arthroscopic transfer of osteochondral...

ARTHROSCOPIC TRANSFER OF OSTEOCHONDRAL ALLOGRAFTS IN A BOVINE ANIMAL MODEL

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

THOMAS SCHIEL

In partial hifilment of requirements

for the degree of

Doctor of Veterinary Science

January, 1998

O Thomas Schiel, 1998

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ARTHROSCOPIC T W S F E R OF OSTEOCHONDRAL ALLOGRAFTS IN A BOVINE MODEL

Thomas Schiel University of Guelph, 1998

Advisor: Mark B .Hurtig

The purpose of this study was to investigate the short-term fate of arthroscopically

implanted 6 . 0 m osteochondral allografts. The experiment was devided into a

preliminary and experimental phase. During the preliminary phase techniques were

developed to asepticaily harvest osteochondral grafis From fresh bovine

metacarpophalangeal (fetlock) joints, and an iuthroscopic approach to the same joint was

developed. In the experimental phase, osteochondral dowels (6.0mm in diameter and

10.0 to 15.0mrn in length) and bone dowel controls were harvested from the lateral and

media1 condyle of cadaver fetiock joints of calves weighing 200kg. These grafts were

refngerated for 12 hours, and then inserted into 6.0mm drill holes in the laterai and

medial condyle of both fiont fetlock joints in six calves using anhroscopic techniques.

One fetlock joint of each calf received two gr& with intact cartilage. wble the

contralateral control received two bone dowels as negative controls. In the six recipient

calves, a total of 24 grafts (12 osteochondral and 12 bone gr&) were implanted

arthroscopically. The calves were recovered fiom anesthesia and aiiowed full activity

during the three month period before they were slaughtered.

Paravital staining, chondrocyte counts. and a modified Mankin scoring system

were used to evaiuate the joint surface in and around the experimental sites. Histology

and measurement of the trabecular bone area under the grah were used to assess the

incorporation of the bony portion of the grrifts. The results were compared to eight

unoperated front fetlock joints of calves weighing 200kg. Overdl, osteochondral g r d s

maintained an articular surface that was inferior to unoperated animals, but were far

superior to bone dowel controls. Osteochondral allografts had good bony incorporation

into the recipient sites, but postoperative congruency with the mounding articular

surface was essential for a good outcome. Five of 12 osteochondral g r a s that were

recessed or othenvise malpositioned became covered with fibrous tissue.

Resurfacing of chondral defects with multiple small osteochondral gnfts

(MosaicPlasty) is currently used in human surgery, and may be useful in selected cases in

animals. The results of this study show that long-term stability and congruency are

essential, particularly in joints were the cartilage is thin. In areas where cartilage is thin.

s m d rnistakes r e d t in exposure of the bony sidewall of the recipient site. New

comective tissue develops from the exposed bone and spreads across the cartilage.

dtimateiy destroying the gr&.

Dedicated to the one meaningful thing in my iife.

My daughter Tara Alexandra Schiel.

Tara,

The trouble with life is that it is too short Alfonso Cabeza de Vaco y Leighton, 17' Marques de Portago

Never forget this.

There is a land where the mountains are nameless and the rivers al1 run god knows where

Robert W. Service "Songs of a Sourdough", Toronto 1907

Never stop lookîng for it.

I love you.

caring. Regina and Klaus Herbstritt, Anne Hanchet and David Boland Carol and David

Atkinson, Kaye and Harold Gnimme. Al1 outstanding human beings. The Tullys who

made me feel very welcorne. I am pnvileged to be surrounded by wonderful people.

Last but not least, tremendous thanks go to Roberta Milite110 for being a good mom for

Tara. Wild Bill (orange #27) is a great uncle, Denice a super aunt!

Often forgotten or denied is the fact that this thesis is only a smail part of my (or

of anybody else's) surgical residency. Years ago, Bemd Maske, M.D. allowed me to

develop an interest in surgery. Natalie Cote, staff surgeon at the Ontario Veterinary

College, taught me most of my surgical skills and becme a close f'riend. Nat. 1 will miss

you. And 1 shall never forget what you did for me. Many thanks to everyone eise who

participated in training me in Ithaca and Guelph over the p s t four years.

The intems Drs. Brin, Parsons, Hewlett, Donovan, Denhaed, Sinclair. Boutros.

King, Richwagen, Nun, Wion and Mak enabled me to take fiequent trips to Ithaca to see

my daughter. Fishing on a weekend for sure beat taking care of horses! When a little

older, Tara will understand what these overworked and underpaid people did for her.

Th& you guys! Some of my fellow residents becarne outstanding fnends that will be

missed. Good luck to you al1 - Chns Boutros, Stuart Munay. Alice Moore-Keir, Johan

Brojer and Simon Pearce. See you wherever.

Finaliy, neither this page or any part of this thesis wouid have been completed

without my very best fnend, Lauren D.Tuily. Lauren, i love you with dl my heart.

Thank you for being there, thank you for everything. Here is to a great funue!

DECLARATION OF WORK PERFORMED

1 hereby declare, with the exception of the items listed below, that al1 worked

reported in this thesis was performed by me.

General anesthesia of the experimental calves was induced and maintained by

Amanda Hathaway, R.H.T.

Microsectionhg and staining of the histological specimens was performed by

Helga Hunter and Jasmine Rendulich.

Dr.Mark Hurtig conducted the cornputer-generated "Paravital Staining" data

collection.

Mohammed Shoukn and Gabrielle Monteith designed and performed the

statistical analysis for this project.

TABLE OF CONTENTS

Dedication

Declaration of Work Pefiormed iii

Table of Contents iv

List of' Tables vii

List of Figures xiv

CHAPTER 1: REVlEW OF THE LITERATURE

1.1 Introduction 1

1.2 Articular Cartilage 2 1.2.1 Composition and Structure of Articular Cartilage 3 1.2.2 Healing of Articular Cartilage 4 1 2 . 3 Repair of Aaicular Cartilage: Current Treatment Options 5

1.3 Joint Debridement and Lavage 7

1.4 Marrow-Stimulation Techniques 8 1.4.1 Abraison Arthroplasty 10 1.4.2 Subchondral Bone Drilling 12 1.4-3 Microf'racture of the Subchondral Bone Plate 14

1.6 Repair of Chondral Defects with Tissue G&s 16 1.6.1 PerichondrialGrafts 18 1-6.2 Periosteal Grah 20

1.7 Chondmcyte Transplantation 3 1

1.8 Osteochondral Gr& 27 1.8.1 Osteochondral Grafting with S m d to Medium Size Gr& 28 1.8.2 0steochondral"Miniature"Grafts 31

1.8.3 Mosaic Arthroplasty in Research and Clinical Application 32

1.9 Arthroscopic Surgery 35 1.10 The Bovine ANmal Model in Orthopedic Research 40 1.1 1 Anatomy of the Bovine Fetlock Joint 40

1.1 3 Aims and Objectives 43

1.14 References 45

CHAPTER U: MATERIALS AND METHODS

2.1 Smdy Design 63

2.2 Prelirnhary Phase 63 22.1 Graft Hanest 64 2.2.2 Graft Testing 68 2.2.3 Arthroscopic Approach 69 2.2.4 Graft Implantation 70

2.3 Experimental Phase 72 2.3.1 Graf? Harvest and Storage 73 2.3.2 Graf? Implantation 73 2.3.3 Collection of Specimens 76 2.3.4 Identification and Histological Analysis of Specimens 77 2.4 Paravitai Staining 80

2.5 Chondrocyte Count 82

3.6 Total Trabecular Area 83

2.7 Modified Manklli Sconng 84

2.8 Statistical Analysis 84

2.9 Sources 86

3.10 References 90

CHAPTER III: RESULTS

3.1 Prelirninary Phase 92 3.1.1 Gr& Harvest 92 3.1.2 Investigation of the Arthroscopie Approach 92 3.2 Experimental Phase 93 3 2 . 1 Donor Animal Examination and Grfi Harvest 93 3.2.2 Gr& Implantation 93

3.3 Collection of Specirnens 97 3.3. L Osteochondral Grafts (Treatment 1): Macroscopic Findings 98 3.3.2 Bone Grafts (Treatment 2): Macroscopic Findings 99 3.3.3 Histological Analysis of Specimens 99

3.4 Paravital Staining 1 O 1

3.5 Chondrocyte Numbers 104

3.6 To ta1 Trabecular Area 1 O8

3.7 Modified Mankin Scoring 1 1 1

CHAPTER IV: DISCUSSION

4.1 Osteoc ho ndral Grafting 1 1 5

4.2 Graft Harvest 1 16

4.3 Gd? Storage 1 17

4.4 Gr& Implantation 1 18

4.6 Summary of Conclusions 126

4.7 References 130

APPENDIX :

Tables 135

Figures 156

LIST OF TABLES:

Page 103. Table 1 . Paravital staining data showing the mean percentage and

standard error of undamaged/totai cells.

Page 103. Table 2. Cornparisons of anatomic sites G (gr&), S (surrounding) and

P (phalam). Analyses of variance for the proportion of damageci/ undamaged cells were

considered significantly different when p<.OS*.

Page 103. Table 3. Cornparison of Treatment 1 (osteochondral grafts) with

Treatment 2 @one grafts) with respect to the graft (G), area immediately surrounding the

gr& (S) and the opposing articular surface (P). Analyses of variance were considered

significantly different when pe.05 * .

Page L06. Table 4. Mean numbers (with standard errors) of Weigart's

haematoxylin-staining chondrocytes counted in the graft (G), area imrnediately

swounding the graft (S) and the opposing joint surface (P).

Page 106. Table 5. This table shows the percentage of chondrocytes in

Treatment 1 and 2 compared to unoperated controls in Treatment 3.

Page 107. Table 6. This table shows comparisons of chondrocyte numbers in the

gr& (G) versus the surrounding area (S), the graft versus the opposing joint surface (P),

and the opposing joint surface (P) versus the surrounding area (S) in the three treatment

groups. Analyses of variance for the proportion of intact chondrocytes were considered

signincantly different when p<.O5 * .

Pige 107. Table 7. The fust and second row show a cornparison of chondrocytes

counted in each anatomic site in the joints that received osteochondral gr& (Treamient

1) and bone grafts (Treatment 2) as compared to unoperated contmls in group 3. The last

vii

row compares chondrocyte numbers between osteochondral and bone gnfis. Analyses of

variance were considered significantly different when p<.05*.

Page 110. Table 8. Mean trabecular area of combined Level 1, 2 and 3 with

standard errors (percentage of bone in the three fields of interest for each anatomic site).

Page 110. Table 9. Cornparison of mean trabecular areas in the gr& (G) venus

surrounding area (S) in osteochondral gr& (Treatment l), bone g r a h (Treatment 2) and

unoperated controls (Treatment 3). The trabecular area in the graft versus the opposing

joint surface (P) and the phdangeai site (P) as compared to the surrounding site (S) are

also included. Analyses of' variance for the tmbecular area were considered significantly

different when pc.05 *.

Page 110. Table 10. The tint two rows show cornparisons of trabecular area in

osteochondral grafts (Treatment 1) and bone gr&s (Treatment 2) with unoperated

nomals (Treatrnent 3). The last row shows cornparisons that indicate there was no

difference in trabecuiar area between osteochondral and bone gr& groups. Analyses of

variance were considered significantly different when p<.05*.

Page 113. Table 1 1. Average combined Modified Mankin scores for the grafi

(G), area surrounding the gr& (S) and phalangeai (P) sites in joints with osteochondral

gr& (Treatment 1), bone grah (Treatrnent 2) and unoperated controls (Treatment 3).

Zero (O) is optimal, higher numbers indicate darnage and degenention.

Page 113. Table 12. Cornparisons for "Ceflularity" scores when cartilage from

osteochondral grafis and bone gafls were compared to operated controls of (Treatment

3 ) Cellularity in the gr& site and surrounding area was different when osteochondral

g&s were compared to bone grah, but not when the opposing joint surfaces were

considered. Analyses of variance were considered significantly different when p<.05*.

viii

Table 13. Scoring for "Erosions and Adhesions". Both types of

gr& had more erosions and adhesions han unoperated controls of Treatment 3 joints.

Joints with bone gr& had some effect on the opposing joint surface that approached

significance. The effect is evident when the opposing surface (site P) are compared

between the two types OF graRs. Analyses of variance were considered significantly

different when p<.05*.

Page 114. Table 14. Sconng for "Matrix" staining scores. Both types of grafis

had significantly less safranin-0 staining than unoperated controls in the gnfi site and

surrounding area. The opposing joint surface in Site P was unaffected. Analyses of

variance were considered significantly different when p<.O5*.

Page 114. Table 1 S. "Subchondral Support" scores for osteochondral gnfis and

bone grafts were significantiy different when unoperated controls in Treatment 3, though

the surrounding and opposing sites were unflected. Analyses of variance were

considered significantly different when p<.05*.

Page 134. Table A-1. Paravital staining results of experimental Cnlf 1 as

percentage of undamaged cells. Colurnn "Samples" indicates the number of evaluated

stained tissue rnicrosections. Some data remains missing.

Page 134. Table A d . Paravital staining results of experimental Calf 2 as

percentage of undamaged cells. Colurnn "Samples" indicates the number of evduated

stained tissue microsections. Some data remains missing.

Page 135. Table A-3. Paravital staining resuits of experimentd Calf 3 as

percentage of undamaged cells. Column "Samples" indicates the number of evaluated


Table A-4. Paravital staining results of experimental Calf 4 as

percrntage of undamaged cells. Column "Samples" indicates the number of evaluated


Page 136. Table A-5. Paravital staining results of experimental Calf 5 as

percentage of undamaged cells. Column "Samples" indicates the nurnber of evaluated


Page 136. Table A-6. Paravital staining results of experimental Caif 6 as

percentage of undamaged cells. Colurnn "Samples" indicates the nurnber of evaluated


Page 137. Table A-7. Chondrocyte numbers of experimental Calf 1.

Page 137. Table A-8. Chondrocyte nurnbers of experimental Calf 2.



Page 139. Table A-1 1. Chondrocyte numbers of experimental Calf 5.

Page 139. Table A-12. Chondrocyte numbers of experimentd Calf 6.

Page 140. Table A- 13. C hondrocyte numbers of normal, unoperated Calf n l .

Page 140. Table A-14. Chondrocyte numbers of nomal, unoperated Calf n2.

Page 141. Table A 4 S. Chondrocyte numbers of normal, unoperated Caif n3.

Page 141. Table A-16. Chondrocyte numbers of normal, unoperated Caif n4.

Table A-1 7. Mean tnbecular area of Level I and standard errors

(percentage per field of interest).

Page 142. Table A-18. Mean trabecular area of Level 2 anci standard errors

(percentage per field of interest).

Page 142. Table A- 19. Mean trabecular area of Level 3 and standard errors

(percentage per field of' interest).

Page 143. Table A-20. Trabecular area (percentage per field of interest; Level I

to 3) of expenmental Calf 1.

Page 144. Table A-2 1. Trabecular area (percentage pet- field of interest; Level 1

to 3) of experimental Calf 2.

Page 145. Table A-22. Trabecular area (percentage per field of interest; Level 1

to 3) of experimentd Caif3.

Page 146. Table A-23. Trabecular area (percentage per field of interest: Level I


Page 147. Table A-24. Tnbecular area (percentage per field of interest; Level 1

to 3) of experimental Calf S.



Page 149. Table A-26. Trabecuiar area (percentage per field of interest; Level I

to 3) of unoperated Calf nl .

Table A-27. Tnbecular area (percentage per field of interest; Level 1

to 3) of unoperated Cnlf n2.


to 3) of unoperated Calf n3.


to 3) of unopemted Calf nJ.

Page 153. Table A-30. Modified Mankin scores (for cellularity, erosions and

adhesions, rnatrix, and subchondnl support), assigned by two investigators (TS and MH).

Experimental Calf 1.

Page 153. Table A-3 1. Modified Mankin scores (for cellularity. erosions and

adhesions, matrix, and subchondral support), assigned by two investigators (TS and MH).





Page 154. Table A-33. Modified Mankin scores (for cellularity. erosions and


Experimental Caif 4.


adhesions, mat* and subchondral support), assigned by two investigators (TS and NlH).

Experimentai Caif S.

xii

Table A-35. Modified Mankin scores (for cellularity, erosions and

adhesions, ma&, and subchondral support), assigned by two investigaton (TS and MH).



adhesions, matrix. and subchondrd support), assigned by two investigators (TS and MH).

Unoperated Calves nl, n2, n3, and n4 received "O" scores for al1 sites.

xiii

LIST OF FIGURES:

Page 157. Figure 1 . Bovine metacarpophalangeal joint (cadaver specimen)

implanted with two 6.0mm osteochondrai allograftS. Gr& are located in the abaxial

section of the medial and lateral condyles.

Page 158. Figure 2. Bone holding device with distal end of donor MCi4. Note

the hacvested gmft seated in MCJ4 condyle. Operator is holding a corhg drill bit (6.0

mm inside diameter).

Page 158. Figure 3. Mitre box and hack saw. Transverse cut through MC3+

releases osteochondral g rd l

Page 159. Figure 4. Bovine metacarpophaianged joint (dissected cadaver

specimen). Forceps (right) are retracting the fibrous band separating the media! and

lateral condyles of MC3+ Arthroscopie sleeve (lefi) is inserted through the fibrous band.

Page 160. Figure 5. A 6.0mm flat bottom drill bit. A cenaal spike allows for

secure seating in the articular surface of (cadaver specimen).

Page 161. Figure 6. A graduated depth measurine gauge inserted in a 6.0 mm

hole in MC3-( (cadaver specimen).

Page 162. Figure 7. Lateral condyle of MC3+ (top; 1 to 3) and opposing articular

surface of the first phalanx (bottom; 2') of unoperated control animal. sectioned for

histological evduation. In the experimental animals, the graft is located in (2). Section

(27 represents the joint surface opposite to the grai?, (1) and (3) are gnft surrounding

sections.

Page 163. Figure 8. Sampling procedure for trabecular bone area measurements

used regions of interest 1 to 3 (10x magnification) under the gr& (right) and in the

xiv

adjacent recipient bone (left). Paravital staining (PVS) used a minimum of three

vibratome sections in the saggital plane extending fiom the lamina splendens to the

tidemark or subchondral bone (shown as n m w rectangles). Areas of interest for the

chondrocyte count were similar to the sites used for PVS. Not drawn to scale.

Page 164. Figure 9. An arthrarcopic view of a gr& (g) being press fitted into a

recipient hole using a gr& passing tool (t). Synovial membrane (sm) in foreground.

Page 164. Figure 10. An arthroscopie view of a recessed bone gnft (arrows)

exposing the sidewdl of the recipient hole.

Page 165. Figure 1 1. A histological section stained witli Safranin-O. Arrows

outline the bony base of an osteochondrd gr& 5x magnification.

Page 165. Figure 12. A Safranin-O stained osteochondral grdt. Note the tilted

articula. graft surface, covered with fibrous tissue. IOx magnification.

Page 166. Figure 13. A Safranin-O stained specimen with dead bone (N) and

cernent Lines (C). Note marrow pthisis. Inser shows polarized microscopy of same site.

Necrotic bone had a different green factor than new bone.

Page 167. Figure 14. A Safianin-O stained specimen of bone gaft showing

extrinsic cartilage repair and marrow pthisis. j x magmfïcation.

Page 168. Figure 15. Paravital staining results (mean values and standard errors)

of cartilage fiom the gr& the area adjacent to the graft and the opposing phaiangeal

surface for Treatment 1 (osteochondrd grafts) and Treatment 2 (bone grafts).

Page 169. Figure 16. Mean chondrocyte counts and standard errors from

Weigart's Hematoxylin stained sections of Treatment 1 (osteochondral grafts),

Treatment 2 (bone grrfts) and Treatment 3 (unoperated animais). Anatomic sites

represonted by columns marked with (*) are significantly different fiom corresponding

sites in unoperated anirnals.

Page 170. Figure 17. Total tmbecular area (mean values and standard errors) of

combined Levels 1,2, and 3 for Treatment 1 (osteoehondrnl grafts), Treatment 2 (bone

grafts), and Treatment 3 (unoperated controls). Anatomic sites represented by columns

marked with (*) are significantly different From corresponding sites in unoperated

mimals.

Page 171. Figure 18. Combined mean Mankin scores and standard errors for

Treatment 1 (osteochondral grafts). Treatment 2 (bone gnfts) and Treatment 3

(unoperated animais). Columns representing anatomic sites marked with (*) are

significantly ditierent from corresponding sites in unopented animals.

xvi

CHAPTER 1: Review of the Literature

1.1 Introduction

The clinical management of articular cartilage defects remains a significant

treatment challenge for orthopedic surgeons, both in human and veterinary medicine

[Hurtig 1988; Beaver, Mahomed, Backstein et al. 1992; Hangody, Szigeti, Karpati et al.

19961. Developmental musculoskeletal defects, osteoanhritis, neoplasia. and trauma can

result in damage to dcular structures, mostly cartilage and subchondral bone Flynn and

Mankin 1 994; Mnaymneh, Malinin, Lackman et al. 19941. Small, untreated defects may

progress to further cartilage darnage and osteoarthritic changes Prittberg, Lindahl,

Nilsson et al. 1994; Jansson 19961. Among a variety of surgical rechniques,

transplantation of large osteochondral gr& is used to resurface human joints ~ u s c o l o ,

Petracchi, Ayerza et al. 1992; Beaver, Mahomed, Backstein et al. 1992; Meyers 1985:

Meyers, Akeson, Convrey et al. 19891. More recently, the use of a multitude of small

osteochondral grafts has been advocated for repair of chondral defects. [Hangody,

Sukosd, SEgeti et al. 1996; Matsusue, Yamamuro and Hama 19931.

Despite the availability of prornising surgical techniques, the loss of articular

cartilage poses a dificult problem due to its limitations of intrinsic regeneration and

healing [Caplan, Elyaderani, Mochiniki et ai. 19971. In a damaged joint surface, rnost

defects are ultimately nIled with fibrocartilage, which often lacks appropriate structure,

composition, and mechanical strength [McIlwraith 1996; Shahgaldi, Amis, Heatley et al.

19911. ln the long term, fibrocartilage degenerates into a mixture of fibrous tissue of

varying maturity, and is subject to reinjury [Aston and Bentley 19861.

Thexfore, surgical resurfacing of damaged articulations focuses on the

establishment and survival of hyaline cartilage woskalewski 1990; Noguchi, Fujino.

Neo et al. 19941. This literature review gives an overview of the most recent

experimental and clinical studies on joint resurfacing in human and veterinary medicine.

1.2 Articular Cartilage

Cartilage is an important tissue in diverse organ systems of mamrnals and other

h a l s [Nickel, Schummer and Seiferle 1986; Buckwalter 19961. The skeleton of

immature rnarnrnals contains an abundance of cartilage because the developing bones

fom fiom a cartilaginous template [Shingleton, Mackie, Cawston and Jeffcon 1997;

Bromer and Worrell 19901. The distribution of articular cartilage in an adult mammal

declines as the cartilaginous template for the skeleton matures to bone Koch 19761.

Hyaline (articular) cartilage, fibrocartilage, and elastic cartilage c m be differentiated

WcDevitt and Marcelino 19941, though, articular cartilage is the pnmary interest for

clhicians and orthopûedic surgeons. [Buckwalter 19831. Cartilage persists on the

proximal and distal ends of certain bones, forming articulations in the head and neck,

spinal column, and especially the lirnbs WcDevitt 1994; Nickel 1986, Buckwalter 19941.

Proper diarthrodial joint function depends on healthy, normal articular cartilage. It

provides an unequalled low-fiction surface, distributes loads, and minimizes peak

stresses on subchondral bone puckwalter and Mankin 1997: Woo and Buckwalter 1987,

Buckwalter 1994; Kempson 19801.

12.1 Composition and Structure of Articular Cartilage

Articular cartilage has an elaborate intemal organization [Woo 1987. Cartilage

consists of a sparse population of mesenchymal cells embedded within an abundant

matrix [Nixon 1993; Brocklehurst, Bayliss, Mamudas et al. 19841. Cells may contribute

up to five per cent to the total tissue volume. Buckwalter and Mankin [1997] report that

human articular cartilage contains only about one per cent of chondrocyte cells, but the

authors state that articular cartilage of other species (small species with a relatively thin

articular cartilage) may have a ce11 density many tirnes greater than humans. Four

cartilage zones cm be differentiated. A superficial (gliding) zone, the intermediate

(middle or transitional) zone, the deep (radial) zone, and the calcified cartilage zone

[McDevitt 1994; Woo 1987; Teshima, Otsuka, Takasu et al. 1995; Wirth and Rudert

1 9961.

'Ihe only type of ce11 in healthy hyaline cartilage is the chondrocyte puckwalter

and Mankin 19971. Chondrocyte formation commences with the differentiation of

stellate-shaped, primitive mesenchymai cells [Nickel 19861. These cells form rounded

cartilage precursor cells called chondroblasts. Subsequent mitotic divisions result in

aggregations of ciosely packed growing chondroblasts that begin synthesis of ground

substance and fibrous extracellular matenal puckwalter and Mankin 19971. Secretion of

extracellular matenal separates the cell clusters and simultaneously embeds the individual

chondroblasts in the ma&. Further mitotic divisions lead to the formation of mature

cells, the chondrocytes [Shida, Singushi, Izumi et al. 19961. They create a molecular

Cramework by synthesising three classes of molecules: collagens, proteoglycans and

noncollagenous proteins. Chondrocytes, once considered metabolicaliy inert, constaotly

synthesize and degrade structurai ma& macromolecules in the process of remodelling

wirth and Rudert 19961.

Structural macromolecuIes include collagens [von der Mark 19771, proteoglycans

[Nakano and Aherne 1995; Hardingharn, Muir, Kwan et al. 19871, and noncollagenous

proteins or giycoproteins [Riley, Lane, Marshall et al. 1996; Cook and Rueger 19961. Al1

are synthesized by chondrocytes fiom amino acids and sugars [Schenk. Eggli and

Hunziker 19861. Matrix macromolecuies form a complex framework which is filled with

tissue fluid [Brockiehurst 19841. This framework acts as a base for the attachment of

chondrocytes. Twenty to 40 per cent of the wet weight of normal cartilage is contributed

by matix macromolecules [Buckwalter et al. 19971. Approximately 50 per cent of the

dry weight of cartilage is collagen. Several distinct types of collagen (arnongst them are

types 1, II, VI, VII, IX, and XI) have been identified in nrticular cartilage [von der Mark

and von der Mark 19771, but 85 to 95 per cent of the total hyaline cartilage collagen is

type II [Wheater 19871.

1.2.2 Healing of Articular Cartif age

Articular cartilage injuries are often due to direct trauma, damage to cells and

structurai macromoiecular organization [Newberry, Zukosky and Haut 1 997; Repo,

Finlay and Eng 1977; Rodkey 1996; M& 1982; Buckwalter 19921. Biochemical

components, stirnulated by mediators, may play an important additionai role for sorne

erosions [Buckwalter and Mankin 1 9971. inflammation leads to enzymatic depletio n of

cartilage macromolecules, loss of vîscoelastic and load-bearing properties, and makes

cartilage more susceptible to M e r injury [Schiller 1995; Buckwalter and Rosenberg

1990; Buckwalter, Einhorn, Bolander and Cruess 19961.

Hyaline cartilage damage has limited potentid for repak due in part to the

avascularity of chonclrai tissue [Stuart 1 9941. Repair of articular cartilage has been

studied and described in several species [Desjardins and Hurtig 19901. Stockwell and

Meachim [1979] described three mechanisms involved in cartilage repair: intnnsic and

extrinsic healing, and matrk flow. Intrinsic heaiing is defined as the proliferation of

chondrocytes in order to produce a new matnu. Neochondroplasia originating fiom

exposed mesenchymal cells in the subchondral bone is referred to as extrinsic repair.

Matrix Bow by wave-like fiow of matrix toward the center of a defect has been described

in the past p e Palma, McKeever and Subin 1966; Ghadially, Ailsby and Oryschak

19741, and was recently reviewed [Bruns, Kersten, Silberman and Lierse 19971. For

cartilage healing, the original defect size is important pixon 19953.

The '%ritical size defect" may vary between locations within the affected joint

murtig, Fretz, Doige and Schnurr 1988; Korvick and Athanasiou 19971 and between

species [Convery, Akeson and Keown 19721. Defects Iarger than 9.0mm in diameter are

unlikely to undergo complete repair [Hurtig 1988; Vachon, Bramlage, Gabel and

Weisbrode 1986; Desjardins and Huttig 19901. Incomplete repair may result in W e r

degenerative changes within the joint, causing pain and loss of motion [Czitrom, Keating

and Gross 1990; Dieppe 1994 and 1995; Moskowitz, Howell, Goldberg and Mankin

1992; Kuettner, Schleyerbach, Peyron et al. 1992; Schiiler 19951.

12.3 Repair of Articular Cartilage: Current Treatment Options

Buckwalter stated in 1990 that physicians and scientists have spent the past 250

years seeking ways to treat loss, damage or degeneration of hyaline cartilage. To date, no

single treatment modality has been identified to regenerate or repair damaged articular

surfaces in adult mammals [Buckwalter 19901. The term "regeneration" refers to the

formation of new tissue indistinguishable frorn normal articular cartilage, while "repair"

refers to the restoration of a darnaged joint surface with new tissue that resembles but

does not dupiicate articular cartilage [Woo and Buckwalter 19881. Most clinical and

scientific progress has been made over the past 30 years, resulting in a better

understanding of hyaline cartilage physiology and pathology, as well as the development

of an array of treatment options winas and Nehrer 1997; Buckwalter and Mankin 1997;

Wirth and Rudea 19961. A variety of methods have been proposed to stimulate the

formation of a new articulv surface in patients with joint disease. These methods include

lavage and superficial debridement [Jackson 199 11, bone marrow stimulation utilizing the

exposure of subchondral bone [Steadrnan 19961, carbon tïber implantation [Brittberg,

Faxen and Peterson 1994; Hemmen, Archer and Bentley 199 1, Valdez 19821, and

biologic repair methods including chondrocyte transplantation [Breinan. Minas, Hu-Ping

et al. 1997; Kandel, Chen, Clark and Renlund 1995; Jackson and Simon 1996;

Hendrickson, Nixon, Grande et al. 19941, artificial scaffolds loaded with chondrocytes

[Sams and Nixon 1994; Sam, Wootton et al. 19951, non-ûrticuiar cartilage implantation

[Howard, McIlwraith, Trotter et al. 19951, transfer of fiesh and fiozen articular cartilage

[Gudler 1 997; Aston and Bentley 1 986; Desjardins, Huaig and Palmer 1 99 1 ; Schachar,

McAllister, Stevenson et ai. 1992; Schachar, Cucheran and Frank 19881 and graftiog with

osteochondral autogenous or dogenic materiai [Hurtig 1988; Hangody, Sukosd, Szigeti

and Karpati 1996; Outerbridge, Outerbridge and Outerbridge 1995; Garrett 1986; L u e ,

Brighton, Ottens and Lippton 1975; Brown and Cruess 1982: Huber, Schrnotzer, Riebold

et ai. 19921. In the recent literature, debridement and lavage, marrow stimularing

techniques, grafting with chmdrocytes, and perichondrial or osteochondral tissue gr&

predominate. These techniques are currently used in clinical trials and treatment regimes

for articuiar defects in humans and deserve a closer review.

1.3 Joint Debridement and Lavage

Arthroscopie or closed-needle joint lavage c m be used to remove free-floating

debris and idammatory mediators, and to relieve joint effision [Chang, Falconer,

Stulberg et al. 1993; Livesley, Doherty, Needoff et al. 19911. Joint lavage represents the

most basic and uncomplicated technique and provides symptomatic short-tenn relief from

pain without addressing the underlying pathology in the affected joint [Rand 1991 and

1985; Richards and Lonergan 19841. Using arthroscopy, a simultaneous debridement of

the articular defect (removal of the damaged hyaline cartilage only) c m be perfomed in

addition to joint lavage ijbloseley, Wray, Kuykendall et al. 19961. A similar but more

invasive surgical technique was fust described by Magnusson [1946] and perfomed by

arthrotomy. This procedure became known as the "houseîleaning" arthroplasty

[Johnson 19861 and consisted of synovectomy, meniscectomy, articula. shaving,

sometimes condylar drilling, and osteophyte resection in the human knee. Despite being

considered a technique that provides only temporriry irnprovement for the patient, some

studies have accredited clinical improvement over severai years to joint lavage and

debridement [Jackson 199 11.

Jackson is credited with the fust description of the benefits of joint lavage. He

noticed a signincant pain relief for patients with intraarticdar knee pathology

immediately after diagnostic aahroscopy. fis 1991 study shows improvement of clinical

symptoms in 45 per cent of the patients at three and a half years postoperatively; in 20 per

cent no improvement was reported. Treatrnent consisted of diagnostic arthroscopy and

joint lavage. Manual or rnotorized instruments c m be used for a mechanical debridement

in addition to the lavage to improve the outcorne. Another study [Jackson, Silver and

Marans 19881 of over 100 patients with knee pathology demonstrated 90 per cent

improvement experienced by the patients immediately after surgery. Three years

postoperatively, the improvement had continued in 70 per cent of the cases.

Hubbard [1996] treated media1 femoral condyle lesions in human knees with

debridement or arthroscopic lavage alone. Al1 76 patients of the randomized, prospective

study had significant chondral lesions accompanied by joint pain, but no significant

radiographie abnormalities. The snidy concluded that joint lavage alone improved

patients initially, but the combination of lavage/debridement was able to provide an

appreciable clinical improvement for up to five years.

Chang [1993] compared the effects of anhroscopic lavage and debridement versus

a closed-needle joint lavage in humans. Both treatment techniques in this randomized.

prospective study resulted in pain relief 12 months postoperatively. The authors

concluded that clinical symptoms may be improved for up to three years, but found

lavage as the only treatment insufncient to enable a patient to rehun to athletic fùnction.

1.1 Marrow-Stimulotion Techniques

Minas and Nehrer [1997] used the term "marmw-stimulation techniques" and

classified abrasion arthroplasty, subchondral driliing and microfracture as surgical

techniques stimdating cartilage repair by penetrating the subchondml bone. Primitive.

pluripotential stem cells capable of differentiating into ail types of comective tissue

colonize the defect [Kim, Moran and Salter 19911. Under ideal conditions, granulation

tissue differentiates into fibrous tissue which in turn becomes fibrocartilage. The ongin

of the celIs contained in the initid blood clot remains a topic of scientific controversy.

Some argue that the blood dot forms immediately after the exposed and debrided bone

starts to bleed, and therefore contains a majority of marrow-derived stem cells F inas and

Nehrer, 19971. Humer, and Rosenberg [1996] suggested the presence of two ceIl types.

They stated that both pluripotential synovium-decived ceils (capable of differentiating

into articular cartilage) and marrow-derived cells (which produce Fibrocxtilage) are

present in the clot. Regardless of its composition, the repair tissue is fragile. Buckwalter

and Mankin [1997] stated that the undifferentiated rnesenchymal cells can only migrate

into the dot, proliferate, and differentiate into cells with the morphological features of

chondrocytes if the surface of the treated defect is protected fkom excessive loading.

Weight-bearing is believed to be one o f the most influentid negative factors aEecting the

outcome of marrow-stimulating techniques. Excessive weight-bearhg of the treated

articular area is iimited in human patients by either wgical techniques (Le. unloading

osteotomy) or restricted postoperative ambulation [Friedman, Berasi, Fox et al. 1982;

Akizuki, Yasukawa and Takirna 19971. Continuous passive motion (CPM) [Saiter

1993; Rodngo, Steadman, Silliman and Fulstone 19941 cm increase the quality of the

repair tissues in articular defects and better overail hyaline cartilage nwival was

demonstrated [O'Dnscoll, Keeley and Salter 1988; Salter, Bell and Keeley 198 1; Salter,

Simmonds, Malcolm et ai. 19801. The correct protocol for postoperative CPM remains

conrnve&ti [Gebhard, Kabo and Meals 19931.

1.4.1 Abrasion Arthroplasty

Abrasion arthroplasty is an Ythroscopic technique that uses manual i n ~ e n t s

or a high-speed b m to remove the defective cartilage and subchondral bone, and to

expose bleeding bone pandy 19861. In hurnan patients. 2.0 to 3.Grnm of subchondral

bone are usually removed and a clean tissue edge is created around the defect [Kim.

Moran and Salter 19911. A blood clot forms over the bleeding subchondral bone

Friedman 19841. The blood clot transfomis into a fibrin clot, and is thought to be

anchored in the defect bed by the exposed collagen. An additional. direct effect on pain

has been suggested [Ficat, Ficat, Gedeon et al. 19791 Subchondrai bone is well

imervated and sensitive to pressure transmitted by softened or incomplete cartilage.

Removal of the softened cartilage will reduce the amount of pressure on the subchondral

bone, thus decreasing the amount of pain experienced by the patient paumgaertner,

Cannon, Vittori et al. 19901. Although widely applied in orthopedic surgery, it cemains

unclear whether or not abrasion arthroplasty promotes cartilage regeneration [Akizuki

1 9971.

A retrospective study reported by Friedman in 1984 included 1 10 human patients

with chondral defects in the knee. Follow-up tirne after abrasion arthroplasty was one

year. Overail, 60 per cent of the patients who undenvent abrasion arthroplasty showed

improvement, 34 per cent were unchanged, and clinical signs or radiographie findings

worsened in six per cent. A second report by Friedman [1986] and a study by Johnson

[1991] described the outcome of abrasion arthroplasty of the knee in human patients.

Friedman noted a signincant age-related ciifference 12 months postoperatively. Patients

less than 40 years of age demonstrated clinical improvement in over 85 per cent of the

cases, while patients with an average age over 50 yem were found improved in only 54

per cent and worsened in ten per cent of al1 cases. Over 35 per cent of the patients in the

higher age group were unchanged after abrasion arthroplasty.

khnson's snidy [1991] included 400 human patients with an average age of 60

years. Ninety-nine per cent of the patients reported a restriction in their activity, with a

loss of mobility in 24 per cent after surgery. Sixty-six per cent continued to experience

pain in the affected joint, and only 12 per cent were reported to be pain free and

unrestncted in their activities. Follow-up arthroscopies in combination with biopsies

found fibrous tissue initially, which becarne fibrocartilage at four io six months

postoperatively. Collagen typing was performed for several patients of this study and

showed increasing arnounts of type II collagen and hyaline cartilage up to 24 months after

abrasion arthroplasty.

An experimental investigation in rabbits by Kim [1991] showed the potential for

regeneration of articular cartilage in defects created by choncirai shaving and subchondral

abrasion performed on the patellae of 40 rabbi& A defect 3.0mm in diameter waç

created with both procedures. Twenty animals of each treatrnent group were allowed

intermittent active motion, with the other anirnals being treated with continuous passive

motion (CPM). There was no evidence of repair tissue in the defects at either four or 12

weeks after chondral shaving. The remaining underlying cartilage, however, had

degenerated. The anirnals treated with subchondral abrasion and intermittent active

motion had healed defects at 12 months postoperatively, although the quality of the repair

tissues varied. Mature, hyaline-like cartilage was found in the defects of the rabbits

treated with subchondrsil abrasion and CPM.

1.4.2 Subchondral Bone DriUing

Drilling is performed through the defect and the subchondral bone plate into

cancellous bone. The holes fil1 with blood clots irnrnediately iifter drilling and anchor the

clots and growing tissues. Further organization of the clots leads to fibrin and comective

tissue formation in the drill holes. Both hyaline cartilage and fi brocartilage eventuaily

develop at the site. Pridie [1959] and Insall [1967,1974] both suggested that, in clinical

practice, multiple small drill holes made through the subchondral plate would result in the

eventual formation of hyaline cartilage. They reported clinical cases in which this

seemed to have occurred, but there was no histological proof. A study of Shamis,

Brmlage, Gabel and Weisbrode [1989] showed histological evidence that holes dnlled

into the third carpal bone of 13 horses contained fibrocartilage and fibrous tissue at 13

months postoperatively.

Mitchell and Shepard [1976] used 25 rabbits in an animai mode1 to study the

repair of articular cartilage in a defect created in the knee joints. The defects were created

by drilling 25 to 30 holes (1.0 mm in diameter) in the femorai condyles and the nbbits

were euthanized in groups at various tirnes a.€ter surgery. Up to four months after

surgery, tissue resembling hyaline cartilage was found in the drill holes and defect. By

eight months, this tissue lacked any hyaline quality, but coverage of the defects was

complete. At 12 months, the tissue was found to be dense collagenous tissue, with a

cornplete coverage of the defects. The authors concluded that this material, although not

cartilaginous, provided a new joint d a c e .

Ficat [1979] coined the term "spongialhtion", based on the exposure of the

spongiosa of the cancellous portion of the patelia after drilling. He reported on four years

of experience with subchondral drilling or "spongiaiization" for patellar defects in

humans. Al1 diseased cartilage was resected en bloc with its corresponding subchondral

bone, leaving a completely exposed cancellous bony bed. Seventy-nine per cent of the 85

patients had a good or excellent result, defined as a reduction in pain after surgery.

Symptomatic relief with this method was observed by Tippet [1991]. He evaluated a case

series of human patients treated with subchondral bone drilling. At 62 months

postoperatively, 70 per cent of the patients showed excellent results. The remaining 30

per cent were equaily divided into good and fair to poor resdts.

Vachon [1986] reported on the effects of subchondral drilling on the cartilage

repoir process in an animal mode1 using six horses. A full-thickness cartilage defect 10.0

mm in diameter was created on the radial facet of the proximal surface of each third

carpal bone. One of the third carpal bone defects in each horse was drilled (five holes.

10.0mm deep, l.Omrn Ui diameter), while the defect in the contralateral limb was left

untreated. Analysis of ce11 numbers and types in the synovial fluid and the mucin

precipitate qudity before surgery and at one and three weeks after surgery revealed no

significant difference between treatment groups. Twenty-one weeks after surgery, the

horses were euthanized and eac h carpal joint was examined radiographical l y,

macroscopicaiIy, and microscopically to evaluate the repair process. Significantly greater

amounts of surface of the defect were covered with dense fibrous and fibrocartilaginous

repair tissue in the dnlled carpai bones. Repair tissue thickness was also greater in the

drilled carpal bones and had an improved attachent to the underlying tissues.

Fibrocarîilage was found covering the drilled lesions, whereas only fibrous tissue was

identified in the undrilled defects. The authors concluded that drilling through the

subchondral bone plate into the cancellous bone irnproves healing of cartilage defects in

the third carpal bone in horses.

1.43 Microfracture of the Subchondral Bone Plate

Small stainiess steel awls are utilized for this arthroscopic technique. The careful

debridement of the defective area, removal of the calcificd cartilage, and exposure of the

subchondral bone is followed by microfncturing of the subchondral bone plate, also

referred to as "breaching*' p i n a s and Nehrer 19971 which creates access to mesenchymal

stem cells by penetrating the subchondral bone plate. Blood clot formation and adhesion

to the rough defect surface occurs, followed by maturation to more dense connective

tissue. Steadman [1996] concludes that the microfiacture technique produces less heat

and therefore less tissue necrosis than subchondnl ddling.

Frisbie, Trotter, Powers et al. [1997] conducted a study to objectively tes the use

of arthroscopic subchondral bone microfiacture in the horse. In ten mature horses, a 10.0

d defect was created arthroscopically in both radiai carpal bones and both medial

femorai condyles using a hmd curette. One radial carpal bone and one femoral condyle

defect of each horse had the subchondral bone plate perfonted using an orthopedic awl.

Treadmill exercise was initiated for dl ten horses. Five horses were euthanized after five

months, the remaining five homes were eutbanized d e r 12 months. On post-mortem

examination, a more uniforni character and greater volume of repair tissue filled the

treated versus control defects at both time periods (74 percent versus 45 per cent).

Histomorphometry of the experimental sites confhned more repair tissue (60 per cent

versus 40 per cent) in the microfractue-treated group, but tissue types were not

statistically different. A greater percentage of type II collagen was present earlier in al1

locations in treated cornpared to control defects.

1.5 Terminology of Tissue Grafting

The terminology of grafting is similar for organ-, soft tissue-, or bone

transplantation, but it has undergone some changes over the recent years. The term

autografl (or autogenous or autologous qufl) refea to transplantation within an

individual Malinin 19901. lsogrufl [Heppenstall 19801 refers to transplantation between

genetically matched individuals (inbred animal strains, identical twins). An cillograt or

ullogeneic gruft, previously called hornogrufl [Heppenstall 19801, describes the

transplantation of tissues between different individuals of the sarne species [Buck and

Malinin 1994; Fitch, Kerwin. Sinibaldi and Newman-Gage 19971. Some authors use the

term ailograft [Gouin, Passuti, Vemele et al. 19961 for a grafi onginating From a living

donor, while alloimplant is used for a gr& kom a dead donor. The term xenograjl

(previously called heterograft) is used for the transplantation between species [Phillips.

Parker and Bloomberg 19881.

The description of the ongin and destination of a graft is equally important: the

orthotopic or isotopic grafl refers to transplantation to the same site while the term

heterotopic gr@ describes the transplantation of tissues to a different anatornic location

veppenstall 19801. The osteoarficular graft replaces a whole or segment of a joint; we

may distinguish between a complete, whole- joint trampfant [Goldberg and Heiple 1983;

Buchmann 19081 including the capsule, from a transplant involving one or two

epiphyseal sections (hemi-joint) including the cartilaginous coven [Roffman and du Toit

1985; Lexer 19081. An intercafary grap describes a bony segment between two

segments of host bone, such as the tibia1 or fernord shaft [Cassotis, Stick and Arnoczky

1997; Mûnkin 19831. The unipolar grog? is a single osteoarticular gnft for a single lesion

in o joint. If the opposing joint surface is affected as well and requires resection followed

by grafting, a bipolar graJ is being placed within the joint.

1.6 Repair of Chondral Defects with Tissue Grafts

Periosteurn, perichondnum, fascia, joint capsule, muscle, and tendon are mong

the sofi tissues that have been used for the repair of articular defects [Niedermam. Boe.

Lauritzen and Rubak 1985; Hoikka, Jaroma and Ritsila 1990; Jensen and Bach 1992:

O'Driscoll and Salter 1986; O'Driscoll, Keeley and Salter 1986 and 1988; Ostgaard and

Weilby 19931. Sofbtissue grafts allow the introduction of a new ce11 population dong

with an organic ma& [Buckwdter 19971. Perichondrîal and penosteal gr& are the two

most cornrnonly used tissues of this group. They are currently used in research and as

well as in ch icd trials.

Penosteum is a multiple-layer comective tissue found on flat bones and long

bones. Perichondnllm is sirnilar to periosteurn but is found at costostemal junctions and

covering bone within synovial joints. Its imer layer contains bone- forming cells

(osteoblasts), which produce a ground substance, osteoid. The osteoblasts subsequently

lose their close association with one another and migrate into the ground substance where

they become bone cells (osteocytes). The end of the perichondrial ossification marks the

transition fiom perichondnum to periosteum. This thin rnembranous fibrous tissue

covers the extemal surface of al1 bones, with the exception of articdar surfaces, in a

sirnilar fashion as described for the penchondrium. Periosteum consists of an outer

fibrous layer and an b e r , more cellular and vascular layer. The outer layer consists of a

dense fibrous tissue matrk and fibroblast-like cells. The imer, osteogenic or cambium

layer contains cells capable of forming cartilage and bone (multipotent cells). The

potential of perichondrial and periosteal grah to produce new cartilage when embedded

in a cartilage defect has been demonstrated in animal models and clinical trials [Wakitani,

Goto, Pineda et al. 1994; Ritsila, Santavirta. Alhopum et al. 19941.

Perichondnum and periosteurn can develop into hyaline cartilage when placed in

a small joint [Skoog and Iohansson 1976; Engkvist and Ohlsen 19791. Woo and

Buckwaiter [1987] demonstrated sirnilar viscoelastic properties of the newly fonned

tissue when compared to hyaline cartilage. Clinicians expressed their concems regarding

a funher differentiation of the grafted tissues, which rnay potentially lead to ossification.

This aspect of the biology of neochondrogenesis using penchondrium or periosteum

became clearer after a study fiom Ritsala [1994]. He stated that the direction of

differentiation rnay be largely determined by the environment rather than by the

phenotype of the transplanted cells. The author suggests that a hi& oxygen tension rnay

trigger a differentiation of the mesenchymal cells to bone, while a low oxygen tension

may favor the development of cartilaginous tissues. However, current theories largely

favor the eventual, unwanted transformation into bone, mostly triggered by the

expression of type X collagen M a s and Nehrer 19971.

The use of periosteal or perichondrid gr& has been h i t e d to defects in smal1

articulations [Skoog 19761 due to the lack of biological fixation for a larger grafi.

Widenfalk, Engkvist, Ohlsen and Segerstrom [1986] reported the use of non-toxic, non-

allergic and biodegradable fibrin glue for graft fwation, allowing graft fixation in larger

joints [Aron and Gorse 1991; Homminga, van der Linden, Terwindt-Rouwenhorst and

Drukker 19891.

1.6.1 Perichondrial G rafts

The first clhical study of perichondnal grafts for defects in the human knee was

performed by Homminga, Bulstra, Bouwemaster and van der Linden [1990]. In 1989,

Homrninga used fibrin glue for the fixation of perichondrid in rabbits. The success

of this expenment led to a clinical trial in which autogenous strips of costal

penchondnum were used to resurface 30 chondral defects in the knees of 25 human

patients. The perichondrial grafts were cut to size in order to match the defect.

sometimes necessitating the use of two gr& per defect. Postoperatively, al1 patients

were immobilized for two weeks, followed by two weeks of continuous passive motion

(CPM). Weight-bearing on the affected joint was not permitted for 12 weeks. The

authors reported that in 28 of 30 cases the defect was completely filled with tissue

resembling articular cartilage ten months &er the grafting (second-look arthroscopy).

The authors concfuded that excellent results were obtainable with this method in the

human knee. No difference was observed in the grafts placed ont0 cancellous bone when

compared to the gr& placed an intact subchondral bed. Biopsies from three patients

with a satisfactory second-look arthroscopy result were obtained. Microscopically. an

intact boue-cartilage junction was seen in one biopsy, with disniptions being present in

the other two samples. V i d l y , the regenerated cells appeared to be chondrocytes and

signs of ossification were not noted. Radiographs were obtained before and after surgery.

Joint space narrowing and the presence of degenerative changes had not increased 12

months after surgery, however, increased radiographie densities were visible in the

regions of the gr&. An increased calcium uptake into the basal layer of the cartilage

may be suggested by this finding. The authoa stated that the g r a h can be placed in

either cancellous or subchondrd bone. This observation differs from a statement made

by Engkvist and Johansson [1980], who suggested that cartilage regeneration from

perichondriurn may only occur when the gr& is placed on cancellous bone. In summary,

they concluded that perichondral @ing of cartilage defects of the human knee gives

good to excellent results, however, the authors admitted that long-term results are needed

for a final conclusion. In 1997, Minas and Nehrer reported on a personal communication

regarding a five to seven year follow-up on the 30 grafls performed by Hornrninga.

Twenty of the 30 grafis undenvent endochondral ossification, associated with pain

experienced by the patient and subsequent degeneration. Sixty per cent of the gaFted

knees had gr& failure as a result of the observed endochondral ossification. Reviewing

this study amongst similar reports, Minas et al. [1997] added their own unpublished case

of clinical perichondnal g r m g in a single human knee. At 54 months. the gr& was

found to be ossifïed and considered a fniled gr&.

To date, the fixation methods of s m d to medium-sized grafts are considered

su£Eïcient [Hornminga 19901. The availabiiity of modern tissue adhesives enables the

surgeon to secure a properly fitted graft. A more important shortcornhg in the clinical

use of perichondrial grafls is the availability of suitable gratüng material. Perichondnurn

is found largely on the s m d cartilage bordes of the rib cage adjacent to the sternum.

Minas and Nehrer [1997] reported the necessity to use several ribs to harvest a graft of

suffcient size. Grdl size is limited by rib size, and a single graft may not be able to

cover a large or multiple defects.

1.6.2 Periosteal Grafts

Osteoperiosteal composite grafts have been used for over LOO years in the

treatrnent of cranioplasties like congenital clefts of the palate [Bemdt 1898; Ritsilae

19721, for tracheal cartilage defects [Ritsilae 19941, for delayed union of fractures [von

Mangoldt 19041, and for the repair of articular defects [Redfern 185 1 ; Bennett and Bauer

1932; Sullins, McIlwraith, Powen and Norrdin 1985; Coutts. Savio. Woo et ai. 1989:

Billings 1 9901.

Penosteum consists of rnultipotent mesodermal cells, and the environmental

influences on the differentiation of free periosteal cells have been dernonstrated warn

1930 and 1953; Rubak, Poussa and Ritsila 1982; Ritsila 19941. Periosteum is believed to

have the capacity to form al1 varieties of connective tissue [Billings. von Schroeder. iMai

et al. 19901. The osteogenic capûcity of periosteum has been known since the classicd

midies of Duhamel in 1739 and Ollier in 1867 [Rubak 19821. Ollier [1867] demonstnted

that the cells of the cambium layer of the periosteum, when detached fiom bone, have the

potential to produce new bone. This finding was confirmed by other researchers passet

1962, Ritsila Alhopuro, Gylling and Rîntala 19721. A study by Ritsila, Alhopuro and

Rintda [1972] found cartilage formation preceding the bone formation fiom fkee

periosteal grafts in a chondrotrophic environment, making it a vaiuable treatment option

for resurfacing of articular defects [Lorentzon 19961. Despite strong advocates of

penosteal grafting the ovedl clinical success with this modality is so variable that it

must still be considered experimental.

1.7 Chondrocyte Transplantation

Interest in the transplantation of chondrocytes has increased over the recent years.

The concept of isolated transplantation of isogeneic and allogeneic chondrocytes to

induce and support cartilage growth is thought to be helpful for patients with chondral

defects. Chondrocyte transplantation has been used in the treatment for human articular

cartilage defects [Brittberg 19941. Problems with chondrocyte transplantation include the

source of cells, in vitro culturing, and foremost, the difficulty of fixation. The alteration,

manipulation, or fabrication of cells, tissues, and organic substances has been referred to

as "tissue engineering" [Jackson and Simon 19961. Several aspects o f tissue engineering

need to be considered for chondrocyte transplantation: (1) The use of tissue cultured cells

to repopulate, remodel, synthesize, and maintain a normal matrix; (3) the use of various

matrices of naturd and/or synthetic origins that act as substrates and may participate in

stimulating a more specific cellular remodeling. (3) a possible addition of bioactive

factors to affect chondrocyte differentiation and growth; and (4) the use of various

combinations of chondrocytes, matrices, and factors to produce cartilage repair [Jackson

and Simon 19961. Incorporation of chondrocytes into an articular defect requires a

protective vehicle [Hendrickson 1994; Wakitani, Kimun, Hirooka et al. 19891.

Chondrocytes can be transplanted as an auto-, allo-, or xenograft [Jackson. Halbrecht,

Proctor et al. 1996; Stone, Walgenbach, A b m s et al. 1997; Gallili, LaTemple,

Wdgenbach and Stone 1997. Allogenic chondrocytes isolated from the matrix and

subchondral bone were demonstrated to be transferable to articular defects [Cook,

Kreeger, Payne and Tomlinson 1997; Aston and Bentley 1986; Hendrickson 19941 with

variable resuits. Xenografts showed immunologic responses leading to graft rejection

wevo. Robinson and Halpenn L 992; Nevo, Robinson, Halperin and Edelstein 19901.

Autologous chondrocyte grafting was described by Grande, Pitman, Peterson et al. in

1989. demonstrating an 82 per cent reconstitution of rabbit cartilage in a defect that had

received an autologous chondrocyte transplant grown in vitro. This compared with a

defect fil1 of under 20 per cent in ungrafted controls. Automdiography was performed on

the tissue filling the original defect. [t showed incorporation of labeled cells into the

repair matrix. The authors concluded that the implanted cells were partly or fully

responsible for the repair tissue. The same transplantation technique was used five years

later in human patients [Brittberg 19941; autologous transplantation is a preferred grafling

method for human patients. Reasons for this preference are the possible immunologic

rejection of chondrocyte allografts [Langer and Gross 1974: Kawabe and Yoshinao 1989

and 199 11 and the transfer of infectious diseases. Fixation of the chondrocytes within the

chondral defect has been achieved by periosteal patches [Grande 1989; Brittberg 19941,

collagen scaffolds mixon, Sams, Lust et al. 1993; Sams 1995; Frenkel, Toolan, Menche

et al. 1997; Freed Grande, Lingbin et al. 1994; Freed, Vunjak-Novakovic, Biron et ai.

19941 and collagen gels pakitani 1989; Freed. Marquis, Nohria et al. 19931. hyaluronan

[Robinson, H a l p e ~ and Nevo 19901, carbon fibre pobhson, Ephnt, Mendes et al.

19931 or fibrin [Nixon 1992; itay, Abramovici and Nevo 1987; Hendnckson 19941.

These carriers not only fhction as a containing vehicle and as a means for the

chondrocytes to adhere to the gmfted lesions, they dso isolate and protect the implanted

cells from an irnrnunologic response as described by Grande [1989] and Wakitani 119891.

As stated by Hendrickson [1994], the use of an injectable carrier-chondrocyte mixture

allows the use of arthroscopie implantation techniques. while the use of sutured periosteal

patches [Brittberg 19941 necessitates an arthrotomy.

Research conducted by Sams [ 19941 and Hendnckson [ 19941 investigated the use

of fibrin and collagen carriers for chondrocyte transplantation. Both studies used an

equine model. Hendrickson [1994] used eight adult horses. which received a chondrocyte

ailograft fiom a nine-day-old foal. The transplanted cells were mixed with fibrin and

arthroscopically injected into 12.0mm defects created in the lateral trochlea of the distal

femur. A similar, ungrafted defect was created in the same location of the contralateral

limb as a control. Groups of four horses were killed at four or eight months

postoperatively. Gross examination revealed improved fiiling of the grafled areas when

compared to the ungraAed controls. The cellular arrangement of the grafted defects

resembled hyaline cartilage, while mostly fibrous tissue was found in the controls. The

authors concluded that isolated neonatd equine chondrocytes c m be embedded in an

articular defect with the use of a polymerized fibrin matrix, resulting in an improved

articular surface compared with ungnhed defects. Fibrin rnatrix provided three-

dimensional support, prevented a dedifferentiation of chondrocytes toward fibroblasts.

and protected the cells fiom a hoa immune response.

The research model of Sams [1995] used commercially available collagen

matrices in the f o m of porous 16.0m.m discs. The coifagen discs were predorninantly

composed of type 1 bovine collagen and the progressive and complete resorption of the

open mesh porous network had been described previously [Nixon 19931. Donor articular

chondrocytes fiom a 12- and a 24-week-old foal were harvested and cultured in the

collagen scaf5olds for seven to ten days. The gnfts were implanted into 15.0 mm defects

in the articular cartilage in the femoropatellar joint of 12 horses. with the contralateral

joint serving as a control (ungrafted articular defect). Gross, histochemical and histologie

evaluations were petformed in groups of six horses euthanized at four and eight months

postoperatively. Although gross differences between operated and control joints were not

present, increased chondrocyte numbers were evident in the deep layers of the grafted

sites. Surface layers of the grafted sites and the entirety of control sites consisted of

fibrous tissue. Sams [1995] summarized that arthroscopie implantation of chondrocyte-

collagen gras improved the cartilage healing in extensive articular defects of the equine

femoropatellar joint. However. the structural organization of the surface layers was

found to be inadequate suggesting a poor long-term durability and survivability.

To date, the most promising chondrocyte transplantation technique is based on the

use of autologous cells. A recent review from Jackson and Simon [1996] gives an update

and overview on the research conducted in the field of chondrocyte transplantation. The

most recent hurnan clinical case report is the work fiom Brittberg [1994]. The author

describes the repair of Full-thickoess defects (mean diameter 30.lmm) in the articular

cartilage of the knee in 23 patients (mean age 27 years). Arthroscopy was used to obtain

300 to 500 mg of healthy hyaline cartilage h m a non-weight beariog area of the affected

knee. This cartilage underwent enzymatic digestion and was cultivated for 1 I to 21 days,

resulting in a ten-fold increase in ce11 numbers. The cultivated cells were transplanted

into the debrided defect by arthrotomy. Fixation of the ce11 suspension was achieved with

a periosteal flap. The periosteum was harvested From the proximal tibia and was sutured

over the drfect using 5-0 Dexon, followed by the injection of the cells undemeath the

periosteal flap. Defects in the patella (chondromalacia) and the femoral condyles (result

of trauma) were repaired. AAer a penod of active knee movement without weight-

bearing, arnbulation was gradually increased during the first two postoperative months.

Patients were followed for 16 to 66 months. Arthroscopy was performed three montlis

after the defect repair, showing regenerated areas of cartilage with visible borders to the

surrounding cartilage. A soft indentation could be felt with the probe in the center of the

graf%ed area. In most cases. wave-like movements of the transplants could be

appreciated. The author concluded that this finding indicated a poor attachment of the

transplant to the subchondral bone. Biopsies were obtained in some cases during the

postoperative follow-up, showinp the formation OF predominantly hyaline-Iike cartilage

containing type II collagen. Most patients were clinically improved (knee pain. joint

effusion, crepitation) d e r surgery. Outcornes were better in cases where trauma was the

initial cause rather than degenerative conditions such as chondromalacia patella.

Malaiignment of the patella or laterd subluxation may have been causes for the

chondromalacia and were not corrected by the grafting, resulting in a continued stress on

the repaired area Brinberg also described the greater contact stress in the patellofemorril

joint when compared to the femorotibial joint as a possible reason for chondrocyte gnft

failure. Brittberg's fmd conclusion stated that cultured autoIogous chondrocytes can be

used to repair articular cartilage defects in the human femorotibial joint.

A report by Minas [1997] p d e l s the fmdings of the Swedish study by Brittberg.

Fi@ patients reported a 50 per cent decrease in pain over four to six mon&. 70 per cent

over seven to nine months, 90 per cent by 12 months, and an aimost cornplete absence of

pain by 18 rnonths d e r autologous chondrocyte implantation in the human knee.

Second-look arthroscopy gives a timetable for the repair tissue maturation and

development. By three months, probing of the implanted area demonstrates "wave-like

motion" of the grafts, by six months the authors describe a %bber-like indentability",

followed by a "unindentable, porcelah-like surface" by 1 8 months. Longest clinical

follow-up was nine years; g r a s were not observed to be undergoing ossification or

degeneration.

Previous reports evaluated allogeneic and autogeneic chondrocyte transplantation

and indicated that both were satisfactory for cartilage grdting [Aston 19861, but recent

reports concentrate on the use of autogeneic tissues. Autologous chondrocyte

implantation is proving to be effective? but s d e cultivation of autologous cells is

demanding. Strict sterile technique in an undeviating aseptic environment for up to four

to five weeks of ce11 processing is mandatory Fiinas 19971. Bacterial contamination has

been reported for other tissues in cell-based therapies and remains as a potential threat in

the field of chondrocyte transplantation. Minas [1997] describes the "ided" human

candidate for autologous chondrocyte implantation; a healthy, Young, active individual

with a significant cartilage injury. The authors d e h e a sipificant lesion as a defect due

to trauma larger than 20.0 to 30.0mm in diameter. Focal degenentive lesions may also

benefit fiom this treatrnent. Human patients with moderate to severe osteoarthritis are

currently no t considered candidates for auto logous chondroc yte implantation. This

technique is not fully evlauated in objective long term studies, and the hgility of the

resuiting tissue as well as its overall maintenance of the chondrocyte phenotype are in

question.

1.8 Osteochondral Grafts

Osteochondral grafting relies on the availability of articular segments for the

repair of chondral defects. The repair tissue originates fkom cadavers, living donors. or

the recipient. Review of the available literature shows the use of varying sizes of'

osteochondral grafts in research and clinical cases [Brooks 1994; Yamashita, Sakakida,

Suni and Takai 19851. Large articular sections up to half- or whole joints. medium size

grafts covering oniy part of an articular surface, and miniature gr& of less than 6.0 mm

in diameter have been used for joint reconstruction. Aside fiom joint resection pexformed

to eliminate joint pain, osteochondral grafting is mong the oldest surgical techniques for

the repair of chondral defects. In order to restore normal joint function in human patients.

both Ench Lexer and Peter 1. Buchrnann used joint transplantations in the early 1900's.

They reported the use of closed and intact human cadaver elbow joints buc ch ma^ 19081

and articular sections of the knee Fexer 19081 in a senes of cases. Surgical

reconstruction of large osteochondral defects is used in humans meaver 1992; Meyers.

Akeson and Convrey 1989; Tsahakis, Beaver and Brick 1994; Flynn 1994; Yamashita

19851, especially for patients with neoplasia. A neoplastic process in, or close to a

human joint, may warrant the excision of articular tissues within healthy margins,

resulting in a large defect maymneh et al. 19941. Total arthroplasties using artificial

joints have eliminated the use of complete cadaver joint transplantations as originally

described by Lexer and Buchmann [1908], and hemi-iirthroplasties have replaced. to

some extent, cadaver boue transplantation [Barrack, Wolfe and Wddman 19971; but

lifespan of a joint prostheses is limited [Barrett, Biswas and MacKenney 19901.

Osteochondral grafting is considered the treatment of choice by many clinicians meyers

1989; Garrett 1986 and 19941. especially for a young patient. Realizing that artificial

materials for chondral defect repair do not have the mechanicd properties and durability

of hyaline cartilage, researchee continued the investigation of osteochondral

transplantation p o f i a n 19851. Attempts to improve osteochondml graft transfer have

been made over time. Provision of vascularity by blood vesse1 anastomoses or

intramedullary muscie flaps is the recent focus of researchers [Brown, Chung. Lantieri et

al. 19971.

An irnrnunologic response to osteochondral allografis has been demonsuated in

human patients as well as in animai models [Stevenson, Emery and Goldberg 1987:

Stevenson. Li and Martin 1991; Stevenson 19831. However. the presence of this response

does not necessarily affect the outcome of osteochondral transplantation [Garrett 1986

and 19941. Large g & s may contain considerably more antigen than smaller

osteochondrai @s murtig 19881. This review OF osteochondrai grafüng concentrates

on midl to medium-size autogeneic and allogeneic grafts and the currently evolving

mosaic arthroplasty.

1.8.1 Osteochondral Grafting with Srnall to Medium Size Grafts

Hurtig [1988] used an animal mode1 compared the use of auto- and allografis.

The author investigated the feasibility of using smail osteochondral gMfts for resurîàcing

the third carpal bone in eight horses. Seventy-five per cent of the radial facet of the third

carpal bone was resected as a 3.0mm thick wedge. The defect was U e d with a fiesh

g d t hmested fiom a similar site in a equine cadaver, while the resected segment was

impianted into a defect created in the contralaterai third carpal bone. Fixation of the

grafts was achieved with Kirschner wires. The experimental design allowed for the

evaluation of srnall osteochondral gnfts implanted as auto- and allografts; al1 eight horses

were euthanized five months after surgery. Technical difficulties with the grafting were

expenenced by the author. Incomplete countersinking of the Kiachner wires was

foilowed by wire migration in some cases. Proper fitting of the graRs to create a

congruent joint surface was found to be challenging, partly due to the hardness of equine

bone. Grafts protniding above the surrounding articular surface were found to be eroded

or produce "kissing lesions" on the opposing joint surface. Recess of the graft below the

surrounding cartilage resulted in coverage of the gafl by fibrous tissue. Hurtig

demonstrated a higher percentage of articular cartilage rernaining in the autografts (mean

82.8 per cent) compared to the allografis (mean 49.1 per cent). In the grafis transplanted

without technicd errors. autognft cartilage appeared to be intact overall. Allografts

demonstrated varying degrees of cartilage loss due to superficial synovial adhesions and

subchondral invasion of the calcifi ed cartilage. Admitting the need of a long-term study.

Hurtig concluded that autogenous osteochondral fragments may be used as a salvage

procedure for horses. The author did not recommend the use of fresh equine allografis.

A report by Outerbridge [1995] describes the use of autografts for osteochondral

defect repair in the knee of 18 hurnan patients with a mean age of 27 years. An

autologous graft fiom the lateral facet of the patella was used to repair a defect greater

than 300mm2 in the weight-bearing surface of the fernorai condyle. Ten patients were

followed for four to nine (mean six and a halo years. All patients were satisfied when

cornparing their preoperative pain and physical restrictions to the postoperative period.

Seven patients reported unrestricted activities including athletic activities like baseball

and hiking, with three patients experiencing some limitations. One patient had restricted

himself to less than hvo hours of continuous athletic activity and remained free of pain.

Two patients experienced mild pain in the anterior knee when kneeling or crawling. Al1

patients had a normal gait and radiographs obtained at various times showed complete

incorporation of d l grfis. Five joints had mild radiognphic signs of degenerative joint

disease (lateral osteophytes). The authors concluded that this type of autogaft is suitable

for the repair of chondral defects. They stated that the use of a fresh autograft has

advantages over the previously reported use OF small allognfts by Garrett [19861 and

McDennott [1985]. Because an autogr& is used instead of an allograft. the risk of

infection and disease transfer is reduced. Outerbridge et al. [1995] stated that this

technique can be used without a specialized and expensive facility for the harvesting and

storage of gras. They found their technique suitable for a srnail hospital with little

special ized equipment.

Garrett in 1994 reviewed 17 patients (mean age: 20 years) treated with fresh

osteochondnl allografts for osteochondritis dissecans defects in the lateral femoral

condyle. Graf3 f~at ion with Herbert screws and follow-up examinations were perfomed

at the tirne of hardware removal (six weeks to six years postoperatively) and at a later

point. Pain, stifiess, swelling, and instability of the knee were eliminated in al1 but one

patient. Screw removal was perfomed arthroscopicalIy, and the grafted sites were

evaluated at that time. Success was denoted by a glistening cartilage surface finnly

f i e d to the subchondral bone. The only failure occurred in an individual who d e r e d

gross gr& fragmentation that lefi a Crater. Two to nine y e m after surgery, 16 out of the

17 grah were considered a success. Garrett found these results (94 per cent success) to

be consistent with those of other investigators. An overall success rate of 77 per cent

with mall, fresh allografts was reported by Meyers in 1989 at two to 10 years

postoperatively. Gross, McNee, Pntzker and Langer [1983] and Locht, Gross and Langer

[1984] reported a positive outcome in 75 per cent of their allotransplantation with small

grafts.

A Failure in 22 out of 100 patients receiving cadaveric osteochondral grafts was

reported by Kandel, Gross, Ganel et al. [1985]. The authors investigated the

histopathology of the failed grafis. Failed grafts were removed between 12 and 84

months after implantation. Findings described in this report include necrosis of the bone

and bone marrow with variable replacement by the host bone. This finding seemed to be

independent of the duration of the gr&. Degenerative changes in the grafted articular

cartilage ranged f?om fibrillation to erosion, but viable donor cartilage was present as late

as seven years after graft transfer. Host bone appeared to interface with the donor

cartilage, but in approximately one third of the gr& a focal invasion of the cartilage

could be demonstrated. Pannus formation was present in some grafts, resulting in

cartilage resorption. Summarising their fmdings? the authors found the results

encouraging. It seemed that some cartilage was viable, and bone was usually replaced in

an organked fashion, but subchondral support could fail catastrop hicall y.

1.8.2 Osteochondral "Miniature" Grafts

C y Iindrical osteochondral grah

referred to as "mini" grafts [Hangody et

smaiier than t0.0mm in diameter are often

al. 19941. In 1993, Matsusue reported a new

treatment for a chondnl lesion associated with antenor ligament dimption in the human

knee. The author used an arthroscopic technique to harvest osteochondral grafts from

non weight-bearing areas of the aFfected knee (femoral condyle), and transplanted the

autografts adjacent to each other into the chondral defect. Although Hangody [1994]

performed a sirnilar grafiing technique by arthrotomy in clinical cases in 1992. the report

of Matsusue and CO-workers has to be considered the first publication of' this new

technique.

1.8.3 Mosaic Arthroplasty in Research and Clinical Application

Hangody [1996] compared the outcome of 64 mosaic arthroplasties performed by

arthrotomy with five arthroscopic gr& implantations. This is the first report of a case

senes of arthroscopic mosaic arthroplasties [Hangody 19961. To date, a primarily

positive outcome with mosaic arthroplasty has been reported for over 100 human

patients.

The technique and instrumentation described by Matsusue [1993] consists of a

standard arthroscopic approach to the human knee, and the use of a chondral shaver to

remove the degenerated cartilage fiom the area of the Iesion. Three holes with a dimeter

of 4.8mm were dnlled adjacent to each other into the Lesion. A cylindrical bone chisel

with an huer diameter of 5.0mm was used for the manual harvest and delivery of three

bone cylinders, each approxllnately 9.0mm in length, into the three recipient holes. The

anterior cruciate defect was repaired, and physiotherapy was initiated starting on the third

postoperative day. The continous passive motion was continued for four weeks, at which

point partial weight-bearing was aiiowed. A second-look arthroscopy was performed 12

weeks post-operatively, and showed that the implanted grafts had united with the originai

chondral defect. Fui1 weight-bearing of the operated knee was achieved four months after

surgery. A third arthroscopic examination of the knee was performed two years d e r the

grafting and showed that the original chondral defect was cornpletely covered with

chondrd tissue; the implanted grafts could not be distinguished from the peripherally

regenerated cartilage. Al1 three donor sites were covered with fibrous tissue. The patient

was pain-fiee and reported no restrictions of his athletic activities (skiing and tennis).

The authors concluded that the initial chondnl defect in this patient was too large to be

simply treated by debridement or subchondnl drilling. Their new method has the

advantage of an uncomplicated alignment of the srnall gnfts to create a congruent joint

surface. Use of a larger. single osteochondral graft can harbor problems in regard to a

satisfactory fitting of curved surfaces, and arthroscopic implantation may be impossible.

A doctoral thesis by Hangody was followed by Curther research reports [1994] and

a clinical paper 119961, describing the use of this surgical technique in the human knee.

A strong emphasis of the Hungariûn researcher was the use of more sophisticated

instrumentation, which had become cornmercially available. At the time of the

conclusion of the dinical trials in Apnl 1996, Hangody was able to review his experience

with 107 human patients. The results of his Iaboratory trials From 1991 were published in

1994 and 1996, describing the operation of 18 adult German Shepherd dogs. Mosaic

anhroplasty was performed via arthrotomy on the weight bearing (18 joints) and non-

weight bearing portion ( 1 8 joints) of the medial kmoral condyle. The articular cartilage

was debrided d o m to bleeding cancellous bone. Holes with a diameter of 4.5mm. 12.0

mm, and spaced 1.Ornrn apart were dnlled into the defect. Gr&s were harvested with

cylindncal chisels fiom the periphery of the femur in the patello£+emoral joint, and were

then press-fitted into the holes. Gross, radiographie, and histologic exminations were

performed four, six, eight, 16,26, and 52 weeks postoperatively.

The author reported survival of the grafied hyaline cartilage in al1 cases. with

significant differences between weight and non-weight bearing areas. Surface congrui ty

occurred in 100 per cent of al1 transplanted areas in non-weight bearing areas.

Radiographically, the grafied bone had incorponted into the surrounding bone. The

cartilage showed no signs of degeneration and was morphologically identical to hyaline

cartilage. Results were less encouraging in the weight-bearing areas. In over one third of

the cases, bone necrosis and subsidence of the gr& occurred. Inegularities in the

articular surface were filled with fibrocartilage and fibrous tissue, but the hyaline

cartilage of the gnfts had survived. Euthanasia of the experimental animais at different

time intervals was used to investigate gr& incorporation. Complete cancellous bony

union was found at four weeks. Fibrocartilage covered the exposed bone between the

gr& and the surrounding cartilage by six weeks. By eight weeks, the fibrocartilage had

grown to the surface, elirninating the small surface incongruities. Specimens collected

16,26. and 52 weeks postoperatively demonstrated a union between the hyaline cartilage

gr&, the fibrocartilage covered areas, and the surroundhg articular surface.

Additionally, Hangody evaluated the donor sites. Four weeks postoperatively, the holes

were filled with bone trabeculae. The nirface was covered with fibrocartilage. Each of

these regenerated components increased in volume and congnllty with time, reaching

preoperative thiclaiess by 16 weeks.

Hangody [1996] reported on the use of üiis new technique in 52 human patients,

suffering From chondropathy or osteochondritis dissecans in the knee. The medial or

laterai femoral condyle and the patella were the aected areas; no other knee pathology

aside Eom the chondrd defect had been diagnosed. The ~ ~ G O A S measured 15.0 to 89.0

mm (mean 45.0rnm) in diameter. Graft harvest and implantation was performed using a

method similar to the one descnbed for the canine trial. Per patient, between five to 19

(mean 10.8) osteochonciral cylinden with a diameter of 4.5 min were harvested and

implanted. The first four patients received grafts cemented with fibrin glue. Al1 later

gr&s were fixed simply by press fitting. Weight-bearing of the operated limb was not

permitted before six weeks postoperatively. Patients in this study were between 17 to 47

years of age (mean 28), and follow-up examinations were conducted nine to 39 months

(mean 29) postoperatively. The 52 patients were evaluated with respect to pain, range of

motion, joint effusion, and radiographic appearance. Forty-five patients were pain free

when weight-bearing, only seven reported mild pain. Forty-four knees showed a normal

rnobility, while eight had a decreased range of motion. Forty-one knees had no joint

effusion, and 11 knees had various degrees of joint effision following exercise. Six

patients underwent follow-up arthroscopies at nhe, 16, 18, 28, 46, or 84 weeks

postoperatively. Hangody reported a sufficient hyaline-like cartilage coverage over the

original defect. The donor sites were covered with tissue similar to fibrocartilage. This

finding was verified with biopsies of the donor sites. 'ïhese results seem promising

because autogenous, mature, My-differentiated cartilage was used to resurface focal

defects.

1.9 Arthoscopic Surgery

The current use of aahroscopic procedures for osteochondrai grafting has been

outlined above, but a closer look at the historical development of arthroscopy is

warranted. Japanese professor Kenji Takagi is generally credited to be the first person to

perfbnn an arthroscopy, using a standard cystoscope at the University of Tokyo in 19 1 8

[Dandy 19841. Takagi developed and refined his own arthroscopes over the following

years, and the efforts produced the fiat clinically practicd arthroscope in 1960. By the

late 1930's Takagi had succeeded in taking still color photographs and a black and white

cinematographic film of the interior of the human knee. In the 1920's in Europe, Eugen

Bircher of Switzerland examined the inside of a human knee using a Iacobeus

laparoihoracoscope and air distension. The report pircher 19211 of this case was

published at the same time Takagi reported his evperiences from 19 18 in Japan. Five

yean later, Geist's description of a synovial biopsy undertaken with the help of local

anesthetics and as an outpatient procedure [Geist 1926; Dandy 19841 was published.

Publications describing arthroscopic surgeries of' the human knee joint appeared in the

literature in 1931 [Bman] and 1934 [Buman, Finkelstein, and Mayer], but failed to

provoke interest amongst surgeons, until finally the topic was declared as %bandoned9'

by the authors themselves. M e r Professor Takagi's retirement in 1949. his successor

Watanabe published an atlas of the human knee as it relates to anhroscopy [Watanabe.

Takeda and Ikeuchi 1969 and 19791 and performed the f ~ s t arthroscopic menisectomy in

1962. With rheumatologists being the fust clinicians to use anhroscopic procedures

routinely in clinical settings [Robles and Katona 1969; Jayson and Divon 19681 for a

large number of clinical cases in the 1960's. the following decade saw the growing

interest of surgeons in this "rediscovered" surgical instrumentation. From 1970 to the

present day, arthroscopie equipment and techniques are rnostly used and developed by

orthopedic surgeons. Orthopedic publications in the early 1970's [Casscells 197 1,

Jackson and Dandy 19721 were followed by the fust course in clinical arthroscopy

(organized by Joyce and Harty at the University of Pe~sylvania in 1973). In 1974. the

International Arthroscopy Association was founded in Philadelphia.

Veterinary surgeons adapted the new technique quickly after it h d becorne

established in human medicine. Among the arthroscopes developed by Professor Takagi

and Watanabe [Watanabe and Takeda 19601 was a small diameter instrument known as

the Takagi 11, which was found to be suitable for arthroscopy of the canine

femoropatellar joint. Experiments conducted by Okarnura in 1945 descnbed synovitis in

the canine knee, using an arthroscope to monitor the changes in the appearance of the

synovium [Okamura 19451. This may be the first use of arthroscopy in animals, followed

by Watanabe's examination of an equine tibiotarsal joint [Watanabe 19491. Large animal

arthroscopy was discussed in the Eumpean literature in the mid-seventies [Smith 1975:

Knezevic and Wruhs 19771, one source mentioning a carpal arthroscopy performed on a

polo pony in Newmarket in 1976 as the first clinical equine arthroscopy in the United

Kingdom [Dandy 19841. The first reported cases in North America were descnbed by

Hall and Keeran [1974] and McIlwraith and Fessler [1978], describing the use of

arthroscopy to diagnose equine carpal problems using a 2.2 mm needlescope.

Arthroscopy not only allowed exploration and treatment of surgicd lesions within equine

joints, but also proved to be an aid in procedures like joint lavage in cases of sepsis and

the treatment of intracapsular soft tissue adhesions.

Severai techniques cm be used alone or in combination to enter a synovial joint

for diagnostic or operative procedures: arthrotomy, arthroscopy [Bardet 19971, and less

invasive procedures like arthocentesis or joint injection will, MacLarnon and Nag

19901. Arthroscopy or arthrotomy are the only available techniques if direct visualisation

of an articula surface and a controlled orthopedic procedure like a gnft implantation are

required. For decades the surgical access to a joint and its intncapsular structures for

diagnostic and corrective orthopedic procedures had been limited to an arthrotomy [Small

19901. The reflection of surrounding tissue, elasticity of the joint capsule, and the need to

gain proper anatomical orientation oRen necessitated rather large incision even for a

relatively minor orthopedic procedure pandy 19841. The arthroscope has dramatically

changed the approach to the diagnosis and treatment of a variety of joint ailmenis in

human surgery [Patel 1990; Dandy 19841, and has been successfully applied to veterinary

medicine WcIlwraith 1984; Hurtig 19851. Diagnostic arthroscopy and arthroscopie

surgery is ofien seen as the outstanding achievement in orthopedic surgery in the p s t 20

years [Smail 19901, although no other advance in human orthopedic surgery was so long

delayed after the initiai reports before it gaîned clinicai acceptance.

The advantages of arthroscopicdly-performed orthopedic procedures usually

outweigh the disadvantages or potential complications [Rozencwaig et al. 1996; Dandy

19841. Arthroscopy has become a cornmonly used surgical technique in hurnan [Curl.

Krome, Gordon et al. 19971 and veterinary medicine [Bardet 19971. Advantages hclude

reduced postoperative morbidity, srnailer incisions, less intense infiammatory response,

irnproved thoroughness of diagnosis, absence of secondary effects, reduced hospital cost,

reduced complication rate, improved follow-up evaluation, and the possibility to perform

procedures that are difficult or impossible to perforrn through an arthrotomy.

Clinically, arthroscopic examination and manipulation is limited to joints large

enough to accommodate the available arthroscopic equipment, thus limiting the use to

larger joints. This limitation may be mostly due to the restrictions experienced with

instruments too large for the exarnined joint. The interest of surgeons and instrument

manufacturee in the development of smailer, flexible arthroscopes and instruments will

most likely allow the examination and treatment of smaller. currently inaccessible

articulauons [Rosenthal 19961. Publications regarding the instnimentation, anatomical

access, indications, limitations, and future usage for arthroscopy in human and veterinary

rnedicine are plentiful, limited mostly to the equine and canine patient in veterinary

surgery F[cIlwraith 1 9841.

Arthroscopy ha been used for bone grafting [Carro. Cimiano, Del Alamo and

Suarez 19961, but literature regarding the use of arthroscopic techniques and equipment

as it relates to osteochondral gr& implantation is spane matsusue 1996; Hangody 1997:

Bobic 19961. The use as a diagnostic tool for initial [Flynn 19941 or follow-up

examinations [Garrett 1994; Outerbridge 19951 is more frequently found in clinicai

reports. Incisions needed for large gr& implantation eliminate the need for

intraoperative arthroscopic examination as the essential areas of the joint are exposed by

the arthrotomy meyers 1989; Flynn 19941. One of the most crucial benefits of

arthroscopy over arthrotomy is a shortened recovery tirne. This factor rnight be a less

important consideration afler massive osteochondral grafüng, since the patient has to

withhold fiom exercise or weight-bearing for the first weeks or months of the

postoperative period ~ c D e m o a 1985]. This penod of rest surpasses the time required

for a proper wound healing of an iuthrotomy incision.

1.10 The Bovine Animal Model in Orthopedic Research

Although ofien used in orthopedic research [Galili 1997; Schinagl, Gurskis, Chen

and Sah 1997; Zimmermann, Pnbhakar, Chokshi et al. 1994; Bryan, Bach. Bush-Joseph

et al. 1996; McMaster 19841, a bovine model for grafi implantation has to the best of Our

knowledge, not been described in the literature. Anatomical and histological

investigation by orthopedic researchen have focused on the collagen architecture of

bovine cartilage [Jefiey, Blum, Archer and Bentley 199 11 and the metacarpophalangeal

joint capsule composition [Kieftogiannis, Handley and Campbell 19941. The bovine

species may have multiple advantages as an animal model: availability of a large number

of genetically different or similar animals, various breeds (sizes) and age groups. and

availability of genetic background information (selective breeding). Cattle are relatively

inexpensive when compared with other large animals. and in case of terminal research

and in accordance with correct drug withdrawal times, the animai carcasses may still be

used for meat consumption.

1.1 1 Anatomy of the Bovine Fetlock Joint

The normal bovine l imb and joint anatomy is relatively well understood and

described in various veterinary anatomy text books and publications @3 aronne 1 9 89;

Nickel 1986; Sisson 1975; Hughes and Dransfield 19531. Pathologie conditions of

bovine joints can be found in the current literahue p a n Pelt and Langham 1970: Bailey

1985; Mogha, Das and Ange10 1972; Bargai 19751. The size and volume of larger bovine

joints is çuitable for arthroscopy Fatel and Guhl 19831, although only a lirnited number

of joints have been descnbed regarding their arthroscopie accessibility and anatorny

[Hurtig 1 985; D u c h m e 19971. Arthroscopy of the anatomically different equine fetlock

joint has been described in the literature WcIlwraith 19841. To Our knowledge,

experimental or clinicd arthroscopy of the bovine metacarpo- or metatarsophalangeal

(fetlock) joint has not been descnbed.

The anatomy of the bovine metacarpo- and rnetatarsophalangeal joints (MCP and

MTP joint) is described in textbooks of veterinary anatomy [Baronne 1989; Nickel 19861.

Alternative narnes for these articulations include "fetlock" and "iuikle" and are commonly

used in veterinary literature. The MCP are almost identical with the MTP joints and they

are classified as ginglymus joints [Nickel 1986; Dyce and Wensing 197 11.

The MCP or MCT joint is forrned by seven bones: a Fused metacarpal or

metatanal bone (proximally), two proximal phalanges (distally), and two pairs of

proximal sesarnoid bones @alma or plantar) [Nickel 1986; Smallwood 19731. The

proximal bone consists of the tiised third and fourth rnetacarpal or metatarsal bones

which remain ununited at their distal end, foming a more or less symrnetrical pair of

condyles. The synovial cavity is divided into a lateral and a medial synovial sac [Dyce

and Wensing 19711. Communication of these sacs can be observed at the palmru or

plantar aspect between the interdigital band of the interosseus muscle and the rnetacarpal

or bones at the level of the proximal sesarnoid bones pesrochers. St-Jean, Cash et al.

19971. Dorsal and palmar or plantar pouches are formed by synovial sacs [Bose and Rao

19831. The distal aspect of the metacarpal or metatarsal bone and the extensor tendons

surround the dorsal pouch. The palmadplantar pouch lies beneath the abaxial and axial

branches of the interosseus muscles and the deep digital flexor tendon. a more proximal

extension has been descnbed for the palmar or plantar pouch than the dorsal pouch

[Nickel 1 9861. A recent study using 1 88 fetlock joints fiom 55 ox cadavers demonstrated

the interior joint anatomy; a communication of the media1 and lateral synovial sac in 98.9

per cent of d l joints was s h o w @lesrochers 19971.

1.12 Summary

The inability of articular cartilage to repak itself has been known for at Ieast 200

years Win= 19971. The preceding literature review describes a number of treatments

that have been used to manage articular cartilage damage. Our current knowledge

indicates that fibrocartilage or tibrous connective tissue rather than hyaline cartilage is the

long t e n result of almost al1 attempts to facilitate repair by recniitrnent and

differentiation of mesenchymal cells fiom the marrow spaces. It is unlikely that the

resulting tissue will have the typical characteristics of healthy, durable articular cartilage.

Studies using perichondrîurn or periosteurn gr& resulted in a hyaline-like cartilage that

degenerates later because of poor g d l incorporation and the nsk of eventual

endochondral ossification. Chondrocyte transplantation has been shown to have

promising potentials for the repair of chondral defects. Studies demonstrating a positive

long-terni outcome are sparse, and the proper delivery and anchoring of the transferred

ceiIs is still under scientific investigation. In al1 these techniques, maintenance of or

regaining the chondrocyte phenotype is crucial but there is strong evidence that this

highly differentiate ceil type tends to dedifferentiate into fibroblasts in a less than optimal

environment. Using adult articular cartilage for restufacing damaged cartilage avoids

many of the above pitfalls.

Massive osteochondral allografts and autografts are considered an aggressive

option for large articula defects. The use of smaller, multiple (less than 10.0m.m)

osteochondnl gmfts in cylindncal shape has been used since 1993 to repair chondrai

defects in humans. Previous experhental and clinical studies demonstrated that small

osteochondral grafls can survive and become incorponted into the joint surface of human

patients [Czitrorn 19861. Osteochondral grafting, especially in form of the recently

developed "mosaic arthroplasty", is therefor accepted as a valuabie surgical technique for

human patients with chondral defects. It is considered to be among the less invasive

surgical procedures if performed by arthroscopy.

Transfer of cyl indrical "miniature" (2.5 to 1 0.0mm) osteochondral autografts is

currently used for the repair of chondral defects in human patients [Hangody 1996: Bobic

1996; Matsusue 19931. The use of small osteochondral gr& in the equine has been

investigated [Hurtig 19881; small cylindrical grafts implanted arthroscopically may be

more stable, less Iikely to become darnaged and suffer the consequences of immune

recognition. While autografls are used for mosaic arthroplasty in humans, the issue of

donor site morbidity has not been resolved [Hangody 19941. The use of allografts

eliminates donor site morbidity [Garrett 19861, and this is particularly attractive in equine

surgery because donon are readily available fiom horses that are euthanized due to

incurable conditions.

1.13 Aims and Objectives

The scope of our study was to establish a basic understanding of small ailogeneic

osteochondral grafts implanted into a defect created in an animal model. The study

investigated the short-term fate of 6.0mm osteochondral allografts arthroscopically

implanted in a metacarpophalangeal (fetlock) joint of bovines weighing 200kg. We

hypothesized that small gr& would survive the handling associated with their intial

harvesting and subsequent implantation. If our allogeneic gr& retained their viability

and support, we would assume that immunogeneic reactions are not significant enough to

damage the cartilage or interfere with bony rernodeling and incorporation. In case of a

positive outcorne, allografts rnay be seen as a valuable alternative to the use of

osteochondral autograh.

We also needed to establish if a 6.0rnm cartilage defect in the fetlock of a 200kg

bovine is beyond the critical size defect that could heal spontaneously. Without this

information, we would be unable to differentiate extrinsic cartilage heding from grafi

survival. We did this by implanting 6.0rnm bone allografts in the second front fetlock of

our experimental animals as a negative control.

For this study we had to develop simple instrumentation to harvest small

osteochondral alIogr& from cadaver joints. We also needed to find an arthroscopie

approach to the bovine fetlock joint that would allow implantation of small grafts.

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C W T E R n: Materials and Methods

2.1 Study Design

This study was divided into two phases. In a first, preliminary phase specialized

instruments for allograft harvest and implantation were developed. We tested the

instrumentation, developed an arthroscopie approach, and refined the experimentai

protocol using bovine cadaveric metacarpophalangeal joints. In the second, expenmentd

phase, we irnplanted orthotopic allografts arthroscopically into the metacarpophalangeal

joints of six calves (see Figure 1). The short-term fate of the implanted grafts was

investigated three months after surgery.

2.2 Preliminary Phase

The allografts used in this study were harvested in our research laboratory from

fresh cadaver joints. The cylindncal gr& were 6 . 0 m in diameter with a length of 15 .O

mm and were harvested as osteochondral gr&. Each metacarpophalangeal joint of the

six recipient caives received either two osteochondral or two bone allografts, implanted

into the weight bearing area of the distai MC,. The bone grafts were used as a control

and were obtained by removing the hyaline cartilage fiom a cylindrical graft. Al1 limbs

used in the preliminary phase were collected fiom a local veal-meat processing plane,

where we had unrestricted access to a large number of fiesh lower front lirnbs.

2.21 Graft Earvest

Severai instruments were fabncated or rnodified lÏom existing equipment in order

to harvest osteochondral cy linders with a 6.0mm diameter. Equipment included coring

bits with plastic ejectors, a bone holding device, and a mitre boxb, a tilting arbour drill

pressc, and metal hack sawsd with cobalt-coated carbide bladese.

Custom-made coring dnll bitsb allowed us to harvest cylindncal osteochondral

gr& with undamaged articular cartilage of a reproducible, constant diameter. nie hollow

drill bits were machined from tool grade steel for economy but also to ensure a durable

sharpness. nie inside diameter was 6.0mm with an outside diameter of 8.Omm, a drill bit

wall thickness of I.Omm, and an ovenli length of 120mm. The cutting end of the drill

consisted of six beveled, chisel-like triangular teeth (see Figure 2). The teeth were

slightly tilted in a counter-clockwise direction.

Nylon rods 200rnm in lengthb and with an outside diameter of 5.9mm were used

as pushrods. Gr&s that had broken off the gr& bed and were lodged inside the coring

drill bits could be dislodged with the pushrods. The soft plastic materiai prevented

damage to the sidewall of the c o ~ g bits. S t e m sterilizationfcaused oxÎdation of the drill

bit sudaces, so ethylene oxide gas sterilizationf was used to sterilize the drill bits and

pushrods.

A tilting-arbour table-top drill press powered by an electric motorc was used For

harvesting grafts. This machine could be set at various speeds (revolutions per minute:

rpm) using a combination of different belts and pulleys. The lowest speed of 580rprn was

used to rninimize thermal damage to the gr&. The drill press was equipped with a

standard Jacob's chuck to accommodate the corhg drill bit, as well as a vise bolted to an

adjustable working surface. This working surface was f i e d to the supporthg post of the

drill press and allowed the sample to be positioned in sagittal and transverse planes. The

tilting arbour feature of this machine aiiowed us to adjust the drill bit in the fiontal plane,

ensuring the coring bit was perpendicular to the joint surface. Coring-bit wobble was

minimized by the accmcy and mass of the drill press.

Unstable fixation of the metacarpus in Our preliminary tests resulted in

uncontrolled vibrations, wobble, or displacement of the bone and slipping of the coring

drill bit. The results were broken bone dowels with damaged articular cartilage, and

broken cot=ing bits due to bending. To avoid this. the joints were dissected and reduced to

the distal third of MC,,. The midshaft of MC,, was approximately 40mm in diarneter in

cross section, and its shape was roughiy rectangular. Stainless steel was used to bbricate

an autocalavable cylindrical bone holding deviceb, measuring 1 OOmm in diameter (inside

measurement) and height. The metacarpus was secured by three screws with sharpened

tips inserted into the cylinder through tapped holes in a tnangular orientation (see Figure

2). This configuration allowed for a simple four-point fixation. The cut surface of the

metacarpus rested on the bottom of the cylinder, with lateral fixation supplied by the

three screws. Securing the bone during drilling allowed the production of stnight bone

dowels. The holding device was secured and positioned on the drill press with the aid of

a vise. The enclosed construction with a closed bottom allowed us to submerge the

articular cartilage in sterile saline solutiod during dnlling to rninimize drying and

thermal damage to the gr&.

Cutîing through MC, required us to hold the metacarpus firmly while

maintainhg the aseptic environment. A mitre box with a triangular shelf and a 2.0m.m

slot was constmctedb of two plexiglas halves, fastened together with stainless steel bolts

(see Figure 3). Steam sterilUationf was used for this device. A standard metal-cutting

hack s a e was used to make a transverse cut through MC, approximately 25.Omm

proximal to the joint s d a c e to release the osteochondral dowels. The use of power

equipment or orthopedic tools was impractical because of the difficulty of sterilization

between uses. Tooi grade hack saws handles and metal-cutting blades produced excellent

results. A stenle saw handled supplied with a new. sterile blade' was used for each cut.

The hack saw handles were re-sterilized by s tem stenlizationf. Saw blades were

sterilized using ethylene oxide gasf and were discarded after a single use.

Al1 of the caives used in this study and their processed carcasses passed the

vetennary meat inspection required in the province of Ontario. The disarticulated lower

tiont limbs from each calf were inspected for scars, skin abrasions or swelling. Only

healthy caives with "normal" Feetlock joints were selected. Transport to the laboratory

used for the bone dowel harvest took less than 25 minutes, and limbs were stored in a

styrofoam cooler.

The hair was clipped, and a routine surgical scrubf was performed (tap water,

chlorhexidine', alcohol", cetrimide"). Al1 steps fiom the surgical scmb to the final stonge

of the harvested cylinders in stenle containers were performed with the operator weanng

surgical scrubs, mask and cap, and sterile surgical glovesw. Gloves were changed

Eiequently to ensure stenlity. The donor limbs, joints, or bones were handled and placed

on sterile disposable surgical drapes'.

Two circumferential skin incisionsy were made appro,ximately 60mm above and

below the fetlock joint. These two incisions were subsequently comected by a

longitudinal incision creating an H-shaped opening over the dorsal aspect of the fedock

joint. Sterile tissue forceps were used to elevate the skin and the circumferential

dissection was continued through the subcutaneous layers. The limb was placed on a

sterile drape, resting on its palmar surface. A sterile hack saw was used to cut MC, and

P, approximately 40.0 mm above and below the fetlock joint articulation. The joint was

removed and inspected for damage to the joint capsule or exposed articular surface. Any

accidentally opened joints were discarded at this point. Each harvested joint was

submerged for ten minutes in a stenle £ive per cent povidone iodine solution', followed

by an alcohol bathu for an additiond ten minutes. We also used the brown color of the

povidone iodine solution as a marker for unintentionally opened articulations. The joint

was discarded if synovial fluid or cartilage were found to be stained. The joints were

removed from the alcohol bath and kept on sterile drapes to air dry.

The joint capsule was incised starting at the medial or laterai aspect of the fetiock

joint. Care was taken not to darnage the articular cartilage and to avoid direct handling of

the joint surface. Dissection was continued until the distal end of MC,, was isolated.

This section of MC, was submerged in sterile salineg until the actual gr& harvest was

performed. The bone section was secured in the holding device. In order to avoid

contaminating contact of fluid or tissues through the open base of the coring drill bit, the

end was closed using a fmger tip of a stenle surgical glovew prier to insertion of the bit

into the Jacob's chuck. The holding device was secured to the working surface of the

drill press in the vise, and was filled with sterile saline with antibiotics. One litre OF

sterile saline solution6 was rnixed with ten million m i t s of sodium penicillin G'. We

harvested one gr& fiom each the media1 and lateral condyle of MC,. If the gr&

separated nom the subchondral base and lodged in the coring drill bit, or if damage to the

articular d a c e or subchondral base was apparent, both fetiock joints fiom this donor

animal were discarded.

The drilled metacarpus was removed fiom the holding device and placed on the

sterile mitre box covered by an additional sterile drape. The sterile hack saw and mitre

box were used to perform a transverse cut at the proximal end of MC,, thus releasing the

cylindrical grafls created by the drilling process. Stede tissue forceps were used to

extract the bone dowels fiom the distal MC, bone segment. A small identification mark

was made to identify the correct orientation of the graft during transplantation. A small

wedge was removed from the dorsal aspect of the bone dowel with a scalpel at the end

opposite to the articula surface. The grafis were placed in a stede container filled with

DMEM tissue culture mediumm and refngerated at 4" Celsius for approximately 12 hours

prior to implantation. The gr&s harvested from the six donor anirnals were removed

fiom the refrigerator and allowed to slowly warm to room temperature approximately two

hours prior to implantation.

2.2.2 Grafi Testing

The stenlity and integrity of the harvested grafts was tested in three preliminary

trials. Five limbs retrieved fiom an abattoir" were used for the collection of ten

osteochondral cylinders. The cylinders were stored in sterile vialsh at 4 O Celsius for 12

hours pnor to microbiological testing in the fonn of aerobic bacterial cultures'.

The improved. second harvest protocol described in 2.2.1 was tested by collecting

ten osteochondral cylinders in two harvest sessions. [mprovements consisted of the

addition of iodine and alcohol bath for the isolated joints, fiequent changes of surgical

gloves throughout the harvest, and the addition of sodium penicillin C? to the sterile saline

used for submersion and cooling of the bone during drilling. In both sessions, the

collected cylinders were stored in pairs in sterile vialsb at 4O Celsius for 12 hours prior to

mimbiological testing in the form of aerobic bacterial cultures'.

In a third trial, eight osteochondral cylinders from six different fiont lirnbs were

harvested to detenine possible thermal or mechanical damage to the osteochondral

cylinders, or metallic Wear particles fiom the coring drill bits. The fresh samples were

examined under a dissecting stereo microscopek and light sourcei. Following refngeration

in tissue culture mediumm at 4 O Celsius for 12 hours, the samples were fixed in 10%

formalin" for 24 hours and then placed in decalcifying solutiono for one week. At the

point of complete decalcification, the samples were again fixed in fornalin" and cut into

5.0 to 6.0pm sections on a microtomeP. Staining was perforrned using standard protocol'

for Safranin-O and Masson Trichrome stains. A microscopeq supplied with a light sourcer

was used to examine the stained sections in order to determine cartilage darnage.

disniption of the cartilage-bone interface, or darnage to the subchondral bone.

2.2.3 Arthroscopic Approach

To the best of our knowledge, an arthroscopie approach to the bovine

metacarpophalangeal or metatarsophalangeal joint had not been described vurtig 1 9851.

The standard insertion technique as described for horses is a diagonal approach to the

dorsal aspect of the fetiock joint WcIlwnith 1996 and 19891. We used ten cadaver joints

to evaluate this approach.

A new insertion technique was developed and tested in a preliminary trial using

45 bovine cadaver fetlock joints. The 5.5m.m-diameter arthroscopy sleeve was inserted

into and through a fibrous capsular band (see Figure 4) situated on the midline between

the two condyles of the metacarpus wckel, Schummer and Seiferle 1986; Desrochers,

St-Jean, Cash et al 1997; Dyce and Wensing 1971; Smallwood 19731. Twenty-three out

of the 45 joints were used for the evaluation of the arthroscopic approach alone. The

remaining 22 joints were used to invenigate the arthroscopic gr& implantation.

2.2.4 Graft Implantation

Instrumentation included a 4.5 mm rigid arthroscope with a standard stainless

steel sleeve and blunt and sharp obdurators, a halogen Iight source comected to the

arthroscope by a sterile fibreoptic cablee, an electric peristaltic fluid purnpu with stenle

~ b i n g comected to a sterile carboy filled with lactated Ringer's solutionm and controlled

by a floor switch, and an arthroscopic video camera connected to a monitor and video

cassette recorder. A steel 6 . 0 m drill bit'" was modified by machiningb the original

standard rounded tip to a Bat one and creating a 1.0 mm spike in the centre (see Figure 5).

The 1.Omm spike was necessary to allow proper seating of the drill bit on the articular

cartilage without the drill bit skating across the joint surface. A stainless steel drill guide

with an inside diameter of 6.0mm and a wall thickness of I.Ornm was fabricated to

protect surroundhg soft tissues during dnllingb. This dnll sleeve also fùnctioned as a

gr&-passing tool in combination with a 5.9mm push rod made of polyvinyl chioride

(PVC). A steel handle was welded at a 90' angle to the drill guide for more control and

as a reference point. Drillhg was perfonned using a standard, nitrogen-driven surgical

drill".

The fetlock joint was distended using 20 to 301111 of sterile salin@ injected through

a 20-gauge sterile hypodermic needlecc inserted hto its dorsal aspect. Care was taken not

to lacerate the d c u i a r cartiIage. The needle was withdrawn and a 5.0mm stab incision

was made over the dorsal midline of the fetlock joint using a #15 scalpel bladeY. An

arthroscope sleeve with the sharp obduratore was inserted perpendicular to the skin

d a c e and, after the sharp obdurator was exchanged for a biunt one, the fibrous band

was penetrated by advancing the arthroscopie sleeve towards the lateral side. The

obdurator was exchanged for the arthroscopeO, and the joint surface was examined.

The axial h d f of the laterd condyle was visualized and a 20-gauge sterile needlecc

was used to determine the proper location for a second stab incision over the recipient

site. A 10.0mm long stab incision was made over the area and the 6.0mm drill bitbv

guarded by the 8.0mm dnll guideb was inserted into the joint and connected to the

orthopedic drill". The proper placement was confirmed with the arthroscope, and a hole

approximately 15.Ornm in depth was drilled. Pnor to drilling, the fetlock joint was flexed

and a vertical orientation of the drill bit to the articular surface was verified by the MO

operating surgeons. The longitudinal axis of MC,, was used as a visual reference point.

The drill bit was seated so that it represented an extension of the lirnb axis, allowing us to

place the 6.0mm hole in the center of the condyle. Sterile salineY \vas applied to the drÏll

guide to ensure cooling during drilling. The drill was removed, and the joint was lavaged

in order to remove bone debris and blood.

Depth measurements of the hole during surgery were made with a graduated

stainless steel 5.9mm rodb (see Figure 6). The rod had circumferential markings at 1 .Omrn

internais, and the chosen 5.9mm diameter allowed for an easy insertion into the recipient

hole.

A cylindrical gr& was cut to the appropriate length using a #22 scalpel bladg or

a hardback scalpeP while supported on a moist, laparotomy sponge. Care was taken to

place the gaft on the sponge with the previously cut notch, indicating the dorsal aspect,

facing upward. This allowed correct orientation afler the rernoval of the notched end.

The handle of the drill guide was used as a reference point for the notch. The graft was

inserted in the drill guide with its dorsal aspect facing the handle and positioned with the

PVC push rod so that 3.0mm of the trimmed end was exposed beyond the drill guide.

The drill guide and gr& were inserted through the stab incision into the joint and, under

arthroscopie control, inserted in the 6 . 0 m recipient hole in MC,. Some pressure was

necessary to fully seat the graCt properly in the hole. The arthroscope was rotated towards

the medial condyle, and a sirnilar surgical procedure was performed to implant the second

gr&*

Bone graRs were produced by removingy the hyaline cartilage cap together with

2.0m.m of subchondrai bone. Othenvise, they were handled identically to the

osteochondral gr&.

2.3 Experimental Phase

Six recipient cdves weighing between 197 to 208kg were used in the study. Gr&

implantation was as described in 2.2. This resulted in a total nurnber of 12 osteochondral

(Treatment 1) and 12 bone gr& (Treatment 2).

For unoperated controls (Treatment 3), eight metacarpophalangeal joints fiom

four calves weighing between 198 and 209kg were collected €rom the abattoir. These

joints were used to establish the normal appearance and histology of the recipient site,

and were assessed by the same cnteria as the experimental joints.

23.1 Graft Harvest and Storage

The six donor animals were male Holstein calves weighing 203 to 217kg,

representing the animal type typically slaughtered as veal calves in large numbers in

Ontario [Tabachnik 19951. The metacarpophalangeal joints were not handled or opened

in the abattoir. A clinical examination was performed at the abattoir" immediately pnor

to the slaughtenng process. Although our midy required only six donor animals. we

selected 12 potential donor animals at the slaughter house. Lateral and dorso-palmar

radiographs of each limb were obtained. Radiographs were evaluated by a clinician.

board-certified by the Amencan College of Veterinary Radiologists5.

2.3.2 Graft Implantation

The six recipient calves undenvent routine clinical examinations 72 hours and 12

hours prior to surgery. Body weights ranged from 197 kg to 208kg. A lateral and dorso-

paimar radiograph of the nght and lefi fore limb of each calf was obtained. Lameness

examination included palpation of al1 four limbs and leading of the animal in a wide

circle on concrete ffoorhg. Calves were housed in pairs and had access to Cree choice

hay and water.

Ail procedures were done in accordance with the guidelines of the Canadian

Couneil of Animal Care". Food was withheld 24 hours prior to surgery, but fiee access to

water was allowed. One hour prior to surgery, an Uitravenous catheteflsL6 was placed in

either the nght or left jugular vein. A venous blood sampleh was obtained fiom the tail

vein 30 minutes prior to nirgery and blood gas values, packed ce11 volume, rotai senun

protein, and electrolyte levels were measuredf. Thirty minutes prior to induction of

general anesthesia, the calves received intravenous sodium penicillin Cr' (20.000

international units per kilogram of body weight; i.u./kg) and intravenous flunixin

megluminec (2.2 milligrams per kilogram of body weight; mgkg). The calves were

premedicated with 0.OSmgkg of xylazine4 intravenously. General anesthesia was induced

with intravenous diazepamy (0.05rng/kg) and ketamine"(3.0mg/kg). The calves were

intubated with a cuffed endotracheal tubet (16.0 to 18.0mm inside diameter) and

anesthesia was maintained with hdothaneq in a semi-closed anesthetic circuitA.

Anesthetic monitoring included direct blood pressure, electrocardiogarn, and blood gas

monitoringf. The calves were positioned in dorsal recumbency on a surgery tablep and

both lower fiont limbs were clipped from the carpus to foot, scmbbed with

chlorhexidinec, alcoholu, and cetrimide', and surgically draped using disposable

impermeable drapes*. The surgical site (nght and lefl fetlock joint) was covered using an

adhesive iodine-impregnated drape". Sterile Cotton hmd towels' were used to support the

fetlock joint and to keep it in a slightly flexed position. Each limb was covered using an

additional sterile drape whenever the opposite limb was operated on.

Arthroscopie implantation of the gnfts was as described in 2.2.4, with the only

difference that subchondral bone bleeding was encountered. Drillhg of the recipient

holes and the gmft implantation were recordedg on audiovisual tapesbb for fuhve

assessments. Grdl implantation technique for each joint was identical. The fust limb to

be operated was chosen randomly. The lateral condyle was ahays operated on first. and

the first operated joint received osteochondral grafts.

The three skin incisions (one over each condyle and the more centrally located

port for the arthroscope) were closed with non-absorbable"" suture material in a simple

interrupted suture pattern after the joint capsule was closed with simple interrupted

sutures using absorbable suture material". A bandage covering the lower limb fiom just

distal to the carpal joints to the fiont claws was used to stabilize and support the limb

during recovery and the irnmediate postoperative phase. It consisted of sterile gauzeff and

a sterile non-adhesive padgY to cover the three incisions in each joint. followed by a

padding layer consisting of sheet cottonhh and a second roll of gauze. The bandage was

secured using two types of adhesive tapeiiY. General anesthesia was teminated and the

calves were placed in stemal recurnbency in a padded recovery stall. The rndotracheal

tube was removed when the caives started to swallow. We assisted the recovery frorn

anesthesia until the animais were standing securely.

M e r recovery nom anesthesia, the calves were transferred into the 3 x 4m

holding stalls and housed in groups of two. Free choice hay and water were offered nhto

hours after the termination of general anesthesia. Al1 calves were closely monitored for

lameness at least three times daily. Vital parameters (body temperature, respiratory rate,

and hem rate) were monitored and recorded once daily. [ntravenous sodium penicillin Crl

(20 .OOOi.u./kg q.i.d daily) and flunixin meglumine' (22rngkg b.i.d daily) were continued

for 48 houn, at which point the intcavenous catheter was removed. Subcutaneous

administration of procaine penicillin G& (20.000i.u./kg b.i.d daily) and intramuscular

Bunixin meglumine' (2.2mgkg s.i.d daily) were initiated d e r removai of the intravenous

catheter and continued for an additional three days. Al1 medication was discontinued

d e r the fifth postoperative day.

The bandages were changed 24 hours after anesthesia and the joints were

evaluated. A similar bandage was applied and changed at 48 hours intervals for a total of

four days. At that point, the fetlock joints were bandaged using gauze" and adhesive

tapeY This type of bandage was used to protect the incisions and the skin sutures, and

was changed every 48 hours until the skin sutures were removed" between days 10 and 14

d e r surgery.

Ten to 16 days after surgery the calves were transferred to an indoor pen

(approximately 12 by 12m) in an animal holding facility. AI1 six calves were housed

together, had fiee access to hay and water, and once daily were fed a standard beef cattle

grain ration. The limbs were not bandaged, and exercise was unrestricted. Daily

inspections were undertaken to ensure a normal attitude and appetite and to detect

lameness .

2.3.3 Collection of Specirnens

The calves were slaughtered 12 weeks postoperatively. A physical examination

and lameness evaluation of the cecipient calves was performed pnor to slaughter at an

abattoir. The caives were slaughtered in a routine fashion (stunning with a captive bolt

gun, followed by exsanguination). During the slaughter process, the fiont limbs were

labeIed and disarticulated at the level of the middie carpal joint, followed by immediate

transport to the laboratory.

Assessment of the experimental sites was started within one hour afler slaughter.

The lower limbs were inspected for the presence of swelling, reduced range of motion.

hair coat, and foot abnormaiities. Dono-palmar and latero-medial radiographs were

obtained from each limb and evaluated by a veterinary radiologist'. Gross examination of

the tissues underlying the healed incisions, the synovial fluid and joint capsule was

performed for each joint. Samples for further testing were not collected. We recorded

the fmdings of the poa mortem examination in a logbook and photographedn" the

articular surfaces of M C , and Pl before dissection was completed.

2.3.4 Identification and Histological Analysis of the Specimens

Evaluation for Treatment 1 (osteochondral g d s , n=12), Treatrnent 2 (bone grafts

n=12), and Treatrnent 3 (unoperated controls n= 16) was similar. In each joint, the distal

end of the metacarpus and the both phalanges were isolated by separating the bones fiorn

the surrounding soit tissues. A meat processing band sa@ was used to cut sarnples into

sagittal slices (see Figure 7). Each slice measured approximately 30 by 40mm with a

thickness between 3 to 8mrn. Three slices originated fiom the articular surface of each

the mediai and lateral condyle. The site with the implanted gnft was identified and

harvested as a slice, with MO similar cuts harvested medial and latetal to this site. One

sagittal section was obtained fiom the joint surface of P,, opposite to the experllnental

site-

A total nurnber of eight sections per limb and 16 per calf were harvested and

processed fkom each of the six experimental animals, giving a total nurnber of 104

"experimental" samples. Sections fiom similar sites and in equal nurnbers were

harvested for each of the four joints serving as normals, giving a total number of 64

"normal" sections. In total, 168 osteochondral specimens were harvested, decdcified.

and processed for histological evaluation.

One hundred and sixty-eight osteochondrai sections required a proper

identification system for processing and for labeling during evaiuation. A simple system

consisting of letters and numbers was found to be suitable. The previously described

protocol for the harvest of the individual bone sections was consistent for each joint. The

calves were nurnbered fiom # 1 through 6 for the experimental mimals, and from #nl

through #n4 For the four unoperated controis. nie second character used for labeling was

a "R" for right leg or "L" for lel? leg, followed by a "C" for condyle or "P" for first

phalanx. " M for mediai" or "Lw for lateral allowed for a proper differentiation.

Furthemore, numbers ranging from 1 to 3 were used to label and identiS> each cut. Cut

#1 was assigned to be the lateral-most cut on the laterai condyle, and the medial-most on

the medial condyle. For either instance, cut # 1 represented the outermost collected

section of a condyle. Cut # 2 represented the central-mon section (between cut # 1 and

#3), which harbored the grafted site. #2 also represented the single section collected fiom

the opposing joint surface on P,. Cut # 3 was the mediai most section on the laterd side.

or the Interal-most aspect on a mediai condyle. With this system, identification of each

specimen or histological slide was guaranteed and easy to maintain and interpret. As an

example, the section #3RCM2 categorizes and identifies this section to be calf number 3.

cight leg (R), condyle (C), medial side (M), cut #2 (which, in this example, represents the

gr& site).

For the statistical evaluation of our redts, we combined al1 joints that had

received osteochondral grafts in one group calied "Treaûnent 1" (n=12). The group

'Treatment 2" included al1 joints with subchondral bone grafts (n=12). The fore limb

fetlocks fiom the four unoperated animais were placed in "Treatment 3" (n=16).

Decalcification was initiated after the completion of the cartilage collection for

paravital staining. The osteochondral sections were fixed in ten per cent buffered

formalin" for 24 hours and then submerged in decalcifying solution' for ten to 26 days.

The solution was exchanged on day five, 12, and 20. The degree of decalcification was

tested with a 20-gauge hypodemic needlecc. "Softness" of the sections was used as an

indicator for the completeness of decaicification on a weekly basis. Decalcified sections

were removed and placed in formalin solution. Individuai bone sections were embedded

in pardEn blocks; microsections were cut on a micr~torne"~ to an average thickness of

5.Opm pignon, Arlot, Meunier and Vignon 19741. The microsections were fixed on

microscopy glass slides and processed for staining with Weigert's Hematoxylin.

Masson's Trichrome, and Safranin-O.

From each grdl site and similar sites in the four unoperated animals three

histological slides were produced and stained. The sections lateral to the gr* were

stained with Weigert's Hematoxylin only, the sections media1 to it with Masson's

Trichrome only. The central section of P, was stained with al1 three stains. The total

nurnber of stains per caif was 12 Weigert's Hematoxylin slides, 12 Masson's Trichrome

slides, and eight Safianin-O slides.

Overail numbers for the ten caives (six experimental and four unoperated

normals) are 120 slides stained with Weigert's Hematoxylin, 120 stained with Masson's

Trichrome, and 80 stained with Safranin-O. The 420 stained histology g l a s slides were

soaed and catalogued for fuaher evaluation.

2.4 Paravital Staining

The osteochondral sections harvested from the experimental animais were held in

sterile saline solutioni until used to produce vibntomePP sections of fiesh aaicular

cartilage. We sectioned the cartilage of the 120 specimens of the six experimental calves

and perfonned the paravital staining within 18 hours after slaughter.

Paravital staining, also called "dye inclusion and exclusion assay'', was used for

the assessrnent of membrane darnage of chondrocytes [Guilbault and Kramer 1964; Bauer

and Vinograd 1970; Nathans and Smith 19751. This test uses ethidium brornideqq (EthBr)

and fluorescein diacetate" (FDA). Staining with FDA and E h B r has the advantage of

direct measurement of intact cartilage cells without harvesting cells from their matrix.

For the FDA stock solution, 40.lrng of FDA were added to lOml of acetone'. FDA

working solution necessitated the adding of 100pl of stock solution to lOml of phosphate

buffered saline (PBS) solution'. The solution was kept in a sterile plastic container and

was protected fiom light. The EthBr stock solution was made by adding 2.5g of

ethylene-diamine-tetra-acetate (EDTA)' to lOOrni of PBS, thus creating a 2.5% EDTA

solution. lOOrng of EthBr were added to lOml of water, and the EthBr solution was

generated by adding l00pl of the EthBr in water to lord of the PBSEDTA solution.

The solution used for the paravital staining required mkhg 100pl of the FDA working

solution and lOOpl EthBr working solution. The solution was kept on ice and protected

fiom light duriog the paravital staining process.

A vibratomp was used to cut tissue microsections from the graft, the surroundhg

area (osteochondral section lateral or medial to the graft site), and the fkst phalanx. The

sections were approximately 80pm thick, and were harvested by cutting From the articulas

surface into the tidemark of the subchondral bone (see Figure 8). Each osteochondral

section was individually gluedU to a polyvinyl base plate for support and ease of

adjustment. The cartilage sections were cut with the vibratome blade moving at a 90°

angle to the joint surface. A # I 1 scalpel b l a d ~ was used to fiee the cartilage sections

fiom the subchondral bone under a stereo microscope'? Between four to six sections

were cut, stained, and evaluated. Three different sites were harvested and stained for

each of the 24 dowels. This included tissue from the articular surface of the implanted

dowel, cartilage fiom the surrounding area and cartilage from the opposing joint surface

(Pl), resulting in a total number of 72 investigated sites. The cartilage samples of each

bone section were placed on a glass microscope slide in a drop of sterile saline solution.

Two to three drops of FDAEthBr working solutions were applied. and the samples were

incubated in the dark for five minutes before examination.

A microscope" coupled to a video imaging system"" and a computer with

imaging software " were used to obtain and store the acquired images for interpretation

and evaluation at a later time.

Color thresholdingw was used to identiQ undamaged, green (FDA-stained) and

damaged, orange (EthBr-stained) chondrocytes. The automated counting facility

included inclusion/exclusion criteria based on ce11 diameter (2 to 20pm) and area (5 to

50pm2). Fragments of stained cells smdler than these dimensionswere not included in

the couot. A rectangular area of interest measuring 4 0 0 ~ by 5 O O p was evaluated at a

125-fold microscopic magillncation. This delayed evduation technique was used

because of the limited viability of chondrocytes and the fading of FDA fluoresence which

prohibit irnmediate evduation of a large number of smples. Data collection (video

imaging and computer genereated ce11 count) included total numbers of cells stained with

FDA and EthBr, total stained cells, and a percentage of undamaged cells. ProcMixed in

SAS" fits a mixed Iinear mode1 and was chosen for this data set. AI1 collected data and

the statistical results were stored in an Excel " file.

2.5 Choudrocyte Counts

Computerized microscopyrhn" generated the semi-automatedw chondrocyte

counts from 80 Weigert's Hematoxylin stained sections' [Arnencan Registry of Pathology

19921. Three sites were evaluated for every glass slide containing the grafted site or a

similar area in the four normal animals (see Figure 8). Site G represented the articular

surface irnrnediately above the gnft, Site S a section of articular surface adjacent to Site

G, and Site P represents a portion of articular surface of the opposing fust phalanx.

Al1 cells in an area of interest measuring 400 by 500pm were counted at a 125-

fold magnification. Color thresholdingw was used to identiQ the chondrocytes stained

purple. The automated counting facility included inciusion/exclusion cnteria based on

ceii diameter (2 to 2 0 p ) and area (5 to 50pm2). Fragments of stained cells smdler than

these dimensions were not included in the count. Counts were expressed as total

numbers. Data were transferred to an Excelw fie, followed by a statistical evaluation in

ProcMixed (SAS)".

2.6 Total Trabecular Area

M i c r o s c ~ p y ' ~ ~ , computer generated bone morphometry" and 80 Safranin-O

stained sectionsi were used to measure the trabecdar area of the grafts, the surrounding

area, and the first phalanx Flint, Lyons, Meaney and Williams 1975; Parfin, Dremer.

Glorieux et al 19871. Four slides were evaluated per limb, resulting in the total of 80

slides for ten calves. Color thresholding"" was used to identify dark pink-stained

subchondral bone. The automated counting facility added the measurements of the

stained subchondral bone areas and expressed it as a percentage of the rectangular area of

interest (measuring 400 by 500pm). i.e. 58%.

Severd areas of interest were defined (see Figure 8). At the g r a site, a

rectangular field (Si te G 1 =Gr&) enveloping the subchondral bone irnmediately under the

articular surface of the grdl site (or a similar site in the unoperated animals) was selected,

followed by two similar rectangular areas deep to Site Gl, called Site G2 and G3. The

three sites combined represented a column-like section of the subchondnl bone at the

gr& site [Johnson, Ewell and Schaeffer 19921. A similar column consisting of three

rectangular fields was evaluated adjacent to the first column (Sites S1 to S3 in the

surrounding area). A third column located at the opposing joint surface of Pl was

evaluated in a similar fashion.

Measurements of the three fields of interest of each evaiuated column were

combined to a mean value (Le. measurements of 45%, 40% and 65% result in a mean

value of 50%). The data was sorted and stored in an Excelu fie. ProcMixed in SAS"

was used for statisticai evaluation.

2.7 Modified Mankin Scoring

We used Safranin-O' and Masson's ric chrome' [Flint Lyons. Meany and

Williams 1975; Rosenberg 19711 stained sections for the scoring. A modified Mankin

scoring system was used ranging from zero (O; no changes), one (1; less than 10 per cent

changes), two (2; up to 25 per cent changes) to three (3; over 25 per cent changes)

[Mankin, Dorfmm, Lippielo and Zarins 19711. We evaluated the gnft sites. the

surrounding area (both originating frorn the distd metacarpus), and the opposing joint

d a c e (first phalam, Pl) in al1 six expenmental calves and four unoperated mimals.

Scoring was performed Wr four different factors: (1) Celluiarity, (2) erosions or

adhesions, (3) matrix staining, and (4) subchondral support. Each factor received one

score for each of the three sites. The scoring was performed independently by two

investigators, Mark B. Hurtig (MH) and Thomas Schiel (TS) using microscopy with

Masson Trichrome and Safranin-O stained histological sections. Seventy-two slides were

evaluated for the experirnental calves, and 48 for the normal, unoperated calves, resulting

in 230 evaiuated slides with 360 different sites. The data were used to create mean values

for each treatment group (osteochondml or bone graft and unoperated animals) by

cornbining the four scored factors (average score). The scores were collected in an

Excel* cornputer file. ProcMixed in SASw was used for the statistical evaluation.

2.8 Statistical Analysis

Our study represented a mked linear model, included fixed and random effects

needed to be evaluated wiliken and Johnson 1994, Noman and Streiner 1994.

Mendenhall l987I. b d o m effects were the experimentai animais? while different

treatments, sites and levels represent the fixed effects. Conventionally, cornparisons

among means are done using the analysis OF variance (ANOVA). Since the experiment

involved repeated measures on the same animal, which leads to correlations among

observations, M O V A is inappropriate. The MIXED procedure in SASm was used for

data anaiysis. The foci of the analyses are the means, variances, and standard errors

observed in each treatment group [Snedecor and Cochran 19891. Data were fitted using

ProcMixed in SAS with the repeated option. ProcMixed can be used for a mixed model

[Littell, Miliken, Stroup and Wolfinger 19961. Details of data anaiysis under the mixed

model have been outlined in textbooks [Littell et ai 19961.

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Department of Physical Resources, University of Guelph, Canada

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Canadian Tire Hardware Store, Canada

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2035 Biocut, Leica, 6707 Nussloch, Germany

Zeiss Junior Microscope with 2Sx, 6.3x, I O X and 40x lenses, Zeiss, Gemiany

Car1 Zeiss Light Source, 6 Volt, 30 Watt, Zeiss, Gerrnany

S. Department of Clinical Studies (Radiology), Ontario Veterinary College, University of Guelph, Canada

E.

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CP-

Stanhexid0ie@ (Chlorhexidine Gluconate Solution 4 %), Novophm, Toronto, Canada

lsopropyi Rubbing Alkohol 70 %, Regal Phmaceuticak, Burlington, Ontario L7N3N4, Canada

Savlon, Pharmacy, Veterinary Teaching Hospital, Ontario Veterinary College, University of Guelph, Canada

TriflexB Gloves, Baxter Corporation, Toronto, Canada

Disposable Surgical Drapes and Gowns, Baxter, Mississauga, Ontario, Canada

Aesculnp AG, 78532 Tuttlingen, Germany

Proviodine Solution (10 % povidone-iodine topicai solution with 1 % Free iodine), Rougier, Chambly, Quebec J3L3H9, Canada

Canadian Council on Animal Care, 1 105- 15 1 Slater Street, Ottawa, Ontario KI P5H3, Canada

14 1 1. 14 Gauge x 5.25 Inches, Mila International Inc., Covington. KY 4 10 1 1. USA

Discofix@, Stopcock with Extension Tube, Burron Medical inc., Bethlehem, PA 18018, USA

DeseretB PRN Adapter, Becton Dickinson Vascular Access, Sandy, UT 84070, USA

Banamine, Schering-Plough, Pointe-Claire, Quebec H9R1B4, Canada

Rompun 100 mglml, Bayer inc., Etobicoke, Ontario M9W1G6, Canada

Diazepam (10 mg per 2 ml vial), Sabex inc., Boucherville, Quebec, Canada

Ketaseî, Bristol-Myers, USA

Tellord, PA 18969, USA

Halothane, MTC Pharmaceuticals, Cambridge, Ontario, Canada

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bb.

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dd.

ee.

ff*

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North Amencan Draeger hc., USA

Kimzey Inc., CA, USA

3M Ioban, 3M Products, St-Paul, MN 55 144, USA

Karl Storz, 175 Cremona Drive, Goleta, CA, USA

Labcor, Anjou, Ouebec HI J2H9, Canada

Sterile Lactated Ringer's Solution, Baxter, Mississauga, Ontario, Canada

Livetec Inc.(Zirnrner), Toronto, Ontario. Canada

6.0 mm Wolfcrafl8 (Length: 139.0 mm, HSS, DM 340), Wolfcraft. 56746 Kempenich, Germany

Synthes inc., USA

VHS Videotape T200, BASF Canada Inc., Toronto, Ontario, Canada

Monoject, Sherwood Medicai, S t.Louis, MO, USA

2-0 Prolene, Ethicon inc., USA

2-0 Monocryl, Ethicon Inc., USA

Surgical Cotton Gauze, Smith and Nephew, St.Laurent, Montreal, Quebec, Canada

Telfa, 7.5 x 20.0 cm, Kendall Healthcare, Mansfield, MA 02048, USA

CDMV, Montreal, Quebec, Canada

3M VetrapTM, 10.0 x 200.0 cm, 3M Animal Care Products, St.Paul, MN 551% USA

Elastoplast, Smith and Nephew, St.Laurent, Montreal, Quebec, Canada

Ethacilin (Penicillin Procaine Suspension), RogadSTB Lnc., London, Canada

Stitch Cutter, Swann Morton Ltd., Sheffield, England

Ident-A-Band@, Hollister Inc., Libeayvüle, IL 60048, USA

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OVC Media, Ontario Veterinary College, University of Guelph, Canada

Slantsaw, Hobart Canada Inc., Don Mills, Ontario, Canada

Vibratome, Cambridge Instruments, Canada

Ethidium Bromide (2,7-Diamino- 10- ethy l-9- pheny 1-phenanthridinum bromide; Homidiurn bromide; approximately 95 %), Sigma Inc., USA

Flourescin Diacetate, Sigma Inc., USA

Krazy Glue, The Borden Company, Willowdale, Ontario M2WV6, Canada

Power HAD, Model DXC-950 (3 CCD Color Video Camera), Sony, Japan

CMA-D2 Camera Adapter, Sony, Japan

Northem Eclipse Software, Canada

Statisticai Analysis Software, SAS@, SAS Institute, Cary, NC, USA

Microsoft EXCEL. Microsofi Corporation (O 1983- 1 996), USA

2.10 References

Amencan Registry of Pathology. A rmed Forces insfifure of Patholugy: Laborntory Methodr in Histotechnofo&v. Washington, D.C., USA, 1992.

Bauer W, Vinograd J. J Mol Bio 1970; 47:419.

Canadian Councii on Animal Care. Guide to the cure and use of experimental animais. Volume 1 (1993) and Volume 2 (1 984), Ottawa, Canada.

Desrochers A, St-Jean G, Cash WC, Hoskinson JJ, DeBowes RM. Characterization of anatomic communications of the fetlock in cattle, using Uitra-articular latex injection and positive-contrast arthrography. Am J Vet Res 1997; 58(7):7 10-7 12.

Dyce KM, Wensing CJG. Essentials of Bovine Anatomy. Lea Br Febiger, Philadelphia, USA, 197 1.

Flint MH, Lyons MF, Meaney MF, Williams E. The Masson staining of collagen. An expianation of an apparent paradox. Histochern J 1975: 7:529-546.

Guilbault GG, Kramer DN. Anal Chem 2 964; 36:409.

Johnson AL, Eure11 JAC, Schaeffer DJ. Evaluation of canine cortical bone gr& remodeling. Vet Surg 1992; 2 1(4):293-298.

Littell RC, Miliken GA, Stroup WW, Wolfinger RD. SAS systemfor mked modds. SAS Institute Inc, Cary, USA, 1996.

Mankin HJ, Dorfman H, Lippielo L, Zarins A. Biochemicai and metabolic abnorrnalities in articular cartilage fiom osteoarthntic human hips. J Bone Jt Surg 197 1: 53-A:523.

Mendenhall W. Introdzîction to probability and statistics. Seventh ediîion. Duxbury Press, Boston, USA, 1987.

McIlwraiih C W. Diagnostic and surgrgrcal arthroscopy in the horse. Lea & Febiger, USA. 1990.

McIlwraith CW in: Nixon M. Equinefiacture repair. W.B. Saunders Company, USA. 1996.

Miliken GAI Johnson DE. Anaiysis of messy data: Designed experiments. Volume 1 . London, Chapman Hill, 1994.

Nathans D, Smith HO. Ann Rev Biochem 1975; 44:273.

Nickel R Schummer A, Seiferle E, Frewein J, Wilkens H, Wille KH. The Lucornutor System of the Domestic iMammnls. Verlag Paul Parey, Berl Wamburg, 1986.

Norman GR, Streiner DL. Biostalistics. The bare essentiais. Mosby, Toronto, Canada, 2994.

Par fh AM, Drener MK, GIorieux FH, Kanis JA, Mailuche H, Meunier PJ, Ott SM, Recker RR. Bone histomorphometry: Standardization of nomenclature, symbols, and units. J Bone Mineral Res 1987; 2(6):595-6 1 0.

Rosenberg L. Chernical bais for the histological use of Safranin O in the study of articular cartilage. J Bone Jt Surg 1971 ; 53-A(1):69-82.

Smailwood JE. An Innoductory Siudy of Bovine Anulorny. J. W. Cain Publisher, Texas, 1973.

Snedecor GW, Cochran WG. Statisticai methoh. Eighth edition. Iowa State University Press, Ames, USA, 1989.

Tabachnik S, Director, DelR Blue Meat Processing, Cambridge, Ontario. Personal Communication, August 1995.

CHAPTER III: RESULTS

3.1 Preliminary Phase 3.1.1 Graft Harvest

Overall, the functionality of the designed or modified instrumentation was

excellent For the harvest of 6.0mrn osteochondral grafts From cadaver joints. Of the ten

osteochondral cylindee that were harvested fiom limbs retrieved from an abattoir and

stored in pairs in sterile vials at 4.0° Celsius for 12 hous, two vials fiailed to show any

bacterial growth, while three vials exhibited a scant growth of Stuphyiococcus

epidennidis. Thus, grafts collected by using our first harvest protocol were unsuitable for

implantation. The 20 osteochondral cylinders collected using the second, improved

technicd protocol for the gr& harvest showed no detectable contamination of the bone

cylinders. Thus, the second protocol that included antibiotics in the lavage solutions was

used to harvest grafts for animal experiments.

Mechanical damage to the articular cartilage, the cartilage-bone interface. or the

subchondrai bone was not observed during the histological examination. The cartilage

and bone interface was intact, the outer perimeter of the grafts showed no signs of

thermal damage, and metal residues were not visible.

3.1.2 Investigation of the Arthroscopie Approach

Arthroscopic approach to the bovine metacarpophalangeai joint using a techaique

descnbed for the equine species proved to be unsatisfactory. Maximum fluid distension

of the joint prïor to instrument insertion resulted in an incomplete elevation of the dorsal

joint capsule fiom the underlying articular surfaces. insertion of the arthroscope directly

into the dorsornedial or dorsolateral aspect of the joint consistently caused signifiant

cartilage damage on the articular surface of the condyles.

Insertion of the arthroscope axially through the skin, joint capsule and fibrous

band between the two condyles prevented the previously observed iatrogenic cartilage

damage (see Figure 4). Visualization of the distal aspect of MC,, and the proximal aspect

of the first phalanges using this arthroscopie approach was adequate for the donal half of

the Fetlock joint. Visualization of the experimental sites (abaxial aspects of the media1

and lateral condyles) could be maintained during the drilling, measuring. and graft

insertion in 22 cadaver Iimbs.

3.2 Experimental Phase

3.3.1 Donor Animal Examination and Graft Harvest

No abnormalities were detected in the pre-slaughter examination of the six donor

animais. Body weights ranged f?om 201 to 232kg. The carcasses of the six chosen

donors passed the official meat inspection. Radiographs obtained of al1 12 limbs were

reviewed by a board certifi~ed veterinary radiologist and found to be fiee of abnomalities.

Visual inspection revealed no apparent darnage of the harvested 24 osteochondral

grafts. The tissue culture medium used For the 12 hours storage of the grafls was

submitted for microbiological testing d e r the gr& implantation. Bacterial growth was

not detected in the 12 satnples.

33.2 Graft Implantation

Physicai examinations were performed 72 hours prior to the experirnental

surgeries. The vital parameters of al1 six animals were within the reference cange for

cattle weighing 200kg. Gait abnormalities were not observed. Palpation of al1 four limbs

revealed no abnormalities in the six recipient animals.

A board certified veterinary radiologist did not detect abnomalities in the 12

metacarpophalangeal joints by reviewing the radiogsiphs of these joints. Physical

examination 12 hours prior to the experimental surgeries found the vital pararneters of al1

six animals to be within the as normal range for cattle weighing 200kg. Venous blood

gas values, serum electrolytes, packed ce11 volume and total serum protein values

obtained one hour pnor to surgery were within the normal range for cattle weighing

200 kg.

No complications were recorded during the induction of general anesthesia

orotracheal intubation, maintenance of anesthesia during surgery, or the postanesthetic

extubation and recovery period. Ai1 six animalç were able to stand between 25 to 15

minutes d e r the terrnination of anesthesia.

The dorso-central arthroscopie approach was used in al1 12 implanted

metacarpophalangeal joints, avoiding damage of the articuiar surfaces. In ail 12 joints.

vinialization of the distal aspect of the fûsed third and fourth metacarpal bones and the

proximal aspect of the frst phalanges was excellent. Visuaiization of the surgery sites

(medial and lateral condyles) could be maintained during the dniiing, measuring, gnA

insertion, and video recording of the implanted grafts. The result of the recipient hole

drilling was excellent in the six calves. The design of the drill bit prevented skidding

across the joint surface. Accuracy of the 24 holes was determined intraoperatively by the

correct location, a clear outline of the hole visible through the arthroscope, accompanied

by undamaged surrounding articular surface and a perpendicular orientation to the

articular surface. These intraoperative observations were at a later date confl rmed by

reviewing the audiovisual tapes. Measuring the depth of the 24 holes with the graduated

depth gauge was easily done under arthroscopie control, and subchondral bone bleeding

was attenuated by insertion of the depth gauge in al1 24 holes. Depth of the drilled holes

ranged from 12 to 16mm.

Gr& implantation necessitated a lOrnrn long, vertical skin incision over each of

the media1 and lateral condyles. After insertion of the gr& passing tool through the

incisions, the limited space in the bovine metacarpophalangeal joint was f o n d to be the

single most limiting factor for alignment over the recipient hole. With the gr& passing

tool aligned with the recipient hole, seating of the graft and ejection fiom the passing tool

was rasily facilitated for al1 24 giifts (see Figure 9). Press fitting of the grafi into the

recipient hole was easily perfomed in 17 of a total of 24 grafts. Only slight pressure was

necessary to eject and seat seven out of 12 osteochondnl and five out of 12 bone grdis.

The gr& surface was found to be congruent with the surrounding articular cartilage. with

no visible recipient subchondral bone. Press fitting was Found to be difficult in seven out

of the 24 gr&. Five of 12 osteochondnl and seven of 12 bone gr& required more

pressure to inîtially seat the graft into the hole. Increased manual pressure was needed to

advance the gr& completely, resulting in crushing of the subchondnl section of the grafi.

This resulted in shortenhg of the gr& length, which led to an incongruent surface (see

Figure 10). A circuiar area (2.0 to 3.0m.m deep) of recipient subchondral bone wail was

cxposed in these cases.

Extravasation of lactated Ringer's Solution fiom the joint into the surrounding

soft tissues during arthroscopie surgery caused edema in the lower Front limbs of al1 6

recipient anirnals. Swelling penisted for 24 to 36 hours d e r surgery. It resolved

cornpletely without any fbrther treatrnent aside from bandaging and anti-inflammatory

medication.

Mild gait abnormalities were observed in al1 recipient animals for 24 hours after

surgezy, consisting of a stiffened gait of the front lirnbs (visible at the walk) and mild pain

on palpation or flexion of the lower front limbs (noticed during the first bandage change).

Pain and stiffness were found to be due to the peri-articular edema surrounding the front

fetlocks and resolved with the swelling over a 36 h o u period.

The metacarpophalangeal joints were ndiographed 24 to 18 hours after the grdl

implantation. Visibility of the grafts varied, only 18 of the 24 surgery sites could be

identified on the radiographs by an outline of the implanted bone cylinder.

During bandage changes, the surgical incisions were inspected every 58 hours for

1 0 to 14 days. No abnormalities were observed. ail 36 incisions healed per primurn. S kin

suture removal was performed approximately 10 days post operatively. At that time.

healing was complete and only minimal scar formation was visible.

No abnormalities were recorded when rectal temperature, heart rate, respiratory

rate, appetite, and ambulation were evaluated on a daily basis for two weeks and weekly

for an additional ten weeks. Al1 six calves had a normal gait from 24 to 36 hours after

wgery until they were slaughtered. A normal growth and weight gain was observed in

the postoperative period.

3 3 Collection of Specimens

Design of the study required termination by slaughtering the calves in the twelAh

postoperative week. The lower limbs were identified and recovered by disarticulation in

the rniddle carpal joint. No health problems were discovered during the slaughtering

process, al1 6 carcasses passed the official meat inspection.

The 12 limbs From the experimental calves had a complete regrowth of the hair.

minimal scar formation in the area of the three stab incisions. no visible or palpable

swelling, and a normal range of motion. Dorso-palmar and latero-medial radiographs

were obtained of al1 12 metacarpophalangeal joints and examined by a board certified

veterinary radiologist. nie experimental sites were visible as an m a of increased bone

density in 14 of the 21 sites. The dorso-palmar ndiographic view proved to b e the most

reliable image to visualize the gr& area. No other abnormalities were found by

reviewing the radiographs of the experimental animals.

Dissection dong the frontal plane of the metacarpophalangeal joint revealed the

absence of adhesions between the skin surrounding the stab incisions and joint capsuie.

The synovial capsules and Buid of the articulation were found to be normal on visual

examination. Al1 24 gr& sites were macroscopically visible. The metacarpophalangeal

joint surfaces were photographed. An error in the processing of the 35 mm diapositive

films caused discolorations of the images, rendering them as black and white images with

poor contrast.

The sarne protocol was used for the collection of the eight metacarpophalangeal

joints of the four animals serving as unopented controls (Treatment 3). Pïe-slaughter

examination confirmed the absence of clinicd abnormalities. Review of the radiographs

by a board certified veterinary radiologist confirmed the absence of abnormalities.

Clinicai exmination of the intact lower Front limbs revealed the absence of lesions, a

complete hair coat, and a normal range of motion. Examination of the joint interior

revealed no abnorrnalities.

3.3.1 Osteochondral Grafts (Treatment 1): Macroscopic Findings

The five osteochondral gatts recessed during the arthroscopie implantation were

covered by a layer of white tissue, that covered part of the surrounding cartilage. These

plaques were firmly attached to the underlying tissues, and their diameter ranged Hom 7.0

to 8.0rnrn. Protrusion from the surrounding articular surface was Iess than 1 .Omm. The

point of highest protrusion in al1 plaques was the cenier, with the edges tapering down

towards the normal articular cartilage. The articular cartilage surrounding these grafls

appeared to be thinned in al1 five gafts, with small areas (diameter between 0.5 and

2.0mm) of subchondral bone visible through the thin hyaline cover. The remaining

articuiar surfaces of the medial and laterd condyles were normal.

The seven osteochondral grafts not recessed during implantation had a clearer

outiine of the round suface. However, in five of these seven gnfts, the interface of

recipient and donor cartilage was hegular, with either a concave or convex appearance

dong one border. Probing of the border revealed F d y attached tissues. Tissue plaques

covering the area of the gr& were absent, and subchondral bone was not visible through

the hyaline cartilage. The remaining cartilage of the medial and lateral condyles was

normal. No macroscopic changes were detected in the articular surfaces of the fint

phalanges, the sesarnoid bones, or the tendons and ligaments surrounding the

metacarpophalangeal joints.

33.2 Bone grafis (Treatment 2): Macroscopic Findings

AI1 12 bone gr& were covered by plaques of tissue similar to the recessed

osteochondral grafts. Diarneter of the plaques ranged From 7.5 to 9.0 mm, protruding less

than 1.5 mm fiom the surrounding articular cartilage. The articular cartilage surrounding

the tissue plaques of the bone gnfts was thin in eight out of the 12 grafts, with small

areas (diameter between 0.5 to 2.0mm) of subchondral bone visible through the thin

hyaline cover. Probing of the plaques and the surrounding cartilage found al1 a res to be

attached to the underlying bone. The remaining articular surfaces of the media1 and

lateral condyles were normal. No macroscopic changes were detected in the articular

surfaces of the fm phalanges, the sesarnoid bones, or the tendons and ligaments

surrounding the metacarpophalangeai joints.

3.33 Histological Analysis of the Specimens

Thickness of the tissue slices varied fiom 4.0 to 1O.Omm. Decalcification of the

cut sections was complete between 14 to 21 days after submersion in the decalcification

solution. Thinner sections were decalcined Taster than the thicker sections. The degree

of decalcification was tested every three days by carefully probing the specimen with a

hypodermic needle. Quality and staining of the histologicai sections was excellent.

At low magnitication, the outline of the donor bone cylinder and the donor

cartilage could be clearly differentiated from the recipient site tissue. This was because

the bony trabeculae were thicker and darker staining in the expenmental site as compared

to the surroundhg bone (see Figure 11). A clef? remained between the donor and

recipient cartilage in al1 cases (see Figure II). M e n a graft was tipped or recessed to

expose subchondral bone in the sidewall of the recipient site, new fibmus tissue

developed and spread across the articular surface (see Figure 10). The Masson's

Trichrome stain outlined the extent of fibroplasia.

The allogeneic donor bone was almost completely replaced by host bone (see

Figure 13). Where remnants of donor bone persisted. they could be identified by

morphous, glas-like trabeculae with no apparent osteocytes. These remnants were

encased in a thick layer of new woven bone. There was some cellular reaction composed

mainly of lymphocytes dong the original hostldonor bone interface but seldom extending

into the deeper areas of the graft. In four of 12 osteochondral gr& and ten of 12 bone

grafts there was marrow phthisis (see Figure 13) evidenced by invasion of new

connective tissue in-between trabeculae. There were no abnormalities evident in the

subchondral and trabecular bone of femoral condyles distant fiom the esperimentd site.

nor were there any such changes in the Fust phalaux.

The £ive osteochondral grah that were not congruent with the articu1a.r surface

were covered with new fibrous tissue. Cartilage covered by this tissue was less cellular

and stained poorly with SaFranin-O. Seven of 12 grafts were congruent and appeared to

have normal chondrocytes and Safiah-O staining. Cartilage on the opposing surface of

the first phalaw appeared normal. Al1 12 bone gmfts were covered with fibrous plaques

(see Figure 14). In ten of 12 cases, the surrounding articular surface of MC,, was thin.

less cellular than normal, and stained poorly with Safkanin-O. There was focal loss of

subchondral bone and replacement with fibrous tissue under the thin cartilage.

3.4 Paravital Staining

Results of the paravital staining performed on fresh microsections of articular

tissues are listed in Tables A-1 through A-6 in the appendix. The statistical analysis is

summarized in Tables 1,2 and 3. Figure 15 in the appendix shows the results as a graph.

Evaluated anatomic sites were the graft surface itself (Site G; n=24), the surrounding

area of the graft (Site S; n=24), and the opposing articular surface of the first phalanges

(Site P; n=24). Treatment groups were split in Treatment 1 (Tl; sites implanted with

osteochondral grafts; n=13) and Treatment 2 (T2; sites implanted with bone grafts;

n= i 2).

Statistical analysis showed significant differences between anatomic sites (p=

0.0001) and between treatments (p= 0.04). Sites were also found to be ~ i ~ c a n t l y

different within the two treatment groups (p= 0.004).

Tabie 1 shows the mean percentages of undamaged ceiis ofeach anatomic site (G,

P and S) derived fiom paravital staining. While between 8 1 and 90 per cent of intact cells

were identined in Sites S and P of both treatrnent groups, only 69 per cent (osteochondrai

grafts; Treatment 1) and 50 per cent (bone gcafts; Treatment 2) were found at Site G.

Table 2 depicts the cornparison of anatomic sites G, S and P within the two treatment

groups (osteochondrd and bone grafts). Statistically significant differences were

observed between Site G and S in both treatment groups. No significant difference was

found in the comp~son of Site P and S. As shown in Table 3, comparing Treatment 1

(osteochondral grafts) to Treatrnent 2 (bone grafis) resulted in a statistically significant

difference at Site G (gr& site) only.

Results-Tables 1,2 and 3

1 1 Site G 1 Sites 1 Site P 1

Table 1 . Paravitai staining data showing the mean percentage and standard error of undamaged/total ceils.

Treatrnent 1 Treatment 2

1 1 Treatment 1 1 Treatment 2 1

69% 4 5% 50% I 5%

1 Site S x Site P I

1 .80 1 .79 1 Table 2. Cornparisons of anatornic sites G (graft), S (surrounding) and P (phalanx). Analysis of variance

90% i 3% 81%k5%

J

.O00 1 *

.O00 1 * Site G x Site S Site G x Site P ,

for the proportion of damagedf undamaged cells were considered significantly different when p<.05*.

88% + 2% 90% k 2%

,004' .002*

1 I Site G 1 Site S I Site P I 1 Treatment 1 x 2

1 1 1

1 .003* 1 .17 1 .85 1 - --- - -

Table 3. Cornparison of Treatment 1 (osteochondral grafts) with Treatment 2 (bone -MAS) with respect to

the grafl (G), area immediately surrounding the gr& (S) and the opposing articular surface (P). Analyses

of variance were considered significantly different when p<.OS*.

3.5 Chondrocyte Numbers

In the appendix, results of the chondrocyte counts are listed in Tables A-7 through

A-12 (Treatment 1 and 2) and Tables A 4 3 through 16 (Treatment 3). Figure 16 in the

appendix shows the results in form of a graph, and the statistical analysis is summarized

in Tables 4, 5, 6 and 7. The evaluated sites were the graft surface itself (Site G), the

surrounding area of the graft (Site S), and the opposing articula surface of the first

phalanges (Site P). Treatment groups consisted of Treatment 1 (Tl; osteochondral

grafi), Treatment 2 (T2; bone graft), and Treatment 3 (T3; uooperahd animals).

Statistical analysis showed significant differences between sites (p= 0.0001) and

between treatments (p= 0.001). Sites were also found to be significmtly different within

the two treatment groups @= 0.000 1). Table 4 shows the mean number of chondrocytes

counted at the three anatomic sites of interest. Table 5 transforms the mean chondrocyte

numbers fiom Tl and T2 into percentages. Site G (&raft site) has 39.7 per cent of the

intact chondrocytes counted when Treatment 1 (osteochondral gr&) is compared to

Treatment 3 (unoperated normals) and 1 1.5 per cent when Treatment 2 (bone grafi) is

compared to Treatment 3.

As s h o w in Table 6, cornparisons of ail sites in Treatment 2 and of Site G with P

and P with S in Treatment 1 and 2 were significant. Only the comparison of Site G with

S in Treatment 1 (osteochondral grafts) and in Treatment 3 (unoperated normals) had no

statisticaiiy significant ciifference.

Table 7 sumrnarizes the comparison of the three treatment groups. A statistically

sigaificant ciifference was not observed for Site P (first phalam) in either treatment group

cornparison and for Site S (surrounding area) when Treatment 1 was compared to

Treatment 3. Al1 other cornparisons have statistically significant differences.

Results-Tables 4 and 5

1 Site G 1 Site S 1 Site P 1 1 Treatment 1

1 I F

1 73.5 + 8.3 1 171.0 f 5.8 1 176.9 + 1.9 1 1 Treatment 2

1 1 1

1 21.4 k 2.1 1 126.5 t 9.7 1 181.0f 2.9 1 1 Treatment 3 1 185.0 f 1.8 1 185.5 f 1.6 1 172.9 f 2.1 1

Table 4. Mean numbers (with standard errors) of Weigart's haematoxylin-staining chondrocytes counted

in the graft (G), area immediately surrounding the grafl (S) and the opposing joint surface (P).

Table 5. The first two rows of this table show the percentage of chondrocytes compared to unopemted

controls in group 3. The last row compares the nurnber of chondrocytes in the osteochondral gnfts

Treatment 1 x 3 Treatment 2 x 3

(Treatment 1 ) to the Cornparison of percentages (intact chondrocytes).

Site G 39.7 % 11.5 %

Site S 92.1 % 68.1 %

Site P 102.3 % 104.6 %


I Site G x P 1 1 1

1.0001* 1 .0001* 1 .96 1 t Site P x s

1 1 1

1 .44 1 .O001 * 1 .87 1 Site G x S

-- --

Table 6. This table shows cornparisons of chondrocyte numbers in the grafi (G) versus the surrounding

area (S), the gnft versus the opposing joint surface (P), and the opposing joint surface (P) versus the

surrounding area (S) in the three treatment groups. Analyses of variance for the proportion of intact

chondrocytes were considered significantly different when p<.OS*.

Treatment 2 ,000 1 *

Treatment 1 ,0001 *

Treatment 3 .95

Table 7. The first and second row show a cornparison of chondrocytes counted in each anatornic site in the

joints that received osteochondnl grafts (Treament i ) and bone ph (Treatment 2) as cornpared to

Site P .10 .4 1 -10

unoperated controls in group 3. The last row compares chondrocyte numbers behveen osteochondral and

Site S .13 .O00 1 * .O00 1 *

Treatment 1 x 3 Treatment 3 x 3 Treatment 1 x 2

bone grafts. Analyses of variance were considered significantly different when p<.OS*.

Site G .O00 1 * ,000 1 * .O00 1 *

3.6 Total Trabecular Area

Results of the trabecular area measurements are listed in Tables A-20 through A-

25 (Treatment 1 and 2 ) and Tables A-26 through A-29 (Treatment 3) of the appendix.

Figure 17 in the appendix shows the results in form of a graph. Three rectangular areas

(one adjacent to the articular surface with the second and third deep to it, each rneasuring

400 by 500 pm) were evaluated at the graft site (Site G), the sunounding area of the graft

(Site S), and the opposing articular surface of the first phalanges (Site P). Treatment

groups consisted of Treatment 1 (Tl; osteochondral graft), Treatment 2 (T2; bone

graft), and Treatment 3 (T3; unoperated animals).

For the statistical analysis we combined the three fields of interest (Table A-17,

A-1 8, and A- 19 in the appendix) of each anatomic site to a column measuring 1200 by

500 Pm. A significant difference in trabecular area was noted when comparing Sites G. S

and P @=0.007) and these sites within treatment groups (p=0.02). The difference

between treatment groups alone was not significant @=O. 16).

Table 8 shows the results of the combined levels. Table 9 sumrnarizes the

cornparison of the combined levels of Sites G (osteochondral grafts) with Sites S (gmft

surrounding area) and Sites P (first phalanx) for the three treatment groups. In

unoperated controls, the trabecular area did not Vary signincantly among the various sites

including the area correspondhg to that grafted, the area surrounding the grdl site, and

the opposing joint d a c e . In osteochondral grafts, the total trabecular area in the grdl

was significantly different fiom the smounding and opposing sites. ui bone gr&, the

gr& was signficantiy different fkom the surrounding site (S) and there was a trend

toward a difference in the phalangeal site (P).

Table 10 demonstrates the diflerences observed within the three treatrnent groups

by site. The trabecular area under both types of grafts was different compared to

unoperated controls, and more so in bone grafts. The surrounding sites were not different

than unoperated controls, though the bone in the phalanges was significantly different.

When considering the two types of grafts, there were no differences in tnbecular area

between any site.

Results-Tables 8,9, and 10

I 1 Site G 1 Site S 1 Site P I 1 Treatment 1

I I I

144.5 % t3.1 % 1 38.1 % t 1.8 % 1 39.3 % f 1.4 % 1 1 Treatment 2

I 1 1

1 45.1 % + 2.6 % 1 36.6 % f 2.3 % 1 40.9 % + 2.0 % 1 1 1 1

Treatment 3 1 35.6 % f 2.6 % 1 37.0 % f 2.2 % 1 33.9 % t 1.8 % 1 Table 8. Mean trabecular area of combined Levei 1, 2 and 3 with standard errors (percentage of bone in

the three fields of interest for each anatomic site).

Table 9. Cornparison of mean trabecular areas in the pfi (G) versus surrounding area (S) in

osteochondral grah (Treatrnent l), bone gnfts (Treatrnent 2) and unoperated controls (Treatrnent 3). The

trabecular area in the graft versus the opposing joint surface (P) and the phalangeal site (P) as compared to

the surrounding site (S) are also included. Analyses of variance for the trabecular area were considered

significantly different when p<.05*.

t

Site G x S

Table 10. The fmt two rows show comparisons of trabecular area in osteochondral grah (Treatrnent 1)

Treatment 1 .O 1 *

Treatment 1 x 3 Treatment 2 x 3 Treatmcnt 1 x 2

and bone grafts (Treatment 2) with unoperated nomals (Treatment 3). The iast row shows comparisons

that indicate there was no difference in trabecular area between osteochondral and bone _mft groups.

Treatment 2 .005*

Site G x P Site P x S

Site G .O 1 * .006* -86

Analysis of variance were considered significantly different when p<.05*,

Treatment 3 .63

.14

.14 .O3 * .6 1

S2 2 7

Site S .65 .90 .60

Site P .009* .O0 1 * -47

3.7 Mankin Scoring System

A rnodified Mankin scoring system was used to score cellularity, erosions, rnatrix

staining, and subchondral bone support. The gr& site (Site G), the surrounding area of

the graft (Site S), and the opposing articular surface of the first phalanges (Site P)

received individual scores. Treatment groups consisted of Treatment 1 (Tl;

osteochondral graft), Treatment 2 (T2; bone graft). and Treatment 3 (T3;

unoperated animals). Tables A-30 through A-35 of the appendix show the results of

Treatment 1 and 2. Table A-36 shows the scores for Treatrnent 3. A graph (Figure 18 in

the appendix) shows the mean scores in form of a graph. Scoring was perfoned

independently by two investigators assigning a score nom O to 3 to each site.

Table 1 1 shows the average scores. Al1 sites in Treatrnent 3 (unoperated normais)

received a "O" score, as did Sites P in Treatment 1 and 2 (no changes). Site G and S

(gr& and surrounding area) in Treatment 1 and 2 (osteochondral graft and bone gnft)

received higher scores, indicating more abnormalities.

Tables 12, 13 and 14 list the results of the scoring for each scored factor and the

statistical ~ i ~ c a n c e when the treatment groups are compared to each other. The

scoring for "Cellularity" is listed in Table 12 and shows a statistically significant

difference for Site G and S when the three treatment groups are compared. Table 13

summarizes the statistical andysis for the scoring for "Adhesions and Erosions". Both

types of grafts had significantly more erosions and adhesions compared to unoperated

controls, though this effect did not extend to the phalanx in osteochondral gr&. Ln bone

gr&, the scores for the op posing surface ap proached statistical difference with

unoperated controls @<0.06). When the two types of grafts were compared to each other,

the graft and surrounding sites were not different with respect to this type of damage,

though the opposing joint d a c e was. As seen in Table 14, the scoring for "rnatrix

staining" shows significant differences for both types of @s and their surrounding

areas when compared to unoperated controls. Osteochondnl grafts had better matrix

staining than repair tissue in the bone graft sites.

Table 15 summarizes the scoring for "Subchondral Support". It shows

statistically significant differences for both types of bone grafts when cornpared to

unoperated controls, but no effects on the surrounding or opposing sites.

Results-Tables 11 to 13

1 1 SiteG 1 Site S 1 Site P 1

1 Treatment 3 1 I 1

l O l O I o 1 Treatment I Treatment 2

TabIe I 1. Average combined Modified Mankin scores for the graft (G), aresi surrounding the gnft (S) and

phalangeai (P) sites in joints with osteochondnl gnfk (Treatment 1), bone grah (Treatment 7) and

1.5 2.4

unoperated controls (Treatment 3). Zero (O) is optimal, higher numbers indicate damage and degeneration.

0.4 1 .O

Table 12. Cornparisons for "Cellularity" scores when cartilage from osteochondnl grah and bone grah

were compared to operated controls of (Treatment 3). CeiluIarity in the grai? site and surrounding m a

was different when osteochondral gratis were compared CO bone grah, but not when the opposing joint

surfaces were considered. Analyses of variance were considered significantly different when p<.05*.

I

O O

Treatment 1 x 3 Treatment 2 x 3 Treatment 1 x 2

I I Site G I Site S 1 Site P I

Site G .O0 1 * .O0 1 * ,003 *

Table 13. Scoring for "Erosions and Adhesions". Both types of grah had more erosions and adhesions

Treatment 1 x 3 Treatment 2 x 3 Treatxnent 1 x 2

than unoperated controls of Treatment 3 joints. Joints with bone grafts had some effect on the opposing

Site S .05* .0007* .04*

joint surface that approached significance. The effect is evident when the opposing surface (site P) are

Site P .3 1 2 4 -68

,000 1 * .0001* .12

compared between the two types of g d l s . Analyses of variance were considered significzuttly different

when p<.05*.

.O1 *

.0004*

.1S

i 1 .O .O6 .O3 *


-- - -

Table 14. Scoring for matrix staining scores. Both types of grah had significantly less Safranin-O

staining than unoperated controls in the gmft site and surrounding ma. The opposing joint surface in Site

P was unaffected. Analyses of variance were considered significantty different when p<.OS*.

Site P .32 .14 .58

Treatment 1 x 3 Tresltment 2 x 3 Treatment 1 x 2

- - - - -- - - -

Table 15. "Subchondnl Support" scores for osteochondnl grah and bone grah were significantly

different when unopented controls in Treatrnent 3, though the surrounding and opposing sites were

unaffected. Analyses of variance were considered significantly difierent when p.Os*.

Treatment 1 x 3 Treatment 2 x 3

Site G .O001 * ,000 1 * .0002*

Site S .006* ,000 1 * -10

Site P .3 1 1 ,O

Site G Site S .O00 1 * i .78 .O001 * 1 .H

CHAPTER IV: Discussion

4.1 Osteochondral Grafting

While a single universally-successful method for joint resurfacing has not been

found, a century of surgical and clinical research has given us several options to choose

from [Tomford, Springfield and Mankin 1992; Lexer 1908; Jansson 1996; Brittberg,

Lindahl, Nilsson et al 1994; McIlwraith 1997; Minas and Nehrer 19971. Osteochondral

grafting was the fist technique for joint resurfacing descnbed in the medical iiterature

[Goldberg and Heiple 1985: Lexer 1908; Roffman and du Toit 19851. This was due to

three factors. First, chondrocyte cultures and artificiai joint surfaces were unknown at

that time. Second, osteochondral allografts were readily available fiom cadavers,

allowing the surgeon to match donor sites with the defective area. Third, replacing

defective tissue with tissue having identical properties has always been one of the main

principles of transplantation surgery [Aichroth, Burwell, Elves et d 197 11. While hyaline

cartilage is an extremely durable tissue, its capacity for repair and regeneration is limited

[Woo and Buckwalter 1988; Mankin 19821. It is logical to attempt to replace damaged

cartilage with an osteochondral gr&, rather than hope for regeneration fkom more

primitive, less organized and less ideal tissues, tissue substitutes or cells. Compared to

state of the art techniques such as chondrocyte transfer [Brittberg et al 1994; Sams.

Minor, Wotton, Mohammed, and Nixon 1995, S a m and Nixon 1995; Stuart 19941, an

osteochondml gr& delivers a complete repair surface (hyaline cartilage) with ideal

attachent to an easily-transplaated, biocompatible base (subchondral bone). If bone

incorporation and cartilage survival of the graft are optimized, osteochondrd graf'ting

results in a functional joint d a c e .

Transplantation of large joint segments to address neoplasia and ankylosis has

been popular since the tum of the century [Buchmann 1908; Lexer 19081, but single

smaller (8-20 mm diameter) osteochondnl grah have been used successfully for

osteochondntis lesions in people [Garrett 1986 and 1994; Meyes, Akeson, Convery

1989; Meyea 19851. Using multiple miniature (2.5-10 mm) grafts implanted by

arthroscopy avoids the morbidity of an arthrotomy, and has the additionai advantage of

more accurate approximation of joint contoun [Bobic 1996; Matsusue, Yarnamuro and

Hama 19931. Instead of attempting to match the donor and recipient joint size and shape

exactiy, many srnall grafts can be used in a "rnosaicplasty" [Hangody, Karpati and Sulyos

19961 to recreate the three dimensional contoun of the joint. This technique is currently

used to fil1 chondral defects in the hurnan knee and d u s as well as in experimental

animal models [Bobic 1996; Hangody, Szigeti, Karpati and Sukosd 19961. Follow-up

examinations of these patients have reported favorable outcornes. but no short- or long-

terni studies have investigated the survival and incorporation of miniature osteoc hondral

grafts. Our study was designed to investigate the short-terrn fate of a small osteochondnl

allograft. implanted arthroscopically into the bovine metacarpophalangeal (fetlock) joint.

4.2 Graft Harvest

The fmt aim of our study was to investigate the harvest of small osteochondnl

orthotopic allografts. Harvest of large osteochondrai tissues fiom cadavers has been

described previously [Garrett 1986; Buchmann 1908; Kandel et al 19851. Harvesting

s m d cylindrical grafts requires more instrumentation and tissue handlhg that might

predispose the grafts to bacterial contamination and damage. Whiie our research protocol

did not have to consider transfer of contagious diseases, this is a real concem in hurnan

surgery. By using relatively simple instrumentation and aseptic techniques we were able

to harvest sterile osteochondral grafts. In Our opinion, this technique is suitable to be

applied for future scientific studies or for the clinicai harvest of miniature allografts.

Further improvements are mggested for the clinical use including graft collection in a

special facility (orthopedic surgery suite) or the use of a larninar flow hood during the

harvest. Our harvested gr& were grossly intact, and our short term data supports the

conclusion that mechanical and thermal damage were minimized. The currently mosaic

arthroplasty instrumentation uses hand chisels instead of powered equipment to harvest

cylindrical grah. In our opinion, the former is a less technical and safer method. For

large animais, particularly horses, this technique needs to undergo M e r testing. In our

clinical experience, equine subchondrd bone is extremely dense and hard compared to

other animais. This may cause problems in the manual harvest of gr&. Hwesting

autografts using our method (drill press with coring bit) is impnctical if not impossible.

4.3 Graft Storage

Prior to implantation, the grafts used in our experiment were sirnply stored at 4"

Celsius for 12 hours in sterile vials containhg tissue culture medium. No other measures

were taken to support or enhance graft viability, yet histology indicated Little or no loss of

cells or matrix. Recent studies have shown that senun-free or senun-supplemented

medium c m be used to store cartilage, and nutrient media helps maintain a steady-state

glycosarninoglycan synthesis [Kawcak. Trotter, Fnsbie and McIlwraith 19961 during

storage. Using media and refngeration, cartilage has been reported to remain viable for

several days. Storing osteochondraI tissues at 4* Celsius for several days has been

reported to have Little effect on the viability of cartilage [Tomford and Mankin 19831.

Storage at -70" Celsius has been investigated and applied in clinicai transplantation,

demonsirathg the survivability of articuiar cartilage fiozen in various nutrient media

[Schachar, Cucheran and Frank 1988; Schachar, McAllister, Stevenson et al 1992;

Schachar and McGann 19861. Cartilage viability in osteochondral allografts after short-

term storage at 4' Celsius is not diflerent fiom that in unrefrigerated grafts, but superior

to cartilage €rom fiozen allografts maiinin, Mnaymneh, Lo and Hinkle 1994; Rodrigo.

Thornpson and Travis 19871. Knowledge gained in the storage of osteochondral tissues

should be reviewed with regards to small g a f t s , but will most likely not be different.

Future clinical use of osteochondral allografts in veterinary surgery could rely on the use

of cadaveric gmAs since donors are easily obtainable. Though we identified some

continuing ce11 reaction in the subchondral bone that was rnost likely immunologically-

based, this did not interfere with incorporation of the allogeneic bone. In fact, we were

surprised by the completeness of allograft bone resorption and remodeling three months

postoperatively. It was dificult to fmd segments of necrotic. acellular bone by this tirne.

We feel that &esh allografts could be valuable for mosaic arthroplasty in order to avoid

the donor site morbidity of autogrofts.

A4 Graft Implantation

The second part of our research investigated the technicd feasibility of

arthroscopie graft transfer using a simple, custom-made instrumentation. Surgical

implantation of the grafts used in our study was associated with minimal trauma. The

only observed postoperative complication was periarticular edema due to extravasation of

irrîgating solution used for joint distension during arthroscopy. While Uivestigating the

anatomy of the bovine fetlock in our preliminary studies, we estimated that a 40mm long

arthrotomy over each the medial and lateral was needed to expose the site chosen for

gr& implantation. We assurned that an experiment similar to ours performed using an

arthrotomy would have caused more postoperative pain and larneness. and would have

made wound dehiscence a serious concem. Many of our technical difficulties were due

to the anatomy and narrow profile of the metacarpophalangeal joint of a 200kg ox. The

dorsal aspect of this articulation is smaller in the bovine species when compared to

horses, partially due to the separation into hvo equd halves [Desrochers, St-Jean, Cash et

al 1997; Nickel, Schummer and Seiferle 19861. This prevents complete elevation of the

dorsal joint capsule during maximum fluid distention. (In the horse, the dorsal aspect of

the joint capsule cm be completely elevated from the underlying bones during

arthroscopy). The intermediate ridges of MC3-( are very prominent in the bovine species

p y c e L9711, and, in the fully distended joint, remain in close contact to the joint capsule.

Insertion of the arthroscope sleeve into a joint requires a varying degree of manual

pressure in direction of the underlying bones, and therefore endangen articular tissues

Iocated immediately under the joint capsule. Insertion of the instrument more parailel to

the articular surface mostly resulted in an advancement of the sleeve into the thick

subcutaneous soft tissue layer, without penetration of the joint capsule. Ou. dorso-central

approach to the bovine fetfock joint through the fibrous band separating the medial and

lateral condyle allowed us to avoid cartilage damage. As compared with the equine

technique, the joint capsule is not penetrated fiom the dorsal aspect in a paLmar direction.

but in a more medial or lateral direction fiom a point deep to the dorsal surface of the

joint capsule. We concluded that this arthroscopie technique codd be used for managing

sepsis and osteochondral fiactures in the bovine.

Our instrumentation was crude compared to the more recently developed and

marketed instrumentation for mosaic orthroplasty. Such instments would irnprove the

accuracy and reliability of delivery of a small osteochondral grafts, and these are

important issues. We have clearly shown that less than ideal delivery of the gmA rnay

result in small incongruities and exposure of subchondral bone in the sidewall of the

recipient hole. The fibrous tissue proliferation that ensues is extremely deleterious to the

articular cartilage of the gr&. Loss of Safranin-O staining indicates proteoglycan Ioss.

This might arise tiom proteolytic enzymes in the fibrous tissue or interference with the

bi-directional flow of solutes during loading that is necessary for articular cartilage

nutrition [Buckwalter and Mankin 1997; Buckwalter 19941.

In cadaveric joints we had no problem inserting the gr&s into the recipient holes.

In seven of the 24 grafts implanted in our experirnental calves increased manual pressure

was needed to advance the gr& into the recipient hole. In retrospect, we feel that this

compressed the subchondral trabeculae, resulting in shorter, recessed grafts. It is possible

that having two surgeons allowed us to more accurately drill the recipient holes without

drill bit wobble resulting in a srnall. more tightly-fitting hole. Given that our recipient

hole and grafts were the sarne diameter (6.0mm) and that the gnfts may swell in a fluid

medium, a graft O. lrnm smaller than the recipient hole would have been more ideal. This

is based on our experience with the depth measuring gauge. This instrument was

fabricated fiom a 5.9mm stainless steel rod, We chose the smaller diameter in order to

prevent damage to the sidewalls of the 6.0mm recipient hole. The rod was easy to insert.

but supplied sorne resistance during the withdrawai due to bone-instrument contact. "Fit"

of the measuring rod was good, indicated by the cessation of subchondral bone bleeding.

We believe that a slightly smaller gr& would be harder to cmsh and still provide

sufficient press-fit friction for stability. A second solution is currently used in human

nugery with miniature grah. The subchondral bone of the hnrvested grafi is compressed

with special forceps. Compression transforms the osteochondral cylinder into a conical

shape, sparing the bone close to the cartilage while being tapered towards the end of the

gr&. Reports indicate good fitting of grafts prepared in this fashion [Hangody et al

19961. We could see three possible disadvantages of this rnethod when compared to the

proposed method of overdnlling the recipient hole. First is the need for additional

instrumentation (compression forceps), followed by an added step in the gr&

implantation protocol leading to more handling and possible contamination. Third, a

disruption of the cartilage-bone interface caused by compression of the subchondral bone

is possible and needs to be ruled out.

Osteochondral gr& cm be secured using a variety of techniques. The use of

interna1 fixation in fom of bone plates and screws [Beaver, Mahorned. Backstein et al

19921, or screws alone Flynn, Springfiield, and Mankin 19941 has been described for

large grah, while tissue cernent or glue [Aron and Gorse 199 11 and pins [Hurtig 19881

have been used for smaller @S. Fixation by press fitting alone has been described in

the literature [Dew and Martin 1992; Garrett 1986; Bobic 19961, and was chosen for our

study. Press fitthg is ideal because it avoids foreign bodies (implants) or materials (glue)

[Hangody et al 1996; Garrett 19861. Delivery of tissue adhesives may also be

complicated in a fluid distended joint during arthroscopy. In our study none of the 24

gr& became dislodged in the postoperative period and cornparison of intra-opentive

videotapes and post-mortem results ruled out the gr& sinking over time. We concluded

that accurate press fitting of a similar graft in a non weight-bearing part of the joint would

also be possible. WhiIe rigid fixation is mandatory for its mechanical stability and

further incorporation by host tissues [Kandel, Gross, Ganel et al 19851, we are relatively

codident in press-fit fixation of small osteochondral cylinders.

4.5 Grafi Histology

We used bone grafls as a worst-case scenario in this experiment instead of leaving

an unfilled recipient hole as a control. This was to ensure that the 6.0mr-n defect we were

resurfacing was clinically significant, and greater than the "critical size defect" that might

heal spontaneously in immature bovines. Our results fiom Mankin scorîng, chondrocyte

counting and paravital staining show that osteochondnl graf?s were much better than

bone grafts. There is a tenfold difference in chondrocyte numbers between bone g & s

and unoperated controls. In this short term experiment, grdi cartilage survived but there

was a 50 per cent decline in chondrocyte numbers compared to unoperated sites. This

loss of cells did not extend to the surroundhg or opposing joint, implying that the

resurfaced site was somewhat functiond. This was not true for bone grafls, which caused

more widespread degenerative changes.

This experiment can not pinpoint whether the decline in cartilage quality in our

osteochondral gafts was due to harvesting, subsequent handling and implantation

technique or postoperative phenornena. We feel that harvesting and storage are not

factors, and although histology is not redly mfXcient evidence for chondrocyte survival.

our techniques are consistent with other successful reports.

Damaged chondrocytes and matrix which dtimately lead to gr& failure have

been reported for osteochondral transplantation as weli as other joint resurfacing

techniques [Kandel et ai 19851. Our analysis used differences observed within treatment

groups and did not consider the differences in the accuracy of the gr& delivery within a

treatment group. We believe that implantation of recessed grafts had a major impact on

the outcome of this expenment. Osteochondral g&s compressed dunng implantation

resulted in exposure of ceci pient subchondral bone, allowing pro liferation of fibrous

tissues that attempted to fil1 the deficit but sometimes covered the articular cartilage of

the entire gr&. We concluded that accurate graft delivery is essential in mosûic

arthroplasty, and as in most synovial joints, the curved surface of the bovine

metacarpophalangeal joint presents a challenge. Poor fitting and instability lead to

serious consequences such as erosive pannus and resorption of subchondral bone [Hurtig

1988; Garrett 19861, but we still think that a good match of donor and recipient surfaces

is easier to achieve with severai small [Bobic 1996; Garrett 19861 rather than one large

gr&. From our bone gr& data it can be concluded that a single poor gr&, or possibly

large gaps between grafts, might have a negative influence on correctly implanted

adjacent gr&, starting a cascade of degeneration, and causing fàilure of the complete

rnosaic arthroplasty .

Articular repair tissues initially consist of a variety of poorly differeotiated cells.

This is in contrast to normal hyaline cartilage where chondrocytes are the oniy one cell

type. Some of the cell types found in fibrous repair tissue (Le. fibcoblasts) are very

similar in size and shape when compared to healthy chondrocytes and are difficult to

differentiate on a morphologicd basis, wÎth or without the aid of computerized

equipment. Despite the fact that we counted a mea. number of 20 cells per visual field of

interest that we interpreted as chondrocytes, in retrospect, the three-month old repair

tissue found in bone grafts probably contained very few chondmcytes. Therefore, we

must assume that the cornputer generated ce11 count used in our study overestimated the

number of chondrocytes in sarnples where repair tissue predominated. To avoid this

error, irnmunohistochemical techniques could be used in future studies to label

chondrocyte-associated antigens.

We had some concems regarding the ability of srnall grafis to maintain adequate

support to the articular surface during the incorporation and remodelling phase. Invasion

of the donor bone and subsequent replacement by host bone has been described for

osteochondral grafts [Kandel et al 19851. Some clinicians consider loss of support to be

the number one Factor responsible for gr& failure [Garrett 1986; Stuart 1994; Tomford et

al 19921. Revascularization and replacement of donor bone is often too slow in large

grdis, leading to a weakened bony scaffold supporting the hyaline cartilage [Brooks

19941. Without proper support fiom its subchondral bone, the repaired articular surface

is unable to withstaud forces experienced during motion and weight bearing and collapse

of the hyaline cartilage or the entire graft can result Flynn et al 19941. We evaluated

~mbecular bone support quditatively by modifjhg the Mankin sconng system and

directly measuring trabecular area. We cm only assume that the small size of the grafts

allowed speedy apposition of host bone on donor trabeculae, followed by orderly

remodelling. Assessrnent of the trabecular area shows no significant ciifferences between

osteochondral and bone grafts. Cornparhg these treatment groups to unoperated

specimens, an increase of 25 per cent is observed in the experimental sites. Increased

trabecular area indicates bone production beyond a simple replacement of donor bone as

a response to the implanted graf?. This increase may be because the three months

postoperative period is suficient time for apposition but remodelling has not yet been

completed, leaving the trabeculae relatively thick due to the combination of allogeneic

donor bone and new host bone. An altemate theory would be that the immune response

to allogeneic bone slowed the initial resorption [Stevenson, Qing Li and Martin 19911

and prevented an abrupt wave of remodeling. The infIamrnatory response could, in some

way, stimulate continued bone production, as could altered biomechanid loading of the

site, but these two factors would be more likely to cause bone loss rather than

accumulation. We concluded that subchondral bone support of the hyaline cartilage was

adequate in our osteochondral g&s at the termination point of Our study. A loss of

mbchondrai support resulting in gr& fnilure can occur at a later point [Garrett 1986;

Kandel et al 19851. A long-term study investigating the subchondral support of miniature

osteochondral allografts and autografts beyond 12 weeks of implantation is needed to

answer the question of long-term outcorne in a similar animal modei. We concluded that

the donor bone replacement by host bone was complete after three months. so a further

support of the articular surface is likely.

Although the nature of these immune responses are diil only partially understood

Fnedlaender nnd Horowitz 1992; Fnedlaender 19831, it is clear that ce11 surface antigens

of the major histocompatibility complex represented in the cellular elements of

osteochondral gr&s cause T-ce11 activation [Horowitz and Friedlaender 19911.

Specifically, cells of the suppressor/cytotoxic phenotype are activated. By using

autologous tissues in orthopedic surgery, such immune responses could be avoided.

Osteochondrzd allognfts diner from other organ transplants by the important biological

Feahire that healing does not necessarily require a vascular anastomosis. Previous

expenmental and clinical studies have show that g&s consisting of a small section of

bone underlying the cartilage c m maintain cartilage viability and heal without collapse

[Garrett 19861. Cartilage implanted as an osteochondrai allograft is known not to be

endangered by the cascade of irnrnunogenic events [Horowitz and Friedlaender 199 1 1,

but ultimate gr& failure will occur after the supporting subchondral bone is affected by

the immune response of the host. A srnail graft has a higher chance of suwivai because

the small amount of donor bone cm be supported and replaced by the ingrowing host

bone [Garrett 1 9861.

The use of autografts for joint resurfacing is technically challenging @3obic 19961.

Tissue harvest causes some morbidity of the donor joint. These grrifts are commonly

harvested from non weight-bearing areas of the patients joint. resulting in multiple small

defects watsusue et al 1993, Hangody et al 19961. These defects eventuaily fil1 with

fibrocartilage mangody et al 19961, and are in effect, trading a large (resurfaced defect)

for several small ones that are hopefully less than the "criticai size defect". Although less

prone to Mmunologic responses, autografts harvested in this fashion may not duplicate

the hyaline cartilage originally found in the area of the defect. Hyaline cartilage

composition and therefore its ability to react to certain mechanical and physiological

stresses Vary within a joint [Korvick and Athanasiou 1997; Finh 19871. It cm be

assumed that cartilage of a "lower quality" transferred £kom a non weight-bearing area to

a weight-beoring section may not be able to adapt to a new microenvironment.

1.6 Summary of Conclusions

Survivai of hyaline cartilage after transplantation of large allograft has been

documented [Czitrorn, Keating and Gross 19901. Our study investigated the short-term

results of small osteochondral allografts transplanted arthroscopically. Grafts with the

dimension used in our study are suitable for mosaic arthroplasty. Currently, only

autografts are used for this procedure in human patients. Gr& used in our expenment

were arthroscopically implanted into bovine fetlock joints using a new surgicd approach

to this articulation. Recovery of the calves was uneventfùl, and the animais remained

free of Iameness. Three months postoperatively, osteochondral graAs implanted in

congniency with the articular s d a c e of the recipient joint had an intact cartilage cover,

while the donor bone was completely replaced by new host bone. These gr&s are

believed to be superior to bone gr& implanted as controls. Both gr& types were

infenor when cornpared to joint sections harvested fiom unoperated calves.

Shortcomings of our study were a low number of transplanted grafts and a low

nurnber of experimental animals. These limitations were largely driven by economic

limitations. The original division into three treatment groups (osteochondral gras, bone

gr&, unoperated animals) designed for our study did not allow for a further subdivision

into grafts implanted in congniency with the recipient joint and grafts recessed fiom the

joint surface. The problem of recessed &s arose From technical issues related to

equipment, and adversely affected osteochondral gr& survival. Assessrnent of

irnmunogenic influences on graft survival, the effect of various storage techniques on

smdl osteochondral &rafts, a direct cornpaison of auto- and allograft and the same

animal model, the effect of weight-bearing on the gr&, and a staged long-term follow-

up were di outside the aims of this study. Further investigations regarding these

questions are necessary and recornmended.

We believe the arthroscopie transfer of srnaII osteochondral grah to be a valuable

surgical technique. Recent reports for the use of autogr& in humans are promising,

dthough they lack a long-term evaluation and histological testing. Because of morbidity

associated with autografl harvest and the possible anatomical difference of cartilage

harvested from non weight-bearing sites could potentially moke allografts a valuable

alternative. The technical protocol used in our s~udy for the tissue harvest was shown to

produce sterile osteochondral grafts and could be used in a clinical setting.

There is potential for the use of mosaic aahroplasty in vetennary medicine.

particularly in the horse. While arthroscopic accessibility WcIlwraith 19841 and

accurate gr& delivery are issues, this technique could be better than medical treatments,

abrasion arthroplasty, and subchondnl dnlling. Resurfacing by tissue graf'ting has only

been performed expenmentally. An equine animal mode1 needs to be established to

investigate the use of mosaic arthoplasty. Aside fiom osteochondritic lesions and bone

cysts. injuries obtained during athletic activity rnay be treatable with this new technique.

Amongst the articulations rnost fiequently injured are the equine carpal.

metacarpophdangeal and metatarsophalangeal joints. Arthroscopie access to injured

aaicular surf'aces in these joints is good for exploration or debridement and fkagment

removal, but may pose a greater difficuity for cylindrical osteochondral implantation

when compared to a large synovial cavity like the t a r s o d or femoropatellar joint.

Some clinically important sites in equine joints have thin articular cartilage, and

we predict that these areas will present a technical challenge for mosaic anhroplasty. A

s m d mismatch between the recipient and donor cartilage in such cases will result in

exposed recipient subchondnl bone. We have shown that this leads to pannus that

desüoys the donor cartilage. A slightly recessed graft in a joint with thick articular

cartilage will ody expose deeper layers of recipient cartilage. Development of

arthroscopie techniques and instrumentation for the horse will be needed before

osteochondral gras cm be used clinicall y.

4.7 References

Aichroth PM, B w e l l RG, Elves MW, Ford CW, Laurence M. Biologicd and mechanical pro blems of osteoarticular ai lografting: The relation to clinicd organ transplantation. 5 West Pacific Orthop Assoc 1971 ; 8:25-70.

Aron DN, Gorse MJ. Clinical use of II-butyl 2-cyanoacrylate for stabilization of osteochondral fkagments: Preliminary report. J Am An Hosp Assoc 199 1 ; 27903-210.

Beaver RJ, Mahomed M, Backstein D, Davis A, Zukor DJ, Gross AE. Fresh osteochondral allografts for post-traumatic defects in the knee. J Bone Jt Surg 1992; 74- B(1):105-110.

Bobic V. Ariliroscopic osteochondral autograft transplantation in anterior cruciate ligament reconstruction: a preliminary clinicd study. Knee Surg Sports Trauma 1996; 3 962-264.

Brittbeq 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; 33 1 :880-895.

Buchmann P. Treatrnent of elbow ankylosis by means of transplantation of the entire joint. Zentalblatt Chi. 1908; 19:582.

Buckwalter JA, Mankin HJ. Articular cartilage. Part 1: Tissue design and chondrocyte- matrix interactions. J Bone Jt Surg Vol 1997; 79-A:600-6 1 1.

Buckwalter JA, Mankin HF. Articular cartilage. Part II: Degeneration and osteoarthrosis, repair, regeneration, and transplantation. J Bone Jt Surg 1997; 79-A: 6 12-62 1.

Buckwalter IA. Musculoskeletal tissues and the musculoskeletal system. in: TwkS Orthopaedics: Principles and Their Application FZth Edition, edited by Weinstein SI and Buckwalter SA. J.B. Lippincott Company, Philadelphia, 1994.

Czitrom AA, Keating S, Gross AE. The viability of articular cartilage in fresh osteochondral allografts after clhical transplantation. J Bone Jt Surg 1990; 72-A: 574- 581.

Desrochers A, St-Jean G, Cash WC, Hoskinson JJ, DeBowes RM. Characterization of anatomic cornmunications of the fetlock in cattle, using intra-articdar latex injection and positive-contmst arthrography. Am J Vet Res 1997; j8(7):710-7l2.

Dew TL, Martin RA. Functional, radiographie, and histologie assessrnent of healhg of autogenous osteochondral gr& and full-thickness cartilage defects in the talus of dogs. Am J Vet Res 1992; S3(l l):214l-Z53.

Dyce, KKM, Wensing CJG. Essentials of bovine anatomy. Lea & Febiger Philadelphia, 1971.

Firth EC. An in vitro study on joint fitting and cartilage thickness in the radiocarpal joint of foals. Res Vet Sci 1983; 34:320-326.

Flynn J, Springfield DS, Mankin HJ. Osteoarticular allografts to treat distal femoral osteoarticular allografts to treat distal femoral osteonecrosis. Clin Orthop Re1 Res 1994; 303:38-43.

Friedlaender GE, Horowitz MC. Immune responses to osfeochondml allogrnnfrs: i%tnre and significance. Department of Orthopedics and Rehabilitation, Yale University School of Medicine, New Haven, Conneticut, 1 987: 1 1 7 1.

Friedlaender GE. Immune responses to osteochondral allografts. Current knowledge and future directions. Clin Orthop Re1 Res 1983; 174:58.

Friedlaender GE, Horowitz MC. Immune responses to osteochondral allognfts: nature and significance. Orthopedics 1992; l5(lO): 1 1 7 1 - 1 175.

Garrett JC. Fresh osteochondral allografts for treatment of articular defects in osteochondritis dissecans of the lateral femoral condyle in adults. Clin Orthop Re1 Res 2994; 30333-37.

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Goldberg VM, Heiple KG. Experimentd hemi-joint and whole-joint transplantation. Clin Orthop Re1 Res 1983; 174:43-53.

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Hangody L, Sükosd L, Szigeti L, K-iti 2. Artroszkopos autolog osteochondralis mozaikplastica. Magyar Tnumatol6gia, Ortopédia, Kézse bészet, Plasztikai Sebésze t l996:49-%.

Hangody L, Szigeti 1' Kirpati 2, Sükosd L. Eine neue Methode in der Behandlung von schweren lokalen Knorpelschaden. Osteo Int 3/1996.

Hurtîg MB, Fretz PB, Doige CE, Schnurr DL. Effects of lesion size and location on equine articular cartilage repair. Cao I Vet Res 1 988; 52: 1 37-1 46.

Hurtig MB. Experimentd use of small osteochondral grafts for resurfacing the equine thkd carpal bone. Equine vet J 1988; Supplement 6(23).

Hurtig, MB. Recent developments in the use of arthroscopy in cattle. Vet Clin North Am: Food An Prac 1985; I(1): 175- 193.

Jansson N. Equine osteoarthritis: A review of pathogenesis, diagnosis and treatment. Pferdehei tkunde 1 996; 1 3(2): 1 1 1 - 1 1 8.

Kandel R, Gross AE, Ganel A, McDermott AGPI Langer MD and Pritzker KPH. Histopathology of failed osteoarticular shell ailografts. Clin Orthop Rel Res 1985: 197:103-110.

Kawcak CE, Trotter GW, Frisbie DD, McIlwraith CW. Maintenance of equine articular cartilage explants in senim-fiee and senim-supplemented media, compared with that in a commercial supplemented medium. Am .l Vet Res 1996; 57(9): 126 1 - 1265.

Korvick D, Athanasiou K. Variations in the mechonical properties of cartilage frorn the canine scapulohurneral joint. Am .l Vet Res 1997; 58(9):949-953.

Lexer E. Substitution of whole or half joints from freshly arnputated extrernities by tiee plastic operation. Surg Gyn Obstet 1908;60 1-607.

Malinin TI, Mnaymneh W, Lo HK, Hinkle DK. Cryopreservation of articula cartilage. Clin Orthop Re1 Res 1994; 303: 18-33.

Mankin, HJ. The response of artîcular cartilage to mechanical injury. J Bone Jt Surg 1 982; 64-A:460-466.

Matsusue Y, Yamamuro T, Hama H. Arthroscopie multiple osteochondnl transplantation to the chondral defect Ur the knee associated with anterior cruciate ligament disruption. I Atthroscopic Re1 Surg 1993; 9(3): 3 18-32 1.

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Mc1lwrait.h CW. In vivo studies of techniques for biologic repair of osteoarthntic choncirai defects. ACVS Proceedings 1996; 128-1 29.

Meyers M, Akeson W, Convery R. Resurfacing of the knee with fkesh osteochondral allografts. I Bone Jt Surg 1989; 7 l-A(j):W-WL

Meyers MH. Resurfacing of the femoral head with fkesh osteochondral dlografts. Long- term results. Clin Orthop Re1 Res 1985; 197: 1 1 1-1 14.

Minas T, Nehrer S. Current concepts in the treatment of articular cartilage defects. Orthopaedics 1997; 20(6):525-53 8.

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R o f i a n M, du Toit GT. Osteochondral hemiarthroplasty. International Orthopaedics (SICOT) 1985; 9:69-75.

Rodrigo JJ, Thompson E, Travis C. Deep freezing versus 4' preservation of avascular osteocartilaginous shell allografts. Clin Orthop Re1 Res 1 987; 2 1 8:268-275.

S a m AE, Minor RR, Wootton JAM, Mohammed H, Nixon AJ. Local and remote rnatrix responses to chondrocyte-laden collagen scaffold implantation in extensive articular cartilage defects. Osteoarthntis and Cartilage 1995; 3:6 1-70.

Schachar N, Cucheran DJ, Frank CB. Viability of Intact Articular Cartilage at Various Times Afier Donor Death. 34th Annual Meeting, Orthopaedic Research Society, Atlanta, Georgia. February 1 -4, 1988. Proceedings.

Schachar N, McAllister D, Stevenson M, Novak K. Metabolic and biochemical status of uticular cartilage following cryopreservation and transplantation: A rabbit model. .i Orthop Res 1 992; 1 O:603-609.

Schachar NS, McGann LE. investigations of low-temperature storage of articular cartilage for transplantation. Clin Orthop Re1 Res 1986; 208: 146- 1 50.

Stevenson S, Qing Li X, Martin B. The fate of cancellous and cortical bone after transplantation of Eesh and fiozen tissue-antigen-matched and rnimiatched osteochondnl allografts in dogs. J Bone Jt Surg 199 1 ; 73-A(8): 1 143-1 156.

Stuart MJ. Treatment of chronic choncirai injuries. Sports Med Arthroscopy Rev 1994; 250-58.

Tomford WW, Springfield DS, Mankin HJ. Fresh and frozen articular cartilage allograh. Orthopedics 1992: L 5(l O): 1 183-1 188.

Tomford WW, Mankin HJ. Investigational approaches to articular cartilage preservation. Clin Orthop Re1 Res 1983; 17422-27.

Woo SLY, Buckwalter IA (editors). In: Injury and repair of the musculoskeletuf sofr tissues. Park Ridge, Illinois. Am Acad of Orthopaedic Surgeons. 1988.

I 1 1 I 1 T2 1 rned 1 G 1 4 1 O 1 1 I I

I I

1 1 S 1 data unavailable

Treatment T l

T l

Table A-1 : Paravital staining results of experimental Calf 1 as percentage of undamaged cells. Column "Samples" indicates the number of evaluated stained tissue microsections. Some data remains missing.

Samples 3

% Undamaged Cells 4 1

Latlmed Lat

Med

data unavailable data unavailable

3 1 50 3 100

data unavailable

Site G S P G S P

1 reatment T l

T l

latlmed lat

T2

Table A-2: Paravital staining results of experimental Caif 2 as percentage of undamaged cells. Column "Samples" îndicates the number of evaiuated stained tissue microsections. Some data remains missing.

med

T2

Site G S P G S

3 1 55 Data unavailable

lat

med

Samples 3

G S


3 3

f

G

Data unavailable 88 21

Data unavailable

3 3

91 55

1 Treatment 1 IaUmed 1 Site 1 Samples ( % Undamagrd Cells 1

Table A-3: Paravitai staining results of expenmental

1

T2

T2

Calf 3 as percentage of undamaged cells. Column "Samples" indicates the number of evaluated stained tissue microsections. Some data remains missing.

G S P G S

Tl

1 Treatment 1 latlmed 1 Site 1 Samples 1 % Undamaged Cells 1

lat

lat

med

T l 1 med

I

Data unavailable Data unavailable

4 3 4

P 1 3

G S P G S P

89 85 85 87

1

T l

Table A4: Paravital staining resdts of experimental Calf 4 as percentage of undamaged celis.

2 1 51 Data unavailable

2 I 93

G S

T l

Column "Samples" indicates the number of evaluated

3 3 4

rat

med

O


76 91 72

3 3

T2

4 0 72

P G S P

G lat 3

T2

2 3

S P

med

90 96

Data unavailable 3 1 96

Data unavailable 2 92

94 98 1 O0

G 1 4 S P

3 4

% Undamaged Cells 87

T l

Table A-5: Paravital staining results of experirnental CaIf 5 as percentage of undamaged cells. Column "Samples" indicates the number of evaluated stained tissue microsections. Some data remains rnissing.

Samples 3

Treatment T l

med

T2

T2

1 1

T l 1 rned 1 G 1 3 1 93 1

latlmed lat

G S P

lat

med

Site G S P G S P


7 6 6

Sample 4 2

Treatment T l

I 1 I

Table A-6: Paravital staining redts of experirnental Caif 6 as percentage of undamaged cells. Column "Samples" indicates the number of evaiuated stained tissue microsections. Some data remains rnissing.

4 3

46 79 89 -

S P

1

-I

89 100

82 95 98

G s P

lat/med lat

3 l 100 data unavailable

Data unavailable Data unavailable

4 I 93

3 3 7

Site G

1

72 97 80 90 96 82

S

T2

T2

G S P G S P

lat

med

3 2 2 6 7 2

med 142

Table A-7. Chondrocyte nurnbers of experimentd Calf 1.

Table A-8. Chondrocyte nurnbers of experimental Calf 2.

Table A-9. Chondrocyte nurnbers of expenmental Calf 3.

Table A-10. Chondrocyte numbers of experimental Calf 4.

Table A- 1 1. Chondrocyte numbers of experimental Calf 5.

17 79

Table A-12. Chondrocyte numbers of experimental Caîf 6.

G S

T2 lat

17 167

G S

T2 lat

--

Treatment 1 latlmed 1 Site 1 CeIl# T3 1 lat 1 G 1 1 7 8

I

T3 1 med 1 G 1 181

Table A- 1 3. Chondrocyte numbers of unopemted Calf n 1.

T3

T3

Table A-14. Chondrocyte numben of unoperated Calf 02.

lat

med

G S P G S P

3 89 190 170 168 172 154

Table A-1 5. Chondrocyte numbers of unopemted Calf 03.

Table A-16. Chondrocyte numbers of unoperated Calf n4.

Table A- t 7. Mean trabecular area of Level I and standard errors (percentage per field of interest).

1 I Site G I Site S I Site P 1

r

Treatment 1 Treatment 2 Treatment 3

Site S 50.3 % + 1.8 % 46.3 % + 2.8 % 51.1 %+?.O%

Site G 44.6 % -t 3.7 % 5 1.4 % f 3.1 % 47.9%I2.6%

Table A-1 8. Mean trabecular area of Level2 and standard errors (percentage per field of interest).

Site P 54.7 % -t 1.7 % 55.5 % k 2.0 % 50.2%&2.7%

Treatment 1 Treatment 2 Treatment3

1 1 Site G 1 Site S 1 Site P 1

47.0 % f 3.3 % 42.8 % k 2.1 % 31,3%f3.0%

Table A- 19. Mean trabecular area of Level3 and standard errors (percentage per field of interest).

Treatment 1 Treatment2 Treatment 3

35.2 % k 1.7 % 34.3 % f 2.0 % 32.6%&2.4%

37.0 % f 1.6 % 39.3 % k 2.1 % 28.9%21.2%

4 1.9 % f 2.4 % 41,1%+2.8% 27.8 % f 2.3 %

28.9 % ,t 2-0 % 29,4%+2.2% 27.2 % f 2.4 %

26.2 % f 1.1 % 27.9%+3.1% 22.4 % 4 1.5 %

I~reatment ( IaUmed 1 Site I TI 1 iat 1 G

Level % Object Area 1 44.00 2 67.55 3 50.53 1 50.08 2 31.13 3 26.40 1 57.98 2 26.14 3 26.35

Table A-20. Trabecular area bercentage per field of interest; Level 1 to 3) of experimental Cdf 1.

% Object Area 50.81

T reatment T2

Site G

latlrned lat

Level 1

Table A-2 1. Trabecular area (percentage per field of interest; Level 1 to 3) of experimentai CaIf2.

Table A-22. Trabecuiar area (percentage per field of interest; Level 1 to 3) of experimentai Calf3.

Table A-23. Trabecular area (percentage per field of interest; Level 1 to 3) of experimental Calf4.

Table A-24. Trabecular area (percentage per field of interest; Level 1 to 3) of experimental Caif5.

1 57.97 2 39.27 3 24.24

Table A-25. Trabecular area (percentage per field of interest; Levei 1 to 3) of experimental Calf 6.

Table A-26. Trabecdar area (percentage per field of interest; Level 1 to 3) of unoperated Caif nl .

Table A-27. Trabecular area (percentage per field of interest; Level 1 to 3) of unoperated Calf n2.

Table A-28. Trabecular area (percentage per field of interest; Level 1 to 3) of unoperated Caif n3.

Treatment T3

Level 1

latlmed 1 Site lat G

X Object Area 53.19

Treatment lathnad Site Level % Object Area T3 lat G 1 53.41

Table A-29. Trabecular area (percentage per field of interest; Level 1 to 3) of unoperated Caif n4.

Table A-30. Modified Mankin scores (for cellularity, erosions and adhesions, matrix, and subchondral support). assigned by two investigators (TS and MH). Experimentd Calf 1.

Table A-3 1. Modified Mankin scores (br cellularky, erosions and adhesions, ma&, and subchondral support), assigned by two investigatoa (TS and MH). Experimental Calf 2.

Erosion Site G S P G S P

Cellularity Treatment

T l TS

3

Matrix 1 Support TS 2 1 O O O O

Laffmed lat

rned

MH

3

MH 2 2 O 1 O O

Erosion Cellularity Site G S P G S P

Treatment T l

TS 3 3 2 2 3 2 2 2 2 0 0 0 0 0 0

TS 5

1 1

TS 1 O

LaUmed lat

med

TS

2

- MH

MH

1

MH 1 O

Matrix

MH 3 O O 2 O O

0 1 1 0 0 0 0 0 0 0 0 0 0

Support TS

0 . 0 0 0 0 0 0 0 0 0 0 0

1 2 2 3

TS 3

MH O

O 2 1 O

MH '

3 O O 3 O O

O 0 0 0 0 0

O 1 1 O

1 O

Table A-32. Modified Mankin scores (for cellularity, erosions and adhesions, matrix, and subchondral support), assigned by two investigators (TS and MH). Experimental Calf 3.

I Cellularity 1 Erosion 1 MatrÎx 1 Support 1

Table A-3 3. Modified Mankin scores (for cellularity , erosions and adhesions, matrix, and subchondrd support), assigned by two hvestigators (TS and m. Experimental Calf 4.

Support Matrix TS 2 O

1 O O

TS 3 2 1

0 0 2 1 0 0 0 0 0

2 ~ 1

MH 2 O O 1 O O

Erosion MH

1 1 O

TS 3

3

Cellularity Site G S

Treatment Tl

Mt(

2 2 2

O 0 0

TS O O

Latlmed lat

MH O O O 1 2 O

med P I 0 G S

2 1

I O

Table A-34. Modified Mankin scores (for cellularity, erosions and adhesions, matrix, and subchondrai support), assigned by two investigaton (TS and MH). Experimental Calf 5.

med G 1 0 1 0 O O 2 S O O O 0 0 1 O O P O O 0 1 0 O O 1 O

Table A-35. Modified Mankin scores (for cellularity, erosions and adhesions, matrix, and subchondral support), assigned by two investigators (TS and m. Experimental Calf 6.

Table A-36. Modified Mankin scores (for cellularity, erosions and adhesions, matrix, and subchondral support), assigned by two investigators (TS and MH). Unoperated Calves nl, n2, n3, and n4 received "0" scores for al1 sites.

Figure 1. Bovine metacarpophalangeal joint (cadaver specirnen) implanted with two 6.0m osteochondral aiiografts. Grafts are located in the abaxiaI section of the medial and Iateral condyles.

Figure 2. Bone holding device with distal end of donor MC34 Note the harvested graft seated in MC34 condyle. Operator is holding a coring drill bit (6.0 mm inside diameter).

Figure 3. Mitre box and hack saw. Transverse cut through donor MCf4 releases osteochondral graft.

Figure 4. Bovine metacarpophalangeal joint (dissected cadaver specimen). Forceps (right) are retracting the fibrous band separating the medial and lateral condyles of MCJ4. Aahroscopic sleeve (left) is inserted through the fibrous band.

Figure 5. A 6.0mm flat bottom drill bit. A central spike diows for secure seating in the articular d a c e of MCw (cadaver specimen).

Figure 6 . A graduated depth measuring gauge inserted in a 6.0 mm hole in MC3-( (cadaver specimen).

Figure 7. Lateral condyle of MCw (top; 1 to 3) and opposing articular surface of the fist phalanx (bottom; 2') of unoperated control animai, sectioned for histological evaluation.

In the experimentai animds, the grdl is located in (2). Section (2') represents the joint d a c e opposite to the p f k , (1) and (3) are graft mounding sections.

Figure 8. The sampiing procedure for trabecular bone area measurements used regions 1 to 3 (10 x magnincation) under the gnft (right) and in the adjacent recipient bone (lefi).

Paravital staining (PVS) used a minimum of three vibratome sections in the saggital plane extending fiom the lamina splendens to the tidemark or subchondnl bone (shown as nmow rectangles).

Areas of interest for the chondroc yte count were shilar to the sites used for PVS. Not drawn to scaIe.

Figure 9. An arthroscopic view of a gmft (g) being press fitted ushg the gr& passing tool (t). Synovial membrane (sm) in foreground.

Figure 10. An arthroscopic view of a recessed bone graft (arrows) exposing the sidewdl of the recipient hole.

Figure 1 1 . A histological section stained with Safranin- O. Arrows outline the bony base of an osteochondnl graft. 5x magnification.

Figure 12. A Sdkmin-O stained osteochondral gr&. Note the tilted articdar gnft d a c e , covered with fibrous tissue. 10x magnifcation.

Figure 13. A Safranin-O stained specimen with dead bone (N) and cernent lines (C). Note marrow pthisis. hset shows polarized microscopy of same site. Necrotic bone had a dBerent green factor than new bone.

Figure 14. A Sananin-O stained specimen of boae gr& showing extrllisic cartilage repair and marrow pthisis. Sx magnification.

Paravital Staining

Osteochondral Graft Bone Graft n=12 n=12

l Graft

8 Adjacent Area

Figure 15. Paravital staining results (mean values and standard errors) of cartilage from the graft, the area adjacent to the g d l and the opposing phalangeal surface for Treatment 1 (osteochondral grafts) and Treatment 2 (bone grafts).

*O0 1 Chondrocyte Count

Adjacent Area

Osteochondral Bone Graft Unoperated Graft n=12 ni1 2 Controls n=16

Figure 16. Mean chondrocyte counts and standard erroe from Weigart's Hernatoxylin stained sections of Treatment 1 (osteochondral grafts), Treatment 2 (bone grah) and Treaûnent 3 (unoperated anîmals). Anatornic sites represented by columns marked with (*) are significantly different from corresponding sites in unoperated anirnals.

Total Trabecular Area

. Graft

5 Adjacent Area

O P l

Osteochondral Bone Graft Unoperated GraR n=12 n=l2 Controls n=16

Figure 17. Total trabecular area (mean values and standard errors) of combined Levels 1, 2, and 3 for Treatment 1 (osteochondral grafts), Treatment 2 (boue graf'ts), and Treatment 3 (unoperated controls). Anatomic sites represented by columns marked with (*) are significantly different fiom corresponding sites in unoperated animals.

Modified Mankin Scoring

2.5

* 2 O CI 0 1.5 El Adjacent Area

E l 8 tî) 0.5

O

Osteochondral Bone Graft Unoperatad Graft n=12 n=12 Norrnals n=16

Figure 18. Combined mean Mankin scores and standard errors for Treatment 1 (osteochondral grafts), Treatment 2 (bone g a f t s ) and Treaûnent 3 (unoperated nnimals). Columns representing anatomic sites marked with (*) are significantly different fkom correspondhg sites in uaoperated animals.

l MAGE EVALUATION TEST TARGET (QA-3)

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