhere to the substratum, only to be replaced in turn by other cells coming from above and behind. The newly adherent monolayer subsequently restratifi es to restore the original multilayered epidermis.

In full-thickness wounds healing by second intention, such as those commonly managed in equine practice, provisional matrix is eventually replaced by a mature basement membrane zone. Repairing epidermis reas-sembles its constituents from the margin toward the center of the wound.3 Epidermal cells then revert to a qui-escent phenotype and become attached to this new basement membrane through hemidesmosomes and to the underlying neodermis through type VII collagen fi brils. This particular aspect of epithelialization is time-consuming, occurring long after total wound coverage is apparent, which may explain the continued fragility of neoepidermis for extended periods following macroscopically complete repair. This is particularly evident in large wounds of the limb, where epidermis at the center is often thin and easily traumatized (Figures 1.7a and 1.7b).

A B

Figure 1.6. Photomicrograph of wound edge biopsy. Normal unwounded skin to the left demonstrating epidermal append-ages (h = hair follicles); new hyperplastic epithelium (EH) to the right, overlying granulation tissue bed. Reprinted from Clinical Techniques in Equine Practice, 3, Theoret CL, Update on wound repair, pp. 110–122, Copyright (2004), with permission from Elsevier.

Figure 1.7. (a) A 5-day-old, full-thickness, experimentally created wound over the dorsal fetlock. Granulation tissue is beginning to fi ll the wound bed. (b) The same wound, 75 days following creation. Neoepidermis is thin, dry, and hairless, and could be easily traumatized. Courtesy of Dr. T. Stashak.

WOUND HEALING 13

Matrix Synthesis and Remodeling

In addition to epithelialization, contraction contributes to the successful closure of full-thickness wounds. Contraction is defi ned as a process whereby both dermis and epidermis bordering a full-thickness skin defi cit are drawn from all sides centripetally over the exposed wound bed.19 This usually occurs during the second week following injury. Wound contraction not only accelerates closure, but also enhances the cosmetic appear-ance and strength of the scar because proportionally less wound area must be covered by newly formed inferior quality epithelium, which is fragile and lacks normal nervous, glandular, follicular, and vascular components (see Figures 4.12 and 1.7b). For this reason, a high degree of contraction is a desired feature of wound repair, at least in the horse.

A number of theories have been proposed to explain wound contraction; however, most authorities agree that it involves a fi nely orchestrated interaction of ECM, cytokines/growth factors, and cells, in particular a specialized fi broblast phenotype: the myofi broblast. These are the most abundant cellular elements of healthy granulation tissue and are aligned within the wound along the lines of contraction. The most striking feature of the myofi broblasts is a well-developed alpha smooth muscle actin (α-SMA) microfi lamentous system, arranged parallel to the cell’s long axis and in continuity with ECM components via various integrins. In addition to these cell-substratum links, intercellular connections such as gap junctions and hemidesmosomes ensure that neigh-boring cells exert tension on one another. Factors producing and regulating contraction are presently unknown, but appear to include various cytokines/growth factors.

Wound contraction is divided into three phases. An initial lag phase (wherein skin edges retract and the wound area increases temporarily for 5 to 10 days) occurs prior to signifi cant fi broblastic invasion into the wound, which is a prerequisite for contraction. Subsequently a period of rapid contraction is followed by one of slow contraction as the wound approaches complete closure. The number of myofi broblasts found in a wound appears proportional to the need for contraction; thus, as repair progresses and the rate of contraction slows, this number decreases accordingly.

During wound contraction, the surrounding skin stretches by intussusceptive growth and the wound takes on a stellate appearance. Contraction ceases in response to one of three events: the wound edges meet and contact inhibition halts both the processes of epithelialization and contraction; tension in the surrounding skin becomes equal to or greater than the contractile force generated by the α-SMA of the myofi broblasts; or, in the case of chronic wounds, a low myofi broblast count in the granulation tissue may result in failure of wound contraction despite laxity in the surrounding skin. In the latter case, the granulation tissue is pale and consists primarily of collagen and ground substance. Wound contraction is greater in regions of the body with loose skin than in regions where skin is under tension, such as the distal aspect of horse limbs. Although it is speculated that the shape of the wound may infl uence the process of contraction, this does not appear relevant in wounds at the distal extremities of horse limbs where skin is tightly stretched and not easily moved.20

As contraction concludes, myofi broblasts disappear, either by reverting to a quiescent fi broblast phenotype or by apoptosis,10 primarily in response to reduced tension within the ECM.21 The myofi broblast persists in fi brotic lesions where it may be involved in further ECM accumulation and pathologic contracture, a condition leading to signifi cant morbidity, particularly when it involves joints or body orifi ces, but is rarely encountered in the horse.

The conversion of ECM from granulation to scar tissue constitutes the fi nal phase of wound repair and consists of connective tissue synthesis, lysis, and remodeling, also referred to as maturation. Proteoglycans replace hyaluronan during the second week of repair, support the deposition and aggregation of collagen fi bers, and provide the mature matrix with better resilience. Collagen macromolecules provide the wound tensile strength as their deposition peaks within the fi rst week in primary wound repair and between 7 and 14 days in second intention healing. Although this corresponds to the period of most rapid gain in strength, only 20% of the fi nal strength of the wound is achieved in the fi rst 3 weeks of repair. At this time, collagen synthesis is balanced by collagen lysis, which normally prevents accumulation of excessive amounts of collagen and forma-tion of pathologic scars.

The balance between synthesis and degradation determines the overall strength of a healing wound at a particular time. The fi rst newly deposited collagen tends to be oriented randomly and therefore provides little tensile strength, whereas during remodeling the fi bers reform along lines of stress and therefore resist dehiscence more effectively. Cross-linking in the later formed collagen is also more effective, although never to the same

14 EQUINE WOUND MANAGEMENT

extent as in the original tissue. The new collagen weaves into that which preexisted and also appears to bond to the ends of old collagen fi bers. These welds are points of weakness, which may rupture under stress.

Collagen degradation within a wound depends on the presence of various proteolytic enzymes released from infl ammatory and mesenchymal cells. Most are of the MMP family of zinc-dependent endopeptidases that are collectively capable of degrading virtually all ECM components. Although MMPs are not constitutively expressed in skin, up-regulation occurs whenever proteolysis is required, such as during cell migration and matrix remodeling. Inactive precursors of the MMPs are cleaved in the extracellular space by proteinases such as plasmin and trypsin, leftover from the infl ammatory and proliferative phases, but also by other MMPs.

To date, more than 20 different MMPs, all distinct gene products, have been characterized (Table 1.1)9 and are generally divided into four major groups. The best-known subgroup of MMPs are the collagenases (MMP-1, -8, and -13), which possess the unique ability to cleave the triple helix of native types I, II, and III collagens, the rate-limiting step of collagen degradation. The fragments generated are thermally unstable and denature into their constitutive polypeptide chains, forming gelatin peptides. Basal epithelial cells at the migratory front of epithelialization are the predominant source of collagenase during active wound repair,9 while the resolution of granulation tissue also depends on the activity of collagenase, in this case expressed by dermal fi broblasts.

Table 1.1. Major matrix metalloproteinases (MMPs) involved in wound repair.

MMP name MMP # Substrates Source

Collagenases

Interstitial collagenase MMP-1 Collagen (I, II, III, VII, IX) Epithelial cell, fi broblast

Neutrophil collagenase MMP-8 Collagen (I, II, III) PMN

Collagenase 3 MMP-13 Collagen (I, II, III, IV) Endothelial cell, fi broblast

Stromelysins

Stromelysin-1 MMP-3 PGs, laminin, fi bronectin, Epithelial cell

Stromelysin-2 MMP-10 Collagen (III, IV, IX, X) Epithelial cell, fi broblast

Stromelysin-3 MMP-11 Collagen IV, fi bronectin, gelatin, laminin Fibroblast

Gelatinases

Gelatinase A (72 kDa) MMP-2 Gelatin, collagen (I, IV), elastin Most cells

Gelatinase B (92 kDa) MMP-9 Gelatin, collagen (IV, V), elastin Infl ammatory cell, epithelial cell, fi broblast

Matrilysin MMP-7 PGs, elastin, fi bronectin, laminin, gelatin, collagen IV

Epithelial cell

Membrane-type MMPs

MT1-MMP MMP-14 Collagen (I, III), fi bronectin

MT2-MMP MMP-15 Vitronectin, pro-MMPs

MT3-MMP MMP-16 Collagen (III), fi bronectin

MT4-MMP MMP-17 Gelatin, pro-gelatinase A

MT5-MMP MMP-20 Collagen (V), aggrecan, COMP Tooth enamel

PMN = polymorphonuclear granulocytePG = proteoglycanCOMP = cartilage oligomeric matrix protein

WOUND HEALING 15

The stromelysins, members of another subgroup of MMPs, possess broad substrate specifi city. Stromelysin-1 and -2 are strong proteoglycanases and can also degrade basement membranes, laminin, and fi bronectin, while stromelysin-3 is only weakly proteolytic. Although stromelysin-2 expression is strictly confi ned to the epidermis, stromelysin-1 is also abundantly expressed by dermal fi broblasts in the granulation tissue associated with wounds. Because of its broad substrate specifi city, it may be important in remodeling the matrix, in particular the newly formed basement membrane, during repair.9

There are two metallogelatinases: the 72 kilodalton (kDa) gelatinase (gelatinase A) which, unlike other MMPs, is produced constitutively by most cells types, and the 92 kDa gelatinase (gelatinase B), produced by most infl ammatory cells as well as by epithelial cells. Both types effi ciently degrade denatured collagens (gela-tins) and also attack basement membranes, fi bronectin, and insoluble elastin. Matrilysin is the smallest MMP (28 kDa) but is a stronger proteoglycanase than stromelysin and also degrades basement membranes, insoluble elastin, laminin, fi bronectin, and gelatin.

Homeostasis between collagen synthesis and degradation during the remodeling phase depends on the simultaneous presence of MMPs and non-specifi c inhibitors such as α2-macroglobulin and α1-antiprotease, as well as natural specifi c inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). TIMPs are a gene family of four structurally related members, TIMP-1 through -4, that inhibit conversion of MMPs from a zymogen to an activated state and that irreversibly bind to the catalytic site of active MMPs. The role of TIMPs in wound repair is not limited to remodeling, since they also promote growth in a wide range of cell types and are thought to stabilize the basement membrane of regenerating epidermis, as well as inhibit angiogenesis and induce apoptosis.

Inhibition of MMP activity during the acute infl ammatory phase enhances the strength of the wound reached during the repair phase despite accompanying decreases in the infl ammatory response and new colla-gen synthesis. This is thought to result from decreased collagen turnover or increased collagen maturation and cross-linking, or both.22 However, under most circumstances, an imbalance between MMPs and TIMPs will lead to abnormal resolution and delayed repair. Indeed, although the presence of MMPs is essential for normal wound maturation, it may also be responsible for the inability of chronic wounds to heal. For example, chronic wound fl uid is characterized by elevated levels of proteinases, particularly MMP-9 and serine proteinases, which lead to excessive protein degradation and the inactivation of critical growth factors. Chronic wounds also contain reduced levels of TIMPs, in particular TIMP-1.23 It is interesting to note that as epithelialization pro-gresses, production of MMPs by epithelial cells is turned off, allowing the formation of hemidesmosomal adhe-sions between cells and the basement membrane.

Wound remodeling continues for up to 2 years. During that time there is no net increase in collagen content, but rather a rearrangement of collagen fi bers into a more organized lattice structure, under the infl u-ence of local mechanical factors, progressively increasing the tensile strength of scar tissue. The majority of type III collagen fi bers laid down early in the healing process are replaced by collagen type I, fi bers become increas-ingly cross-linked, and the normal skin ratio of 4 : 1 type I to type III collagen is re-established. Glycosamino-glycans are steadily degraded until they reach concentrations found in normal dermis. The duration of the maturation phase depends on a variety of factors including the patient’s genetic makeup, age, location of the wound on the body, type of injury, and duration of infl ammation. At maximum strength, cutaneous wounds remain 15–20% weaker than the normal surrounding tissue, although this varies markedly among species (Figure 1.1).24

Mediators of Wound Repair

Wound repair relies on a complex amalgamation of interactive processes involving formed elements of blood (e.g., erythrocytes, platelets, leukocytes), ECM, and mesenchymal cells. Although histological and mor-phometric observations have permitted a detailed description of the kinetics of cellular and macromolecular components involved in repair, much remains to be learned about the regulation of such activities. Restoration of structural integrity and partial functional properties appears to rely on soluble mediators synthesized by cells present in the wound or in the surrounding tissue which coordinate migration, proliferation, and protein syn-thesis by the various cell populations involved in the repair process.

Cytokines, defi ned as 4–60 kDa signaling glycoproteins released by most nucleated cells, are among the most important soluble mediators regulating wound repair. They act in concentrations of 109–1012 M in an auto-crine (same cell), paracrine (adjacent cell), or endocrine (distant cell) fashion. For cytokines to exert an effect,