s.-ì E

EPIPHYSEAL PLATE REPAIR USING FAT INTERPOSITION

TO REVERSE PHYSEAL DEFOR}IITY An experimental study

JI I j I I l l I Thesis submitted in March 1989 for the degree of Doctor of Medicine in the University of Adelaide

by

Bruce Kristian Foster, M.8., B.S. (Adel.), F.R.A.C.S.

The work described was performed within

the Department of Pathol ogy of the University of Adelaide

I TABLE OF CONTENTS

TITLE I

TABLE OF CONTENTS II

TABLE OF FIGURES IV

TABLE OF TABLES VII

SUMl'IARY X

DECLARATION XII

ACKNOl.lLEDGEMENTS XIII

Chapter I GLOSSARY I

Ghapter 2 AII'ls 2

Chapter 3 NOR]'IAL PHYSEAL FUNCTION 3

3.1 INTRODUCTION 3 3.2 MECHANICAL PHYSEAL INJURY 7 3.3 CLASSIFICATION OF FRACTURES l0 3.4 ANATOMY & PHYSIOLOGY 18 3.4. r Zone of Reserve Cel I s 3.4.2 Prol i ferati ng Cel I s 3.4.3 Hypertrophic Cells 3.4.4 Mi neral i zati on 3.4.5 Metaphysi s 3.4.6 Ci rcumferenti al Structures 3.4.7 Arti cul ar Cart'il age 3.5 BLOOD SUPPLY 30 3.6 BIO1'4ECHAN ICS 32

Chapter 4 DEVELOPMENT OF CLOSURE ]'IODEL 36

4.1 EXPERIMENTAL AIM 36 4.2 REVIEIil OF THE LITERATURE 44 4.3 MATERIALS & METHODS 4.3.1 Animal s 4.3.2 Operative Technique 4.3 .3 Radiol og'ical Techniques 4.3.4 Deformity Index 4.3.5 C.T. Measurement 4.3.6 Spec'imen Measurements at Autopsy 4.4 RESULTS 58 4.5 D I SCUSS ION 7l 4.6 CONCLUS IONS 74 II Page No.

Chapter 5 FAT INTERPOSITIONAL REVERSAL 75

5.1 EXPERIMENTAL AIM 75 5.2 REVIEI^, OF THE LITERATURE 75 5.3 MATERIALS & METHODS 84 5.3.1 Bone Brjdge Resection 5.3.2 Radiologìcaì Techniques 5.3.3 C.T. Measurements 5.4 HISTOLOGICAL QUANTI FICATION 93 5.5 RESULTS 0F l SQ. CM. DEFECTS 108 5.6 RESULTS OF 2 SQ. CM. DEFECTS t2t 5.7 RESULTS 0F 3 SQ. CM. DEFECTS 134

Chapter 6 DISCUSSION 145

Chapter 7 CONCLUSIONS & FUTURE DIRECTIONS 158

Chapter I APPENDICES t62

Chapter 9 BIBLIOGRAPHY 169

III TABLE OF FIGURES

Paqe No

3.1 Growth arrest following trauma. 5&6

3.2 Poland's classification of physeal fracture. ll

3.3 Salter and Harris' classification of physeal l3 fracture.

3.4 Rang's classification of physeal fracture. 16

3.5 Ogden's classificatjon of physeal jniury. t7

3.6 Normal physeal anatomy. l9

3.7 Physeaì zonaì function. 20

3.8 Blood supp'ly to the epiphys'is. 31

3.9 Normal physeal radiology. 35

4.1 Summary of growth djsturbance caused by surgical 42 trauma to the ep'iphysea'l plate in immature dogs.

4.2 K-wi re marker pì acement. 47

4.3 Ep'iphyseal arrest by phys j odesi s, accordi ng 49 to Phemister.

4.4 Radioìogical bone deformity. 52

4.5 Measurement of bone lengthening ratio. 52

4.6 Computerized tomographic section of the tibial 54 physis.

IV Page No.

4.7 Measurement of pin distances at autopsy. 56

4.8 Preparation of histologÌcal section. 57

4 .9 Macroscopi c vi ew of a prox'imal ti bi al fusi on . 59

4. 10 Radi ol ogi ca'l variati on of fusi on. 6l

4.ll Histologica'l evidence of fusion: bone response. 66

4.12 Histological evidence of fusion: physeal response. 67

4. 13 Histological evidence of non-fusion: physea'l 68

transpl antatj on.

4.14 Histological evidence of non-fusion I. 69

4.15 Histologica'l evjdence of non-fusion II. 70

5.1 0perati ve deta'i I s of physeal bridge resect'ion 85 and fat interposition.

5.2 Quantification of physeal area by computerized 94 tomography.

5.3 Hjstologicaì assessment of host tissue response 95 to implant - capsule formation.

5.4 Histological assessment of host tissue reaction 97 to implant - epiphyseal bone response.

5.5 Hjstolog'ical assessment of host tissue reaction l0l to'impìant - cortical bone remodel'ling.

V 5.6 Hjstological assessment of fat autograft.

5.7 Histological assessment of bone brìdge reformation. 104

5.8 Hjstological assessment of host physeal response. 105

5.9 H j stol og'ica'l assessment of host physea'l 109 response - polarized light examination.

5. l0 Hi stol og'ical assessment of host physeal ll0 response - peripheral physis.

5.ll Rad'iolog'icaì evidence of fusion and reversal ll4 of deformity.

5.12 Bone bridge reformat'ion - peripheral . 120

5.13 Interst'iti aì physeal repair. t22

5. 14 Attempt at medial bone bridge reformation - 132 low power examination.

5. l5 Attempt at medial bone brjdge reformatjon - 133 high power examination.

5.16 Bone bridge reformation I. 742

5.17 Bone bridge reformation II. 143

6. I Physeal defect. t5l

6.2 Physeal repaÌr t52

6.3 Physeal growth. 155

VI LIST OF TABLES

Page No.

I Rel ative incidence of physeal 'iniuries 8

4A Provocation Series - Deform'ity index at 3 months 62 (i) bone lengthening ratio (i i ) pin angul ation

4B C.T. scan results 63

4C Direct measurements at 3 months autopsy of: 64

Bone p'in distance versus bone length difference

4D Radiology & histology correlatjon of fusion 7t

5A I sq. cm. defect - 3 month survival tt2 Deformity index at 3 months

5B I sq. cm. defect - 6 month survival ll3 Deformity index at 3 and 6 months

5C C.T. scan results for I sq.cm. defect 116 at 3 month and 6 month survival

5D H'istopathol ogy I sq. cm. reversal s - sheep tt7 autopsied at 3 months

VII Page No.

5E Histopathology I sq. cm. reversals - sheep ll8 autopsied at 6 months

5F 2 sq. cm. defect - 3 month survival 124

Deformity index at 3 months

5G 2 sq. cm. defect - 6 month survival t25

Deformity index at 3 and 6 months

5H C.T. scan results for 2 sq. cm. defect t27 at 3 month and 6 month survival

5I Hi stopatho'logy 2 sq. cm. reversal s - sheep t28

autopsied at 3 months

5J Histopatho'logy 2 sq. cm. reversals - sheep t29

autopsied at 6 months

5K 3 sq. cm. defect - 3 month survival 135

Deformity 'index at 3 months

5L 3 sq. cm. defect - 6 month survival 137

Deformity index at 3 and 6 months

VIII Page No.

5M C.T. scan results for 3 sq.cm. defect - 3 month 138 and 6 month survival

5N Histopathology 3 sq. cm. reversals - sheep 140

autopsied at 3 months

50 H'lstopathology 3 sq. cm. reversals - sheep 141 t 1 autopsied at 6 months I 1

;

I i

,trl '1

I

I I

l

I

I

I I

I

IX SUMMARY

A predi ctabl e method of reversal of physea'l (ep'iphyseaì pl ate) damage at the level of deformity is not current'ly avajlable.

A reproduci bl e model of a periphera'l part'ia'l bone bridge to the proximal medjal tjbial physis v',as created jn lambs at the age of 6 weeks. Three months after the initial procedure, reversal of the bone bridge lesjon u,as achieved by excjsion of the bone bridge and autologous fat interposition from the ipsilateral knee fat pad.

There v',ere 3 groups of animals with lesions to the phys'is. At reversal in the first group of animals, C.T. scans determined that the size of defects was 17.2 + 2.2% of the total physeal area. For the second group of anjmals, the size of the defect was 28.0 + 6.0%.

For the thjrd group of animals, the lesion was 33.7 + 7.2%. Us'ing an unpaired Student's ú test, there u,as a statjst'ica'lly sign'ificant increase ìn sìze of lesion between groups (p < 0.0001). ldi th 'interposi t j on of fat radi ol og'ical cniteri a j ndi cated success j n the resumption of physeal growth jn 85.7% of cases in the first group, 54.5% in the second group, and 50.0% jn the third group.

The survjval of the fat and its maintainjng of pos'ition I'rere critical factors to the success of the reversal procedures. For example, 'in the first series of experiments, success js 100% if two animals unable to satisfy both criteria are elimjnated from analys'is.

Measurements of the areas of the physea'l defects us'ing C.T.'images between the initial (reversa'l) procedure and autopsy at three and six

X months showed a decrease ìn size of the defects. These, hov'lever, failed to reach a statistically sign'ifjcant level of change in the size of the defects. This is presumptive evjdence of the ìnabiìity of physeal to repaìr.

Histolog'ical assessment showed that there bras minimal evidence of physeal stimulation or resumption of a normal physis in the defects. A spur of cartilage cou'ld be seen to arjse centraì'ly from the phys'is and to point towards the metaphys'is. In this regÌon there was loss of normal physeaì organ'ization. Under polarized light mìcroscopy the phenotyp'icaì expression showed metapl asi a of the hyal 'ine cart'il age to f i brocart'il age. Chondrons of physeaì di sorgani zati on occurred.

It is postulated that correction of deformity occurs by a non-union effect, which allows the rema'ining normal physis to functjon and groþ,, rather than by a process of interstitial physeal repa'ir.

In the first group of animals where the area of defect was 17.2 +

2.2%, the defect may be resected wi th a 100% success rate. ldi th larger defects, reversal of deformity may be partìal or may not occur. Success was determined by the size of the lesjon and the absence of bone bnidge reformation.

The cljnical significance of thjs study js that peripheral defects of < 17.2% of growth plate area will respond successfully to physeaì interpositional surgery.

XI DECLARATION

I declare that this thesis contains no material which has been accepted for the award of any other degree or diploma 'in any University and that to the best of my knowledge and bel ief, the thesis contains no material previously pub'l ished or written by another person, except where due reference is made in the text of the thesis. I further consent to the thesis being made available

for photocopyi ng and I oan as appl i cabl e, i f accepted for the award of the degree.

BRUCE K. FOSTER

XII ACKN0t'ILEDG EMENTS

The facilities of the Flinders Medical Centre Animal House were made avai I abl e wi th approval by the Fl i nders Medj cal Centre Ethj cs Committee. Mr. Ray Yates and staff (particular'ly Mrs. Christjne Hill) were always prepared to put in an extra effort to care for the animals. Mr. Yates and his family provided an animal husbandry system to obtain lambs of known age. Through the Institute of

Medical and Veterinary Science Animal House, Mrs. Alma Bennett and

Mrs. Anne Schloithe gave additional anjmal care facil itjes as 'log'istics sometimes required. 0ther personne'l who assisted at operatìons were Mr. Phil Hutchins, and Mrs. Alana Hansen. Mrs. Hansen a'lso gave exceptional additional technjcal assistance.

Dr. Michael Sage, Director of Radiology, agreed to the use of C.T. and x-ray facjl ities at the Fl inders ]'ledical Centre. The C.T.

Technical Staff Officer, Mr. Edmund Arozoo often worked after-hours to receive the necessary anaesthet'ised lambs and to obtain excellent quaì i ty scans.

Computerized planimetry of the C.T. images after selection of the most appropri ate cut v',as undertaken 'i ndependentl y by Mr. lJi nston Franc'is (RadÍotherapy Department, Royaì Adelaide Hospital.) Dr. Njck Fazzalari (Institute of Medjcal and Veterinary Science) and Dr. 0le I'liebkin (Department of Medicine, Royaì Adel aide Hosp'ita'l , University of Adelaide) gave statistical advice.

Dr. M'ichael Rozenbjlds (Pathoìogy Department, Fl inders Medical Centre) superv'ised the production of the histopathological specimens and together with the author examjned and re-examined specimens us'ing

XIII ljght microscopy before the author's final independent assessment. Mr. Rikki Kerr expertly cut and decalcifjed the large tibjal bone specimens. Professor Barry Vernon-Roberts (Department of Pathology, University of Adelaide) gave additional ass'istance as supervisor.

For the production of the manuscript, the Department of Orthopaedics (The Adelaide Children's Hospital ) Secretarial Staff, particu'larìy Mrs. Ronda Saint, h,as responsìble for an exceptional effort. Mr. Dale Caville (Department of Pathology, Un'ivers'ity of Adela'ide) produced the excel lent photographs to support the written and diagramat'ic work of the Medical Il'lustrator, Mrs. Colleen L'ìoyd at The Adelaide Children's Hospitaì. Ms. Ann Noble of the Student Counsell ing Servìce (University of Adeìaìde) and many col'leagues assisted by proof-reading the manuscript.

Financial support came from many sources: Adelaide Bone and Joint Research Foundat'ion, Adelaide Children's Hospital Research Foundatìon, Australian Orthopaedic Association Research Foundation, Channel l0 Medical Research Foundatìon, Fl inders Univers'ity (Department of Surgery), and the Roya'l Australian College of Surgeons

Research Foundation.

DEDICATED TO

Penny and my famity for providing an emotionally supportive

envi ronment.

XIV Chapter I GLOSSARY

ARTHROTOMY: Opening of a joint by an incision.

DIAPHYSIS: The main shaft of a bone, between the two

physes which mineral izes from a primary centre.

EP I PHYS I ODES I Creation of a partia'l or complete bone

PHYSIODESIS: bridge to join across the physis between the

epiphys'is and metaphys'is.

EPIPHYSIS: The juxta-articular bone, ossified from a separate ossjfication centre.

INTERPOSITION: Pl acement of mater i al between the ep'i phys i s

and the metaphysÍ s at the I evel of the physis.

METAPHYSIS: The region of the of tubular immedjate'ly near to the phys'is, where

mi neral i zati on of the growi ng cart'i1 age takes

p'l ace.

PHYSIOLYSIS: To rel ease or di ssol ve an abnormal bone bridge across the growth plate.

j PHYS I S/ The d i sc of spec'i al zed hyal i ne cart i 1 age

EPIPHYSEAL PLAT E/ that lies at the ends of the tubular bones

GROVJTH PLATE: providing for ìongitudinal bone growth.

I Chapter 2 AII'IS

The physis (ep'iphyseal plate) lies at the end of the long bones and 'lengtheni duri ng growth 'i s responsi bl e for bone ng.

The compl jcation of I imb shortening and/or anguì ation due to premature growth arrest of the physis presents a challenge to orthopaedic surgeons. Such arrest of bone growth may be partìal or complete, central or peripheral, and g'ive a variable degree of deformity as a consequence.

The hypothesis is that the physis has an internal mechanism of repair to restore physeal function.

The aims of thi s experimental study v',ere to establ i sh a def i ned degree of deformity by partial growth plate excis'ion, and then to examine different methods of reversal of such deform'ity to observe the process of growth p'late repa'ir. The secondary a'im was to defjne the percentage of phys'is that could be resected yet still enable reversal of deformity.

2 Chapter 3 N0RÎ'|AL PHYSEAL FUNCTI0N

3.1 INTRODUCTION:

There are many causes of physeal deformity.

Congenital absence may be partial as in Madelung's (1878) deformity of the radius, Blount's (1937) disease of the ' and Kirner's (1927) deformjty of the terminal phalanx of the little finger or it may be complete, as in Proximal Focal Femoral Deficiency IReiner

1901, Shands & MacEwen 19621 or Hemjmelia [Thompson, Straub & Arnold 1957, ldestin, Sakai & l,'lood 19761. In addition, there are

i I I -defj ned j nborn errors of metabol i sm such as achondropl asi a

IParrot 1878] and the mucopolysaccharidoses that cause dwarfi sm [Hunter 1916-1917, Hurler 7920, Morquio 1929, Lamy & Maroteaux 1957'

Scheie, Hambrick & Barness 1962, Sanfilippo, Posod'in, Langer & Good

19631.

There are many acqu'ired aetioìogies. The commonest cause of physeal deformjty is injury IHutchinsen 1894, Bowen 1915, Bergenfeldt 1933'

El iason & Ferguson 1934, Bisgard 1935, 1937, Ruckensteiner 1947, Harsha 1957, Budig 1958, Cassidy 1958, Dale & Harrjs 1958, Harris 1958, Brasheer 1958, 1959, Sakadida 1964, Aìtken 1965, Steinert 1965' 0'Brien, Morgan & Suter 1971, Sussenbach 1972, Tachdiian 1972, Rang

7974, Ogden 7978, Bouyal a & Rigaul t 1979, Lovel ì & [¡ljnter 1986' M'izuta, Benson, Foster, Paterson & Morris 19871.

3 A cl i ni cal exampl e of a physeal Ì n jury produc'ing deformi ty 'i s

illustrated in the case of K.M. an 8 year old female who sustained a

distal femoral fracture. 12 months after injury she developed a 10" valgus deformity of the distal and knee compared to the normal 3" of va'lgus. There was 2.1 cm. of shorten'ing (Fig. 3.1).

Infect'ion, ei ther fo'l l owÌ ng a compound fracture or as a resul t of

sept i c arthri t'i s or osteomye'l 'i t'i s , may al so cause physeal damage [Duthie & Ferguson 1973, Letts 1988].

Vascular occlusion causes physeal damage. This may result from:

(i) frostbite [Straub 1929, Löhr 1930, Bennett & Blount 1935, Greene 1943, Crismon & Fuhrman 1947, Thelander

1950, Shumacker & Lempke l95l , Pi rozynski & t¡lebster

1953, 1954, Bl austein & Siegl er 1954, Dreyfuss & Glimcher 1955, Bjgelow & Ritchje 1963, Morscher 19671.

(ii) burns [Evans 1959, Frantz 1966].

(iii) irradiatjon damage IStevens 1935, Judy 194I, Spang'ler

1941 , Barr 1943, Langensk'i öì d 1953, Baserga l96l , Barnhand 7962, Argüelles 19771.

(iv) electrical injurjes IKolar & Vrabec 1960, Brinn & Moseley 1966, 0gden l98ll.

Vascular occlusjon may also be a result of treatment, as in the

treatment of sl 'ipped upper femoral cap'ita'l epi phys i s ft'Ja'ldenström 1940, Ponseti & McCintock 19561 or the treatment of congen'ital

4 P!.g¡r¡rc 3.1,¡ 6nGÑ """"""'t å*RS,S|! FO[,X,4@ffm llwff,' ¡lrPandlatcrrn.x-rÉy¡ehouarcl.ütivoly Salter-Harris typG 2 fracture of thc

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236

369 34 B Cl'l t2a

64

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31 5 Cll 3t 5 Cl'l -r92 R

-236

32ø R + - 4 øø øø ¡1¡1 1å,,,.",;rt;"i -19? 1 5 R LEG68.4cm LLEG 66'3cm Figurg 3-1.3: ÀP x-ray shoss the increased.valgus anguration of 10" compared to the normal valgus of 3.. dislocation of the hip ISalter, Kostuik & Dallas 1969' Bucholz & Ogden 1978, Kalamchi 1978, Kalamchi & MacEwen 1980, Cooperman 19801.

The author's own'interest in physeal damage was stjmulated in 1980/81 when, as a Research Fellow at the Alfred I. duPont Institute in ldilm'ington, Delaware, U.S.A., a review of pat'ients wjth Perthes

Disease was undertaken IBowen, Schreiber, Foster & ldein 1982, Bowen, Foster & Hartzell 19841. The review showed a sign'ificant incidence of physeal i nvol vement as wel'l as ep'iphyseal avascul ari ty. Thi s was prognosticaìly important since the degree of femoral head deformity and probab'i'l i ty of I ong-term hi p degenerati ve arthri ti s correl ated with the degree of physeal damage.

This thesjs is concerned with the reversal of a mechanically induced

I es i on to the perÍ pheral zone of the phys'i s .

3.2 MECHAN CAL PHYSEAL INJURY:

0f alI mechanical iniuries to the Iong bones duning chiIdhood approximately 15% jnvolve the physìs IBowen 1915, Bergenfeìdt 1933' Eliason & Ferguson 1934, Bisgard & Martenson 1937, Neer & Horwitz 1965, Salter 1970, Peterson & Peterson 1972, Tachdiian 1972, Rang 1974,Ogden 1982a, Lovell & lilinter 1986, Mizuta et al 19871.

The relatjve inc'idence of specific areas of physeal jniury is shown jn Table I [Neer & Horwitz 1965, Peterson & Peterson 1972, Ogden 1982a, Mizuta et al 19871.

7 TABLE I Relative Incidence of Physeaì Iniuries

Neer Peterson 0gden Hi zuta & PHYSEAL & & Benson SITI Hortitz Peterson Foster et. al .

No % No % No % No T,

3 0.9 0 0 t 0.3 3 0.9 72 3.0 22 6.7 27 6.1 7 2.0 332 14.0 20 6.1 56 t2.6 24 6.9

t24 5.2 I 0.1 5 1.2 l6 4.6 2L 0.9 3 0.9 I 0.4 1096 46.2 98 29.8 114 25.7 100 28.4 136 5.7 t2 3.7 ll 2.5 l6 4.6 l0 3.0 I 1.8 l5 4.3 39 ll.8 4l 9.3 9l 25.8 23 5.2 0 0

7 2.2 9 2.3 0 0 4 1.0 0 0 28 l.l l8 5.5 t7 3.8 I 0.4 L7 0.7 6 1.8 2 0.6 4 t.2 2 0.2 2 0.6 0 0 l2 2.7 0 0 238 10.2 59 17.9 60 13.5 33 9.3 302 12.8 2l 6.4 15 3.4 l2 3.5 6 1.8 3 0.9 5 1.5 ll 3.7 2l 4.7 25 6.2

2368 100% 330 100* 443 100% 353 100%

8 In 1965 a revjew by Neer and Horwitz covered 2'368 consecutjve physeal injuries. This and other reviews showed that males sustain physeal injurjes more frequently than females. The ratio is variably reported as rang'ing from 4:l to 9:1. It has been proposed in the I iterature that the s'ign'ifjcant aetiolog'ical factor here is the greater exposure of boys to trauma from athl eti c acti vi tj es . 'longer Additiona'lly the physes of the male also stay open than the physes of the fema'le, extending the peniod of possible trauma. The study by Mizuta, Benson, and Foster et â1, in 1987 confirmed these findings, and suggested that the site of iniury v',as an important prognostic factor. It also confirmed that for a hospital of primary referral (The Adelaide Children's Hosp'ita'l) the 'incidence of physeal injury was 77.9%.

There may aìso be different responses of the physes to trauma durìng the adolescent growth spurt. Morscher et al (1965' 1968) has shown that there are hormona'l1y medjated differences in the physeal response to experimentally applied stresses.

All studies have shown that jn all the long bones the distal physes are injured more commonly than the proximal physes (Table 1). This higher incidence of iniury to the distal radius, dista'l tibia and

pha'langes may result from the increased exposure of these more distal regi ons to trauma rather than from any uni que physi ol ogi ca'l

suscepti b'il i ty of these part'icul ar physes.

These studies also show that there are 2 age periods of rap'id skeletal gro!',th: the first year of 'l'ife, and adolescence (9-12 in g'irls 12-13 in boys). Fractures involving the physes commonly occur

9 during these periods. This suggests that at the same physiolog'ical age the physes of mal es and femal es are equa'l'ly suscept'ibl e to i nj ury.

3.3 CLASSIFICATION OF FRACTURES:

In 1863 Foucher first proposed a classification of physeal 'injury. It recognised that the level of the line of separat'ion between the phys'is and metaphys'is is consjstent. This acknow'ledged the recognition in the early lgth century that thjs was a different type of fracture to that of the diaphys'is.

In 1894 Hutchjnson wrote in the Eng'lish ljterature of an increasing awareness of these i niurj es. In 1898, Pol and advanced a classification of 4 types of fractures, based on a revìew of museum specimens and experimental cadaver work (Fig. 3.2).

Aitken (1965) presented a classjfication which designated 3 types of fractures. He was the first to emphasize that deformjty as a result potent'i growth di sturbance was rare of i nj ti al mal uni on or al ' although the degree of jnitial d'isplacement v',as significant. Aitken however, emphasized that the compressìon iniury wh'ich was extremely difficult to diagnose, and was generaìly thought to be of little sign'ificance, yêt could easjly lead to maior growth deform'ities.

A deta j I ed cl assi f i cati on of fracture types i nvol v'ing the phys'i s b,as proposed by Salter and Harris in 1963. This classificatjon was based on a combination of the mechanism of iniury, relationship of the fracture line to the various cellular ìayers of the physìs, and the

prognos'is fol I ow'ing subsequent di sturbance of growth.

-10- r ilOlm'r'8 CL^&3[flerNl,il W ,mY$EÃû, fKßUilJ. . illh¡,rr ¡rc 1 ttlroû. øÊ infruryr ¡ ¡t I tt ( ño¡rroduecú rúth ¡nmi,rrirom f,rm FojLmü, J. ¡ frlmtû'e toplratÅo,n eû tùr ryi¡ñtyril. !ot&n, ¡ili,th, úniücc ¡,nd C@., Lt'}t Þy co'urtcry Frætumr ir¡r €httrófGu, iloekwod et tl, J.¡.Li¡ryl,ne,oüt, 3r tlt4. t Pu¡e ¡nil complete P¡rti¡l seporation, with oe¡nrrtion lr¡cture ol the iliaPhYnis

Prrtirl scptrrtion, ritb lrro' Completc roPrrrtion, wi3h tu¡c ol tbe cPiPbYrir frroture ol 3hc cPiPhYsir

ll Salter and Harris recognised five radiologically definable groups (Fis. 3.3).

Type I: There is complete separation of the epiphysìs from the without any osseous fracture. The growing cells of the physis remajn with the .

This type of injury is common in early childhood when the physìs is thick. It is also seen in patholog'ica'l conditions wjth physeal separation, for example in scurvy, rjckets, osteomyel itis and endocrine imbalance lsalter 1970]. This last factor is thought to be s'ignificant in sl ipped upper femoral capita'l epiphys'is IHarris 1950].

Wide displacement is uncommon because the periosteal attachment remains jntact. Reductjon is not diffjcult, and the prognosis concerni ng future growth j s excel I ent unl ess the epi phys'i s i s intra-artìcular IDale & Harris 1958], in wh'ich case the blood supply may be damaged resulting in premature closure of the physis.

Type II: The injury occurs where the line of separation extends along the physis to a varjable distance and then out through a t¡iangle of metaphyseal bone. This is called Thurston Holland's s'ign (1929). It is the commonest type of fracture.

This type usualìy occurs in children over l0 years of age. The is torn on the side oppos'ite to the angulation, but is intact on the concave side.

The grow'ing cartìlage cells of the physìs remain with the ep'iphysis.

-t2- Púür'Eûtil, flgro^ro 3. 3 ¡ õttffit tm H RRIS| cfås,El meâîrilt¡ oùr rnnetum . I ll (ilc!¡nodu'e,cd rl-th pcmf,rsi,o"n fron SlLter, n. l. a¡ld [i¡rrl'¡, f. R. fajrrrri,ca involvLng thG spiphylorl plete . J. l@'ûe "loint Surg., 454¡ 587, 1963. ) I r0,

0

U

'iû, Type I 'l¡v.tnô Type ll A. B.

Type lll ïype lV c. D.

o \

Type V

E.

b. t. l\\"{\2 ol A'( q ôt'

\

Har'^is- classification of epiþhyseal þløte injuries øccording to salter and

t3 The prognos'is concerni ng growth i s excel I ent, prov'id'i ng the circul ation to the epi physi s remai ns i ntact, wh'ich is usually the case.

Type III: The fracture, which is jntra-art'icular, extends from the joint surface to the level of the hypertrophic cells of the physis' and then extends along the growth p'late to its periphery.

This type of injury is uncommon. The commonest sites are either the upper or lower tibial physis, and iniury'is due to intra-articular shearing forces.

Accurate reduct'ion is essential to prevent bar formation across the phys'is and to restore a smooth ioint surface.

As with Type I and II 'injuries, provided the bl ood supp'ly to the separated ep'iphys'is i s i ntact the prognos'i s i s good wi th accurate reducti on.

Type IV: The fracture, which is intra-artÌcular, extends from the joint surface through the epiphysis across the full thjckness of the physis and through a portìon of the metaphys'is producing a complete discontinuity of epiphysis, phys'is and metaphys'is.

Perfect reductjon of a Type IV epiphyseaì plate jniury js essential for function of the epìphysea'l plate, and for the restoration of a smooth ioint surface ISaìter & Harris, 1963].

Unl ess the fracture i s undi spl aced, open reducti on i s a1 ways necessary. The phys'is must be accurately real'igned to prevent bony

-14- union across the pìate with resultant local premature cessation of growth. These fractures occur common'ly at the lateral of the and the distal femoral physis.

Type V: This relatively uncommon type of iniury results from a severe crushi ng force appl 'ied through the epi phys'i s to one area of the physÌs. It occurs jn ioints that normalìy move in one plane only, such as the ankle or knee. At the ankle a severe abductjon or adduction injury to a foot whjch normally on'ly f'lexes or extends is likeìy to produce crushÌng of the phys'is which may or may not separate. Displacement of the epiphysis under these circumstances 'is unusuaì, and the initial radìograph gives little indication of the serious nature of the iniury. Indeed, the iniury may be dismjssed as a sprain. One must suspect compression of the phys'is under such circumstances, and hope to prevent premature cessation of growth by protection from we'ight bearing for three weeks ISalter 1970].

Thus, the prognosis for Type V physeal injury is poor and difficult to pred'ict. A type V injury may occur jn association w'ith any of the other more defined types of iniury.

Rang (1969) has added another peripheral physeal iniury in which the Ranvier Zone is iniured along the immediately adiacent metaphys'is and epìphys'is. It may arise as an extension of a traumatically induced infectjon or severe burn, or as a result of direct trauma to the p'late, such as from a lawn mou,er iniury or bike wheel spoke iniury.

This has become known as an additjon to the Salter-Harris group to be called a Type VI iniury (Fig. 3.4).

-15- ! lmÛ Frcnî n i'niury tll'¡r n t ¡rüc,o¡t halioû,. ( &moúrcod ri,tù ¡nrnt¡r!,on frør @, Ë. fiü. ¡rroüth pJlntr a',¡¡d I'tr dl¡orC¡r¡. irltümæ, Ftl.Iln f üf.Iki'ml, 1,9Õt. , Type I Type II Type III

+ c

Type IV

Type V Type VI

ò

¿ I

1ó Figure 3.5: OGDEì¡S CLÀSSIFICÀTION OF PHYSEÀL INJURY- 1A: Propagati.on across the physeal- cartilage- 18: Propagation across the diseased primary spongrosa (e-g- Ieuke¡nia, thalassermia) with the physeal interface varj ably invo-l-ved. lC: Disruption of a localized segrnent of the physis.

2A= Partial propagation across both the physj.s and rnetaphysis.

28z Free and attached rnetaphyseal fragnents-

2C¿ Propagation across both the prinary spongi-osa and rnetaphysis - 2D= Localized disruption of the physis at the point of propagation into rnetaphysis. 3À: Epiphyseal fragrnent with propagation through the physis- 38: Epiphyseal fragment with propagation through the primary spongiosa- 3C: Crushing injury to peripheral- physis.

: _ : I JL': Nonarticurar cartii-age iivIIISIUTT te-9- rb\-¡rrqr-L - tuberosity) - -epiphys 4À: Conù ined eat-phys eal-irrc t aphys eal- fragment. 48: EpiphyseaJ.-physeal-nret aphyseal f ragnent conbined with Tlpe 3À or 38 lesion- 4c: propagation through a nonarticular epiphyseal regron (e.g-intraepi-physeal cartilage of the developing femoraJ. neck- ) 5: Longitudinal growth retardation of a major physeal segment - 6: Àvulsion or crushing of the peripheral physis ( zone of Ranvi-er). 7A: Osteochondral fragment involving the physis of the secondary ossification center. 78z Chondral fragnent involving hlpertrophic cells of the physis of the secondary ossification center. 8: A rnetaphyseal fracture tenporarily cuts off the nutrient artery (N), causing transient ischenia to the nretaphyseal segrment betveen the fracture and the physis- 9: to periosteu¡,t1, rith or sithout discrete osseous ?.T.gFln1urìr, disrupts no¡naJ- lhembranous ossificaüion- (Reproduced with permission from Ogden, J-: J- Pediatr. Orthop., 2z L9E2.l t l_ \ !ì t

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17 Certain types of epiphyseal and physeal iniuries cannot be readily classif ied by the Salter-Harris classif icat'ion, and comp'l'icated combination iniuries occur. Furthermore, other growth mechanjsms such as the zone of Ranvier, epìphyseal and peniosteum are not included. ggden (1982a, 1982b), proposed an enlarged classification to cover the other growth mechanisms. This classification 'is more complex and has yet to stand the test of time (Fig.3.5).

3.4. ANATOMY & PHYSIOLOGY:

The phys j s i s a comp'l ex structure wi th a high degree of cellular organÌzation. It consjsts of cells whjch are anatomically and functionally different, but which merge into each other (Fig. 3.6, 3.7).

The immedi ately adiacent metaphysi s Ì s 'important as a funct'ionaì'ly and anatomicalìy totally independent structure. The metaphyseal blood supply contributes the vascular buds and pluripotential cells responsible for cartilage resportion and new bone formation.

The blood vessels to the physis must pass through a supportìng bone plate to reach the cartilage basal cells. Immediate'ly below the bony ep'iphysÌs one finds the zone of reserve or rest'ing cells. These merge consecuti ve'ly i nto the zone of proì 'iferati on, the zone of maturation or hypertrophic cells, the zone of calcificat'ion, and finally the metaphys'is, where vascular invasion and bone formation occur (Fig. 3.6).

-18- f.[ru$ j},ér reme amV€ü&, rmn*ru. & À qú oclll,u&¡r oæ¡roùmsüü'q år ¡fditrr ûil a&!, {n¡Nc"

De*o @,ü th rywÐe t. l,* M nü,*to. D- em of ro*üûng €tÙl[û. G' üfl1 of nrua&ùûcmüflm. D. ütm d UrymtuqÈtø slll.l[¡'. I U. &@ga øt eoùI mfn¡npr,ltf,"on olr prwùeùon m$,eùfir'c'rüüm. F. I'"û!Pü of b,orc fo.'¡rn¡tioa- (x 5' 0) (Rsp¡nqdueed rùth ¡¡¡4¡ÈtEion fron lruQüa amd ltorgen' l'9ÓO. )

f,rtoilLoqú tiloq @f t'ht I c|¡ lt' ia tibi,r Pathrc- It tbt otrrre.ü,uril[ q3¡úarü&ou oû tbc n¡r¡eirrliad ]rynl,ir,m ç*rtri.trtgG ¡ü¡ thro rdütnt ¡frrynir. (r 2$'CI1 I ?:, I ,t" .t I.:,d' '¡* 6 ,q ru .l {, a'1, ô. c 2 'U4

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V I . @fthü üb f t@ñ.û oG th qlt¡ftnrÅ1, lnd t'h.ûr twtûoni t (mpmorûw! lf,th prrnû¡¡.ilæ ûoon CJ¡o¡r, t. &. (¡ú.l ffi. ml'wl,æÈcl.rt¡tr, rtrtm. ñr¡rol@UÍr bi,ooil¡d¡tr¡¡ td pft¡rrtolo¡r¡tr trtil torft, Ckt¡rehi,!.f, l"ivt"ognt'@m, tr93, Itt2. I M MATRlX METABOLISM ZONE FUNCTION CALCIU ACTIVITY SYNTHESIS

:T' -n, â Boney Plate Support I' l ? r Aerobrc Reserve 0 tensroll 2 I Prolif erative Cell Division ct-)

-L Mitochondnal o- Hypertrophic ATP ¡-!J calcium upper accumulalion (t') accumulalion O= t9 z. ]J.J glycogen o CD mid Matnx vesicles -) slorage cc Anaerobic C.) Calcif ication calcifying calcif ication

0steoid formation Metaphysis Vascular invaston Calcification of þ ¡ A. 1o spongiosa *t t ,ffi The functjon of the different zones of the phys'is can be summarized diagramatically (Fig. 3.7). The following describes the characteristjcs of each physeal zone.

3.4. I Zone of Reserve Cel I s

The cells in the reserve zone are approximately the same size as those 'in the prol iferating zone IBrìghton, Sug'ioka & Hunt 1973' Bnighton 1978, Hunziker 19871. They are spherical in outline and exjst alone or jn pairs; they are not arranged in columns as are the cells of the other zones. Although named "rest'ing cells" morphol og i ca'l I y and bi ol og'i cal ì y, the cel I s are not rest'i ng because they have abundant rough endoplasmic reticulum and are rich'in 1ìpìd [Brighton, Sugioka & Hunt 1973]. However, Kuhlman jn 1960, 1965 and ¡lray & Goodman in 1961, have demonstrated that their enzyme content as well as their content of ions, jncludìng calcium is the lowest of the entire physis. The intercellular matrix in this area is more abundant than in any other zone. It is h'igher in col'lagen content than those of other zones, and the collagen fibrils are random'ly distrjbuted IIrving & Wuthjer 1968]. The proteoglycan, fip'id, and water whjch constitute the other components of the intercellular matrix are present in smaller amounts than in the rest of the physís 'low, ILindenbaum & Kuettner 1967]. Qxygen tension is [Anderson' Cecil & Saidera 19751'indicating in all probabiìity that the blood passìng through the bony end p'late to supply the reserve zone does not I ose much of i ts oxygen to the cel I s . Autorad'i ographi c techniques demonstrate that cells in this zone divide onìy occasionally IKember 1960].

-2t Brighton in 1978 has suggested that these celIs store Iip'ids and other nutrients for subsequent activjty but this is not certain.

3.4.2 Zone of Prol i ferati nq Cel I s

The haphazard arrangement of of the reserve zone 'long gradually I ine themselves up into vertical columns, w'ith the axes of the columns being paral'lel to the'long axes of the bone.

The cells have a flattened appearance and there is a great increase in rough endoplasmic retjculum IHoltrop 1972a, Brighton, Sugioka & Hunt 19731. Synthesis of proteoglycans in this region is the lowest in the entire physis IGreer, Janicke & Mankin 1968]. But 35S incorporation has been demonstrated to be maximal in this zone ISchmidt, Rodegerdts & Buddecke 1978] . The intercel I ul ar matrix contains the h'ighest content of proteog'lycans' IGreer, Janicke &

'l 'i Manki n 19681 and therefore i t i s ogi cat to propose that there s either a low rate of breakdown [Brighton 1978] or a real increasein proteoglycan synthesis wjth an increase in lysosoymal act'iv'ity lSchmidt et al 1978].

The role of the pro'l'iferating zone is cell division, and jt 'is in

'l th i s I ayer that growth of ong bones takes p'l ace . The prol 'i ferat i ng cells are the only cells of the physis that normally divide, and growth in 'length is a direct result of that division. In 1960

Kember demonstrated by autoradiograph'ic uptake of tritiated thymidine that the prol'iferating zone selective'ly takes up the nucl ide.

-22- 0nce cell divisjon has occurred with each cell being ljned up in a coìumn, a change in intercellular act'ivity occurs. The cell enters the hypertrophic zone as the cells more proximal to it' grow a$,ay from it [Hunz'iker 1987].

The oxygen tension in the zone of proliferation is the highest in the physis, which, when coupìed with accumulation of glycogen jn the cells, makes it ìikely that aerobic metabolism and storage of glycogen is occurring IBrighton 1978].

3.4.3 Zone of Hvpertrophic Cells

From the proximaì (epiphyseal) end to the djstal (metaphyseal) end of the hypertrophic zone each cell diameter enlarges to five times its orÌginal size IBrighton, Sugioka & Hunt 1973, Buckwalter 1986' Hunzi ker, Schenk & Cruz-0rive 19871 . The ì arge quantities of glycogen of the proliferative zone disappear in the middle of the hypertrophic zone IPritchard 1952, B¡ighton, Ray, Soble & Kuettner

19691 .

Morphologica'l'ly, the cells that jnitially have well-formed ultrastructural elements gradually fjll wjth holes that take up more than half the volume of the cell [Ho'ltrop 1972b, Brighton, Sug'ioka & Hunt 19731. At the metaphysea'l end fragmentation and loss of subcellular detail occurs.

The oxygen tension is extremely low as a result of the jncreased distance from the epiphyseal blood supp'ly, and Shepard and Mitchell in 1976 proposed that thjs relative anoxia may be responsible for the orderly sequence of cell activity.

23 The zone is rich in enzymes of the Krebs Cycle [Kuh'lman 1960' 1965] and in I ipids IIrv'ing & tduthier 1968]. The matrix contains the lowest content of both colìagen and proteoglycan IGreer, Janicke &

Man ki n 1968, I rv'i ng & tluth i er 19681 .

Electron microscopy has shown dense granules in the mitochondria of cells in this zone. Biochemical and histochemical evidence IMartin & Matthews 1970, Brighton & Hunt 19761 and x-ray djffractjon studies have shown these to be calcjum and phosphorous ISutfjn' Holtrop & Ogi'lvie l97l]. The mitochondria are most abundant at the epiphysea'l end of the hypertrophic zone, but in the midportion they rapìdly lose their calcium 'ions, IBrighton & Hunt 1976) and by the metaphysea'l end both the mitochondria and cell membrane show no evidence of calcium retention [Arsenis 1972].

As the mitochondria lose calcium, matrix vesicles beg'in to appear outside the cells. These have been suggested as the initial site for mineral depositìon lAnderson 1969, 1973, Boskey 1979, Arsenau]t

1e881.

3.4.4 Mineral ization

The details of calcification of the intercellular matrix of the phys'is are obscure. lilithin the physjs there is a progression from a mjneral-free state to the point where the cells gradually load their cellular membranes with calcium and then with calcium phosphate IAnderson, CeciI & Sajdera 1975, A]i, [rlisby, Evans & Craig-Gray 19771, until the entire matrix vesicle and'its other surfaces are encrusted with mineral. This mjneral spreads through the matrix

-24- [Anderson 1969, Anderson, Matsuzawa, Sajdera & Al i 1970, Al i, Anderson & Sajdera l97l].

It has been proposed that matrix vesicles promote calcification by concentrati ng the transportati on of cal ci um and phosphorous and perhaps by remov'i ng the i nhi bi ti on of cal ci fi cati on. The phosphol jpids of vesicles show a h'igh affinity for calcium and phosphorus IBoskey 1978]. Matrix vesicles are present at sites of mineralizat'ion in the body; however, their association with mineral has almost always been observed when aqueous preparative techn'iques have been used. If the experìmental techn'iques are altered to jnclude only organic solvents, the vesicles appear to be mineral-free ILandis & Glimcher 1978]. l'ljneral ization of involves the depos'ition of mjneral 'in type I bone co1ìagen, whereas physeal cartiìage conta'ins type II collagen. The organization of the mineral in the physis appears to be random rather than well ordered. There is, therefore, no reason to believe that the process of mineral deposition must be jdentical. It is log'ical to propose totaìly separate mechanisms. Gjbson, Bearman & Flint in 1986 have proposed that type X col'lagen may be the injtiator of the mineralizatjon process.

There 'i s a gradua'l change i n appearance and acti vi ty of cel I s from the ep'iphyseal to the metaphysea'l end of the phys'is. In the prof iferative zone where oxygen tension is high, aerobic metabolism occurs and g'lycogen is stored. The mitochondria appear to form ATP and store calcium - these processes are mutual'ly excìusive ILehn'inger 19701. In the hypertrophìc zone where the oxygen tension js low, anaerobic metabolism appears to occur, and the glycogen js utilized

-25- and d'isappears by about the middle of this zone. This calcium accumulation requires engergy, ILehninger 1970] and it may be that ATP is used in the process [Shap'iro & Lee 1978].

At about the midportion of the zone of hypertrophy calc'ium accumulation by the mitochondria ceases, perhaps because available energy ìs used up, IBrighton 1978] and extracellular matrix vesicles appear, wjth the calcium and the phosphorous eventually appearing as hydroxyapatjte crystals. It is not clear whether the initial form of mineral in the matrix and matrix vesicles is hydroxyapatite or an amorphous phase, (Brighton 1978) or whether another type represents the fjrst deposit of calcium and phosphorous. hlhat is certain however, is that there is a regular shift to the extracellular fluid from the mitochondria of the matrjx (perhaps through matrix ves'icìes) and that this can be folìowed through the varìous zones of physeal chondrocytes from both anatomical and temporal points of view IHowelì le71 I .

Cell death occurs as calcification takes place in the ìongitudinal septae. The horizontal septa dividing chondrocyte from chondrocyte do not ca]cify, and thus the mineralization js also columnated.

The organ'ization of the chemical constituents of the physis is 'important. The col'lagen of the physis is type II Ilduthier 1969, Ph'il ì 'ips & Young 1976, Reddi , Gay & Mi l l er 19771. Its chemi cal makeup'is such that jt should not calc'ify as readily as type I collagen of bone IGlimcher 1976]. The proteoglycans of the physis are well characterized IRosenberg 1980, Poole 1982, Buckwalter 1983, Horton 19881. The physjs must be able to absorb energy in a fashjon ana'logous to that of articular carti'lage. The physis is part of the

-26- load-bearìng apparatus. The proteoglycan link to physeal collagen

is very regu'lar [Shepard & Mitchell 1976] and similar to that seen in articular cartilage. It is possible that some of the bjomechanical properties of articular cartilage are derived from this link and from the relationship of proteoglycan to water molecules. It is logìca1 to presume that this bjomechanical advantage occurs in physeal cart j'lage as wel I .

Proteoglycans have a more important function jn the physis. They are synthesjzed in all physeal zones, but there is a decrease in the amount and in the poìymerization of these proteoglycans IHung,

Llilkins, & Blizzard 1962, Jibril 1967, Matukas & Krikos 1968, Howe'l'l , et al 1969, McConaghey 1972, Kan & Cruess, et al l98ll. There are a'l so enzymes 'in physeal carti'l age for degrad'ing proteogìycans ILucy, D'ingle & Fell 1961, Dziewiatkowskj 19661 that are primariìy lysosomal

jn nature [Greenspan & Blackwood 1966, Bnighton, Sug'ioka & Hunt 1973] and are found in the hÌghest concentration in the zone of hypertrophic carti I age.

It has been demonstrated that proteoglycans inhibjt mineral'ization, and it was thought that mjneralization of carti'lage could not proceed until they were removed or altered IEhrlich 1985]. The observation that they disappear from the longitudjnal septa as mineralization occurs has been made, IBayì'ink, Wergedal & Thompson 1972, Brìghton, SugÌoka & Hunt 19731 but immunohistochemical studies indicate an alternatiVe aggregation rather than a true removal IRosenberg, Poole & Pidoux 1980, Poole 1982, Horton 19881. It has been suggested that

thj s change i n the aggregrati on of the proteogìycan makes the carti'lage more permeabl e, and it i s thi s that 'is responsi bl e for the

-27- rapid spread of mineral from the matrìx vesicles, permitting calcification in the metaphyseal end of the zone of hypertrophy.

The metaphysis is thus presented with a honeycombed surface with a low oxygen tension, few l'iv'ing cells, and a cartilage matrix h'igh in calcium phosphate that appears to be hydroxyapatjte.

3.4.5 Metaphysi s

The metaphysis beg'ins at the last transverse septum in the epiphysis IBrighton 1978]. The loss of the transverse septae leaves the metaphys'is with a tjssue structured like a honeycomb. The vertjcal septae persist, channelling vascular invasion jnto the areas prev'iousl y occup'i ed by cel I s .

Intense cellular prolìferation is seen in this area. Tritiated thymidine is taken up by endotheljal cells, and by osteoprogenitor cells immediateìy behind the cap'i'llary buds. The capillaries invade the empty spaces and there is evidence of vascular stasis [Brighton

19741. Occasional ruptures of capiìlaries are seen [Ham 1969] and electron microscopic inspect'ion shows precip'itates of materjal that are also consistent with stasis ISchenk, l,'liener & Spiro 1968, 'low, Arsenault 19881. 0xygen tension is IBrighton & Heppenstaìì l97l] and enzyme studies indicate that anaerobic metabolism is possib'ly tak'ing pl ace.

0steoblasts are found immediately behind the invad'ing and div'iding capi'llary buds. The'long'itudjnal septae from the physìs are almost completely calcified and are called the primary spong'iosium IBrighton 19781. Rapid endochondral ossjfication then occurs, with the layìng down bone on the cartilaginous bars. This is the secondary spong'iosum [Mclean & Urist 196l].

28 Resorption of metaphyseal bone must begin immediately for the metaphysis is funnel shaped and narrows to become dìaphyseal bone.

3.4.6 Circumferential Structures

Two important structures surround the cartilaginous physis.

The groove of Ranvier (1873) lÍes at the level of the zone of reserve cells and flows into or merges with that zone. Tonna in 1961 proposed that the cel I s of thi s zone provide cel I s for the perìchondra'l ring and cells and matrix for the lateral growth of the physi s. The groove al so contai ns cel I s that di fferenti ate as fibroblasts, attaching the groove to the physìs iust proximaì to the zone of reserve cells [Shapiro, Holtrop & Glimcher 1977].

The perichondral ring of Lacroix (1951), js a fibrous tube that enci rcl es the phys'is at the physeaì bone iuncti on. At the prox'imal end it is continuous with the cells and collagen of the groove of Ranvier and this serves as an anchor point. It merges with the periosteum distally. It therefore limits the physis and provìdes lateral support, resist'ing the compressive forces across the phys'is [Shapìro, Holtrop & Glimcher 19771.

3.4.7 Arti cul ar Carti I e

Endochondral ossification takes place at the basal layer of articular cartiìage throughout the body. This prov'ides some growth in length, but its major functjon i s to al low for the growth of the j cartj'l agi nous porti on of ioi nts. When the epi physeal I i ne s obliterated at the time of physea'l closure, these basal articular ìayers retain some capacity to respond to stjmuli and can' under patho'l og'ica'l cond'iti ons, resume thei r acti vi ti es.

-29- There is an inherent polarity to articular cartÍlage. If a segment of articular cartilage'is reversed, and its ioint surface edge placed on the interior with the base of that segment facing the ioint cavìty, the roles of the art'icular chondrocytes will not reverse ll'lcKibbjn & Holdsworth 1967]. Repair potent'iaì js limited and arti cul ar carti I age repai r becomes fi brocart'i I age under I oad ICampbeìl 1969, Bentley, Smith & Mukerjhee 1978, Trippel, Ehrlich, Lìppieì'lo & Mankin 1980, Zaleske, Ehrlich, Piliero, May & Mankin te82l.

3.5 BLOOD SUPPLY OF THE PHYSIS

The three major structural portì ons of the phys'is have thei r own independent blood suppìies, IDale & Harris 1958, Trueta & Morgan

1960, Brookes 1971, Chung 19761 consi stent wjth their separate functions (Fig. 3.8).

The epiphyseal artery supplies the epiphysis (or secondary centre of ossification). Vessels entering the bony epiphysis at right angles to the surface send branches through the bony plate support'ing the phys'is. They then branch like roots of a tree to supply the tops of the columns of cells in the prol'iferative zone. These vessels appear to pass through the reserve zone, and each small arterjole supplies between 4 and l0 cell columns [Trueta & Morgan 1960].

The metaphysjs receives its blood supply from end arterioles that have in'large part arisen from the nutrjent artery. About 80% of the metaphysis is supp'l'ied by the nutrient artery IBrighton 1978]. The peripheral metaphyseal vessels supply only the outer portìons.

There are no anastomoses between epiphyseal and metaphyseal vessels. It is possible to'interrupt one or the other experìmentally, [Trueta

30 SO ün ni}j[Etrlt$ts. tlbm¡c æc 2 b*lie .$ümlPìn"ü lhn cp irc ir ont:lrcny blr ùú'tieuù*ù @rurtúL¡gp- yrc.tùe ter tho opi.pùç* ranüvGrting th Pl"r ùu ¡t tb t¡ç¡ri,¡ùory o,f tü¡ä qDi¡db!ru'ocJ' ryLeto. Nm'ing tÅq¡ülCInpaü ttËr(r vçúÜe&ç ror vuil,mrcb[c te rqøt'mro-

mc ûr Drrttr¡r covr,cnû b¡ lrtùc,r¡L¡r crrtù.ùaEo- l.¡ þr prmtúûtl,tü tlln soctÕx rt thr oü& ø,f t,h¡ çû¡b¡nirl. In th¡,ü t¡r¡n qrôlùlraeoL ar¡nrlti.@tr eorrùd @G€ur üûü.hoüt ¡cni,ous qü¡¡n tø yrrc.¡çI¡. ( uúth parnú¡¡ûon frm DnLc, G. G. and l{¡rri¡' n. n. Progurecll i',n qp:ü¡ÑU¡loljt rrynratúon¡. i¡. D@ttc n¡@i"ût l}trrt., *,Sr [I7, 195'û.) cA. pøríelr.ondrir¡rrl o.r tic'LrIa.r cc.rtilcrg ø

a. e'E)iprn¡sio-I pJ-o-tø

(,

p ør Lo-qtøurn-

pørLostøtrrrr- {v t' 6' & Morgan 19601 and to observe the effects on function. Interruption of the epiphyseal vessels causes death of the physis and stops the proì i feratj on of these cel I s. Interrupti on of the metaphyseal vessels causes fajlure of jnvasion of capil'lary loops from the metaphysis and a widening between the zone of mineralization and the zone of pro'l 'i ferat i on .

The groove of Ranvier and perichondraì rÍng of Lacroix receive their nutrition from a separate set of perichondral arteries.

3.6 BIOMECHANICS:

The intrinsic strength of the physis is provided maÌn1y by the jntercellular cartilag'inous matrix. In the fjrst two zones the matrix is abundant, and the growth plate is mechanìcally strong. In contrast in the third zone the hypertrophic chondrocytes en'large and there js decrease in the intercellular matrix, mak'ing this zone the weakest portion of the epiphyseal plate. Thjs weakness js to shearing, bending and tension stresses, and not to compressìon [Bassett 1962, Bright, Burste'in & Elmore 1974, Moen & Pelker 1984].

The fourth zone js reinforced by the additjon of calcification but it i s sti I I weaker than the fi rst and second zones. Fractures genera'lly involve the third and fourth zones propagatìng a variable djstance into each, not cleavjng neatly between the two zones.

The weakness of the hypertrophÌc zone was demonstrated by Haas (1919' l93l) who found that if the periosteum of the periphery of an epiphysea'l plate lvas incised, then the epiphysìs could be detached relatjvely easily from the metaphysis by gentle pressure. He felt that the plane of cleavage was constant, and that jt went through the

-32- layer of hypertroph'ic cartiìage. Amam'ilo, Bader and Houghton in 1985 reported on the role of periosteum in growth plate failure.

Harris (1950) and Harrjs & Hobson (1956) des'igned an apparatus to determine resistance to shear of the upper tibjal epiphysis'in rats, and confirmed that when the ep'iphysis separated from the metaphysis the pl ane of cl eavage consi stently passed through the thj rd (hypertrophìc celì) layer. The clinical sign'ificance was that the basal physeal cells remained with the epìphyseal fragment.

The region of trabecular bone formation in the metaphysis also contributes to physeal strength. The thinness and fine structure of the metaphyseal cortex in children makes it susceptib'le to certain kinds of injuries (e.g.compression or torus fracture) that are not seen in adults.

The cartilage of the epiphysis provides a certain shock-absorptive effect as long as it is primari'ly cartìlaginous. In this situation the fracture producing forces are transmitted more directly into the metaphys'i s , resul t'i ng i n torus fractures . As the epi physeal ossjfication centre enlarges, thjs resi'liency and ability to absorb stress probably are I essened, and the deformi ty forces are transmjtted d'irectly into the growth plate which may be sheared through the third and fourth cellular zones.

The specìfic three dimensional configuration of the phys'is in mammals 'is extremely variable and probably develops in response to the forces applied across each physis [Karahariu 1967, Bechtol 1973, 0gden 1979, le80l .

Because the human body is not h'ighly spec'ial ized for rap'id twist'ing locomotion, the confjgurat'ions of the epiphyseaì plates of the distal

-33- femur and proximal tibia are not as geometrically complex as they are in anjmals specialjzed for rapid running. The distal femur of the artiodactyls represents a portion of a h'inge ioint, and the epiphysis contains four cone-shaped projections from the metaphyseal side whjch fit jnto appropriate concave shaped areas of the epiphysis and physi s. These prevent d'i spì acement i n an antero-posteni or/ med j al -l ateral di rect'ion, as wel'l as preventi ng epi physeal rotat j on.

There is also a cone shaped lappet formation around the periphery of the epiphysis to further resist d'isplacement. The importance of this js that the epiphys'is is mechanical'ly protected by these cone-shaped project'ions, part'icularly from shearing [0gden 1982a].

However, these are secondary stabilizers to the jntrinsic physeal stabjlizers, and are affected by the growth pìate undulations in add'ition to the many smaller mammillary processes. They cannot be appreciated on standard two dimensional radiographs (Fig. 3.9).

In human adolescence the cone shaped projections are more pronounced, and thus shear fracture and transverse di sp'l acement may i nduce fractures through multip'le levels of the growth p'late metaphysis and epÍphyseal centre, rather than through the hypertrophic cellular zone. This irregular fracture line may pre-dispose the growth plates about the knee having a hìgh incidence of traumatica'l1y induced physeal arrest.

34 sI@ü4,[,PßXYgüAn"nA,D'IOúO6Y-ÀrPandlatcnel normJ. knoe in a child 10 yGars oì.d' fh.e udulatione otr physoal comtourr of co,EDG s,he¡md projoctio'ns tnd tmllo¡r nrntll.ary P,r@i¡tetJ.@'ù8- @f thc t¡hyrir elnngt b: ca¡fly ¿pPrêeiateü. tm,o¡¿ atructureÚ provl'fic *G'eonf,lüürlr stabitity to th.e pihyais to reciat sho¡r foree'

Chapter 4 DEVELOPMENT OF THE GROWTH ARREST I'IODEL

4.1 EXPERIMENTAL AIM:

To confjrm that the technique of Phem'ister (1933) bras appropriate to produce partia'l growth arrest in a sheep tib'ia model .

4.2 REVIEI,' OF THE LITERATURE:

In 1727 Stephen Hales discovered that the long bones grew in'length on'ly at their extremities. By tracing markers in the bones of animals he, and Du Hamel in 1742, and Haller jn 1757, and Hunter jn 1760 demonstrated a lack of an interstitial mechanism of diaphyseaì bone lengthening in the normal process of bone growth.

The understanding of the mechanism of bone growth by the mid e'ighteenth century may be iudged by the description given'in the l74l edition of Cheselden's Anatomy: "The cylindricaì bones and all others whose fibres are nearly paraìleì, begin (to ossify) about the middle of each fibre, and thence shoot forth to their extremities; not always in continued ljnes, but frequent'ly beginn'ing new , whìch soon join the former; and by the continual addition to this ossifying matter, the bones increase till their hardness resists a farther extension; and the'ir hardness aìways increasing while they are growìng, the increase of their growth becomes slower and slo!{er, unt'il they cease to grow at all."

Endochondral ossification in grow'ing bones u,as not described until 1836, when l'ljescher made reference to this basic growth process.

Todd and Bowman in 1845, followed by Sharpey (1848), Leidy (1849),

-36- and Tomes and De Morgan (1853) also studied the anatomy of the physis.

In 1858 Müller described the microanatomy of the physis, and this llas further clarified by Fell (1925), Ham (1932)' (1969), Dodds (1934)'

McLean (1940), Streeter (1949), Lacroix (1951)' (1971), Maximow &

Bloom (1954).

Leser (1888), Retzius (1888-89), and subsequentìy Retterer (1900), Dubreuil (1913), Lacroix (1951) and Sissons (1956) believed that the physis greu, by the increase in size of individual cartilage cells of the zones of the pìate nearest the epiphysis, where the existence of mitoses was noted.

The contrary view that interstitial growth wjthin the bone itself was respons'i bl e for the ì engthen'i ng v',as proposed by vari ous authors [Hellstadius 1947, 1951]. An increase in the length of the mass of a spine fusion had been noted in immature animals and also in children IBisgard and Musselman 1940]. In experimental studies on rabbits Odelberg-Johnson (1939) showed micro fractures of the fusion mass wi th subsequent fracture heal i ng to expl ai n thi s mechani sm of interstjtjal growth.

It is now fjrmìy established that cells of the resting zone closest to the ep'iphysis replicate, and as these cells mature and increase in s'ize they pass from the matuning zones to the hypertrophjc zone and become mineraljzed by endochondral ossifjcation lHunziker et. al re87l .

-37 - The mechanism of transverse, or diametric growth of the epiphyseal carti'lage plate has received little attentjon in the literature.

Ranvier in 1873 proposed that the width of the ep'iphyseal plate was increased by interstitial growth. This uras the same mechanism as that for long'itudinal growth. In 7920 Keith, oh radjolog'ical evjdence from severaì cases of diaphyseaì aclasìs, supported the view of Ranvier.

In l94l Policard and ìn 1947 Langensk'iö1d made similar conclusions after examining patients with 0llier's djsease. Langenskiö1d and Edgren in 1949, after inducing radiation'iniury to the epìphysea'l plate confirmed these observations.

Solomon jn 1966 postuìated that the transverse diameter of the epiphyseal cartilage plate increased by appositional growth from the overlyÌng perichondrium, and that the same source is responsible for lateral extension of the articular cartjlage during growth. Rang in 1969 agreed with that conclusion and described the type 6 physeal i nj ury.

Shapi ro (1977) studi ed the peri phery of the ep'iphysea'l carti I age of rabbits using light and electron microscopy after label'ling wjth 3U-thymidine, 3H-proline and 3H-glucosamine and histochemical stains for proteogìycans and alkaljne phosphatase. By these methods three groups of cells in the zone of Ranvier were identifìed. The second

I evel group of cel I s were chondrobl ast precursor cel I s that contributed to appos'itional growth of the physis. The other cells were either fjbroblasts merg'ing with the periosteum and provìd'ing a periosteal tethering mechan'ism, or osteoblastic progenitor cells deep

-38- 'in the groove prov'id'ing a cuff of bone surrounding the epiphyseaì growth plate and the adiacent metaphysis.

In 1867 0llier performed a series of experiments to observe the effects of iniury on the epiphysea'l p'late. Different degrees of j njury were created. He found that superf i ci a'l I i near i nci s'ions across the epiphyseaì p'late of skeletalìy immature rabbits and cats had little influence on bone growth, but that deep incisions led to growth arrest. Multipìe needle punctures of the p'late did not affect growth. 0llier also showed that experimental separation of the epìphysis did not influence growth if the epiphysis u,as immediate'ly rep'laced.

In 1873 Bi dder proved that a part'ia'l I esi on of the epi physea'l pl ate was followed by the formation of a bone bridge between the metaphysis and epiphysis. The bone brìdge caused retardation and degenerative changes in the phys'is starting from the side of the iniured area. His observations were confirmed by Nové-Josserand (1894).

In 1878 Vogt d'id not find any retardation of growth'in goats and sheep if epìphyseal separation occurred at the natural I ine of cleavage of the ep'iphyseal and metaphyseal iunctjon. However, if the ep'iphysis was deeply lacerated at the time of separation, then growth retardatjon could occur.

Usìng rabbits Jahn noted in 1892 that if transverse'ly oriented discs of the epiphyseal p'late 0.5 to 1.0 millimetres in diameter were removed from the p'late at the ep'iphyseal end of the physis columns' then these columns were unable to reproduce themselves.

-39- Haas j n l9l7 and 1919 analysed the growth di sturbance i n the metacarpal and of dogs produced by surg'icaì 'incisions, crushing, and other injuries of the centre of the ep'iphyseal pl ate.

Gatewood and Mullen (1927) and Banks (1941) showed that jf the damage h,as confined to the epÌphysis and did not involve the epiphyseal pìate, then there was no effect on growth.

Friedenberg in 1957 found that, in attempts to prevent bone deformity, he could not control the reactive osteogenesis secondary to the trauma. Resection of sectjons of epiphys'is in rabbjts resulted jn premature arrest, probably as a result of the resectjon of the regenerative zone of Ranvier and ossifjcation of the ring of Lacroix. He noted that, in anjmal s with minor peripheral deform'it'ies, micro-fractures of the osseous bridges were constantly induced. Johnson and Southwick in 1960 confirmed this micro-fracture effect as a form of metaphyseal remodel ì i ng.

Ford and Key in 1956 perforated the epiphyseaì cartilage of young rabbits with a l/8" drill. Thjs did not cause major shortenÍng in 'larger non-osseous or fjbrous bnidges. tJhen drill sjzes were used shortening became more marked as a result of osseous bridges.

Harrjs and Hobson (1956) studjed the displaced proximal femoral ep'iphysis in rabbits, finding a line of cleavage in the zone of hypertrophìc cart'ilage cells. In monkeys, Dale and Harris (1958) confirmed that separation occurred through the junction of hypertrophic cartilage ceìls and the calcified port'ion of the growth

40 plate. This substantìated the work of Haas (1931)' in which he attempted ep'iphyseal cartjlage plate transfer.

Campbel I , Gri sol i a and Zanconato ( 1959) studi ed the hi stol ogi ca'l effects and growth djsturbance caused by surgica'l trauma to the epiphyseaì plate in immature dogs.

The summary of the findings is represented 'in Fig. 4.1.

It was shown that small diameter central defects to the physis up to 5 x 0.045 centimetre drill sizes either with or without pins caused minimal growth retardatjon. If a threaded pin or larger diameter hole of 5/32' was used then physeal arrest occurred. t'lith peripheraì defects, i f the metaphysi s and ep'iphysì s of the di sp'l aced bone segments became avascular or denatured by alcohol, there was maximal growth retardation.

The effect on long'itudina'l growth of the bone by metal p'ins and wires transversing the epiphyseal p'late has been investigated by several authors [Haas 1945, 1950, Gelbke 1951, Siffert 1956, Key and Ford 1958, Campbell, Grisolia and Zanconato 1959, Ford & Canales 1960' Kolesnjkov 19671. It has been shown that smooth p'ins cause least di sturbance.

Many authors have exp'laìned the vulnerability of the physis to other than mechanical trauma. Temporary ischaemia caused by moderate deviatjon of the ep'iphyseal plate may lead to , thereby causing bone bridge formation between the epiphys'is and the metaphysis ITrueta & Amato 1960, Troupp 1961, Brashear 1963' Young 19661. It has also been possible to create physeaì iniuries by using

-41 P4qn$gr_.*.1r EEtAñy Of 6ñ@ DISW'mAüCß Cå[t6ß'lÐ tT ¡ffiXcAL $nûüA'ÎO tmn ED¡FHr'tÛâ[¡ nlfrm Iñ ImnF¡üm q¡GS. For e.ntral grwth amort tl¡c es-i,tle.rl (nlnlmll i¡tjury Í'aa ú 5/'32' d,rnll hole, úlroal for ¡nriphcral rctrrôltion tha roqmirommt r,aa ava¡c'ulari.ty of thc dirplaeoú bo'¡n aognrontr. (Rcpr@lrcod rlth pcnriari,on fror Clryuboll, J. C. , Gri'¡olla, A. tûd .8anc@'ntto, G. Tho of,foetr ¡lroduccd I'n t¡ùG ¡ cartil,agl,n'olrr cpLphysceL platc of, l"qraturu deq¡c Uy ¡ ' ex¡nrimr'rtal rurgl,cal trarmte. J. Èonc JoLnt 8utr9. , t llA¡ 1221, 1959. I Lòne Drauöng No. of General Efect on Grouth Grouþ Oþeratìon of Oþeratòon Oþerøtions

angulation I Resection of the margin of the epiphy- 6 Minimum seal plate, epiphysis, and adjoining metaphysis

2 Longitudinal osteotomy through the distal radial and femoral epiphysis crossing the plate in a plane perpen- dicular to it Minimum retardation a. No internal frxation 5 Minimum retardation b. Internal frxation - one screw trans- l5 versely across the metaPhYsis 5 Maximum retardation c. Detachmentof all soft tissues from fragment, with rePlacement and fixation Maximum retardation d. Detachment of soft tissues, inser- 3 tion in 90 Per cent alcohol for ten minu tes, rePlacement, and fi xation l5 Variable retardation 3 Longit Y in the *"!1- physìs tending distallY (none to moderate) io it e with seParation across line of cleavage

4 Drilling of holes longitudinally across the disial epiphyseal plate of the radius and femur Maximum retardation a. One hole , one-quarter of an inch 2 diameter in l3 Minimum retardation b. One hole, five-thirty-seconds of an inch in diameter 5 Minimum retardation c. One hole, five-thirty-seconds of an inch in diameter, with the inser- tion of beeswax No retardation d. Eight to ten holes,0.45 millimeter 5 in diameter Arrest 5 Longitudinal insertion of cortical-bone 8 grafã thomogenous), fr ve-thirty-second.s ðf an inch iñ thickness, through a drill hole across the distal epiphyseal plate of the femur

6 Longitudinal insertion of smooth metal- lic p"ins extending from the articular ,,t.fuce of the epiphysis into the meta- physis of the distal part of the femur and radius t4 Minimum retardation a. Steinmann pin, five-thirty-seconds of an inch retardation centi- 4 Variable b. Five Kirschner pins, 0'045 (none to minimum) meter r7 Arrest 7 of threaded er of an inch of an inch) rface of the epiphysis into the metaphysis of the distal Part of the femur

42 x-ray irradiation IPerthes 1903, Engel 1938, Rejdy et al 1947]. Bone-seekÌng radio-isotopes result in simil ar changes IRay & Thompson, êt ô.l, 19561. A diet rjch in Strontium90 is able to disturb ossification and cause degenerative changes Ín the physis IStorey 1965]. Externaì physical changes such as frost bite probabìy cause damage as a result of vascular occlusion ILindho]an' Nilsson &

Svartholm 19681.

Growth arrest produced by a bone bridge between the epiphysis and metaphysis is used in clinical pract'ice after the method of Phemister (1e33).

Irrespective of the aetiologÌcaì factor causing a bone bridge' it is evident that it constitutes a mechanicaì 'impediment to growth' and the size and densjty of the bone bridge are in direct proportion to the growth disturbance [0ll ier 1867 , Bjdder 1873, Nové-Josserand 1894, Nakahara 1909, Haas 1919, Phemister 1933, Ford and Key 1956, Friedenberg and Brashear 1956, Friedenberg 1957, Campbel'l, Grisolia and Zanconato 1959, Johnson and Southwick 1960, Heikel 1960' l96l' Barash and Siffert 1966, österman 19721.

The percentage of physeal area that has to be damaged to create a fusion has not been documented. For this reason, for the purpose of this work, it was decided to use an animal model of peripheraì growth p'l ate damage by creat'i ng bone bridges after the cl 'i n i cal method of Phemister. A piece of pe¡ipheral bone, including epiphysis, phys'is' and metaphysis is reversed. It was recogn'ised that small bone bridges have caused only minjmal growth djsturbances.

-43- 4.3 MATERIALS AND METHODS:

4.3.1 Animals:

Th i s ser j es of experi ments uras undertaken on Meri no/Suf fol k cross-bred sheep since s'imilar experiments on I imb lengthen'ing have been performed us'ing sheep as a model IMontjcell i l98la, l98lb, DePablos 19861. Hecker in 1983 indicated that possibly only for the dog does there exist a wider range of experimentaì surg'icaì techn i ques .

An animal of suffj ci entìy ì arge skel eton i s 'important when considering the need for reversal procedures of deform'ity. In 1978 Sledge and Noble reported that, experiments failed in a rabbit model with a distraction frame to reverse deformity, due to fractures of the tong bones through the pin sites on the bone, and so recommended studies on larger animals.

Bisgard and Martenson (1937) showed that in goats a Phemister technique (1933) of induc'ing a bony deformity could be satisfactory' but had some difficulty in induc'ing a compìete growth arrest if the remnant pìate was left und'isturbed at the time of reversal of the

epi physea'l/physeal/metaphysea'l bone segment.

Bright (1972,1974, l98l) used pupp'ies for a series of experÍments

i nvo'l v'ing reversaì of bone arrests . Du¡ing the course of the author's experiments, it was consjdered that the use of puppies would

not perm'i t compl eti on of the expeniments because of the anti -vivi section i ssue.

44 Other larger animals such as dogs and pigs have a longer age to maturity (up to 6 years), but sheep rapidly mature to adult height by

9-10 months even though fu'll skeletal maturity does not occur until 2 l¡2 years [Newton-Turner 1953, Allden 1965]. Sheep have a potentia'l 'l'ifespan of l5-20 years in the laboratory. 0n farms 5-8 years is suitable for long term experimentation after surgÌcal modification. The relatively rap'id maturation would allow study of the long-term effects of any physeal manipulation on premature physeal closure.

The pig is an alternative'large laboratory animal. Two books on this

subject have been pub'lÌshed "The Pig as a Laboratory Animal", lMount and Ingram 19711 and "Swine in Biomedical Research", [Bustad and McClellan 19661. Pigs have many s'im'ilarities to man, particularly in their digestive system and sk'in, but they can grot{ to an unmanageab'le size and can be most unp'leasant and uncooperative animals in the ìaboratory. These attributes restrict their use.

Histological'ly, the characteristics of sheep bone have been found to be similar to man IPerren 1969, Monticelli l98la, l98lb, Peltonen 'larger 1984, DePablos 19861. Experiments on animals having ìonger peniods of immaturity are more applicable than experiments on rabbits or mice. This is because there are risks of failure by producing a fracture in smaller bones and also the growth potentia'l of the physis can continue throughout adult life in some species such as the rat.

In l98l Seinsheimer and Sledge showed that physeal activity was age and site dependent. Therefore, to obtain lambs of known neonatal ôge, a program of animal husbandry !'ras establ'ished IFoster et al 19341. This ensured consistency between experimental animals for comparative rate of physeal activìty.

-45- For the provocative injtial operative procedure of physeal damage at sjx weeks of age the lambs were transported with the ewe and weaned in the post-operative period.

The hind limb was selected as it has no fibula. It represented the tibia as a s'ing'le bone IS'isson and Grossman 1953]. The t'ibia has a s'ingì e physi s proximal ly to contri bute to angul atory and I ong'itudi na'l growth potenti a'l . The forel i mb bones !',ere not used because the lambs would be hjndered in mobility, particuìarìy in standing from a

ìy'ing posi tì on where kneel i ng i s often requi red.

4.3.2 0perative Technique:

6 }leek old Iambs had a generaì anaesthetic induced by Pentothal and

Fl uothane/Nitrous Oxide technique.

The experimental hind limb u,as random'ly chosen. The contralateral limb had a sham operatìon as a control. A sterile free drape of the 'longitudinal I imbs was done. A medjal incision exposed the

subcutaneous tissue, and then an arthrotomy of the knee joint v',as performed to determjne the level of the prox'imal physis of the tibia.

The physis t^,as inspected d'irectly using binocular loupe magnification of x 2.5.

A 0.062 mm. Kirschner wire (K-wire) was placed in the ep'iphys'is and a K-wire marker was a'lso placed in the diaphysis a known distance from the proximal pin. In earlier experiments this djstance varjed from animal to animal . After the init'ial pi'lot group of 5 animals an F-shaped template of confjrmed 20 mm. separat'ion also ensured para'lle'l placement of marker pÍns (Fig. 4.2).

-46- Fi.gn¡ro ,1. Z I ß;tf,'B XAnmf, .

Ftgmrr {.3. I ¡ A.p. *-ra!r ¡bsur t-rire mrh¡r pü,m ir lùtn oo Ì,oüt rd rlgült tl,Þtl ¡roct o¡nrmtivol.y. !ü'hc ß-uûrer rcnain in ¡i.tr¡ and tluc pi¡r ¡o-grr¡.Latiotr gryo a mtturc of bo¡ro varu¡ ançulatio,n" F tçdr&.tå ln'Gl'Nd to eürtÉG of, r pf.üe at a k¡rom lO n .gc¡l. It eonfirrcd that tlrc r¡to of, proxLml tibtll. gh¡noat grouth ü¡¡ ln/u¡oh. (u,ru¡t¡bllrDod drta) METHOD OF PIN PLACEMENT

20mm

"F" ternplate

47 These radi o-opaque markers remai ned i n pì ace throughout the

'l experi ment to aì I ow measurement of ength and angul ati on . Intra-operative use of the Image Intensifier (Toshjba 6XT) confirmed the pin pos'itions in each case.

Using the technique of Phemister, a s'ingle partial peripheral growth arrest uras undertaken i n each experimentaì ti bi a. A I sq. cm. defect of the growth pìate was excised en óloc using a I sq.cm. box chjsel and transposed (Fig. 4.3). To quant'itate the effects of 'larger bone bridges, 2 sq. cm. and 3 sq. cm. defects v'rere created by the same method.

In alt experiments no further physis was exposed or removed other than the s'ingle operative field. The soft tissues were sutured over the bone, and the skin closed with a contjnuous 0 dexon suture (3.5 metric green braided polygìycolic acid sutures coated with polaxamer 188 Dav'is & Geek) after Betadjne (Pov'idone - iodine solutjon B.P. Delta-t,lest Ltd., l,lestern Australia) solution was placed in the wound to decrease the risk of infection. Also to limit the risk of wound infection a 2 ml. b.d. intramuscular 3 day peri-operative course of Streptopen antibjotics t{as g'iven. (Procaine Penjcillin 200 mgs/ml and Dihydrostreptomycin 200 mgs/mì, G'laxo.)

0n the contralateral limb a control sham operation t¡,as performed. Thjs involved an jncisjon of the skjn and a knee arthrotomy to expose the prox'imal tibja. A longitudinal singìe incision of the periosteum between the ep'iphys'i s and the metaphysi s u,as done and K-wi re pi ns were p'laced into the metaphysis and epiphysis as previousìy

descri bed.

48 &Fimrr l. 3 r üD'[DËt8tñ[, }mmt? ût tffiütr@t3tt8, åßrc@mÛre @ Pm[üfljt. À I a'E- ct- frrgrr*nt of g'routh Px.rt¡ utr exet¡'aá cn bLoe l*llng a bo* chi¡on. a.¡rd t¡ra,ncpl.antod vLtù s,nêc rcvçrsed- ( h¡lroöuord rlth ¡nem.irci.on fron Pù¡cn"i.¡tcr, D. D. (trcr¡tive rarcüürmû.t @f, l"omgnülÂfran.I goøuth ûr M.e f,n thn tr.rt.rtBt

of dofor¡ûtl,c¡. , ¡. Eo,ne JoLnt $urg,, tr5¡ I, 1t33. ) , I Transplant ready for insertion with Area of ends reversed transplant ì \ outlined I I r¡ I { ¡ t, a r ,t

a À \o 'Epiphyseal cartilage plate ü i ìi ¡, I ¿ ¡l I I d \ I ' Transplants re- 'ì t versed and in situ .'' ,l t. ¡ t: t ,.i ,i l, I At the finish of the operative procedure under anaesthetic the male lambs were castrated, to allow ease of handlÍng post-operat'ively and all animals v',ere crutched to dim'inish risk of stock loss.

0n recovering from the anaesthetic, the lambs walked fully weight bearing and v',ere transferred to the farm for on-going observation after a 3-5 days post-operat'ive care in the animal house.

At three months post-operation, anima'ls brere killed by nembutal overdose and fusjon confirmed by histological examinatjon.

4.3.3 RadiologÌcal Techniques:

To avoid any variatjon in magnification all x-rays were taken in the same standardized manner. The hind I imbs u,ere held in neutral rotation for the antero-posterial view, and full external rotation for the lateral view. The djstance between the tube and the x-ray p'late was standardized by us'ing a perspex x-ray cassette ho'lder, upon wh'ich the lamb l'imbs were held, and the focal distance of the tube was 80 cm. Post-operatjve x-rays confirmed the marker pos'it'ions.

Initialìy for the p'ilot animals the x-rays brere repeated in all lambs at 2 week'ly 'intervals. The sheep were given a Pentothal anaesthetic for these x-rays. This frequency of radiological confirmation of fusjon proved unnecessary and x-rays brere then taken at 6 weekly intervals to monitor the progression of peripheral physeal fusion.

The state of the epiphyseaì plate, the longitudjnal growth of the bone, changes in varus deformjty, and the site of the provocation

procedure !,Jere recorded on the x-rays.

-50- 4.3.2 Bone Deformity Index:

(i) Bone Lengthening Ratio:

In al'l x-rays the pin distance and pìn angìe trlas measured, as was the bone length and bone angle (Fig. 4.4). Serial measurements of bone length, pin-separat'ion distance, bone angle and pin angle were undertaken (see Appendìx I).

Desp'ite using a standard radiographic technique there were variable magnification effects from each measurement taken at different times. The magnification effects remained consjstent for the same day when both I imbs were rad'iographed in the same I'ray. Thus, to e'liminate the magn'ificat'ion errors, a ratjo of bone length and pin distance hras calculated as suggested by Dr. N. Fazzalari (Fig. 4.5).

The bone I engthen i ng rat i o 'i s equal to Pin Distance Bone Length

Therefore each bone may be cal cul ated as;

Pin DjstA!çe = Çtlt X nification factor I Eõñe-tenstF- ÃiBi magn ca on ac r

El iminates magnification I

i.e !rDl BONE LENGTHENING AtBt RATIO TIBIA I

AND CtDz x magnification factor 2 AZBZ magnification factor 2

El iminates magnification 2

i.e. 9ZDZ = BONE LENGTHENING AZBZ RATI0 TIBIA 2

The ratio of: BONE LENGTHENING RATIO OPERATED LEG

-51 ?ig¡rncr ¡1. ¡l ¡ R¡NDX@,@GICA& B@m DßfomilTtr '

¡ totturÖmütt of booe rcogth ( A-D I and pûn D). :lfeasuronontof,BoncAngles?hatlon'êanrEle . t th. ühûft/.rti"er¡lrr rr¡rflco 3nElc' ¡Ëoasi,rreimntof,Ptrn.ànglèrEithardi'reetly' Cob,b ætLrcd ( 19{t ) -

Iñe R*ç[Or ?o r¡tûo sf, of thr of¡entod Log rrrr dividod by: tho rati'o l of @f, tne co,ntràJ. log. trnitiall!¡,'thi¡ ratio ra¡ 1.@O'@ a,u¡d fell to 0.50'0 aa arroatmnt occurrgd. MEASUREMENT MEASUREMENTS OF OF BONE LENGTH BONE ANGLE PIN ANGLE AND PIN DISTANCE

c

D

B were proiected through lhe pins On the X-rays l¡nes were projected along L¡nes ¡n order to measure the pin angte [col A-B=Bonelength the shaft of the bone and across the t¡b¡al (Atter method of Cobb) C-O=Pind¡stance plateau, in order lo measure the bone angle the C - D = 20mm with template measurement

BONE LENGTHENING RATIO Elimination of Magnification Effect TIBIA 1 TIBIA 2 Ar Az Cl Cz Dr Dz

Br B2

52 defined the parameter of final bone 'length. Initially this ìs 1.000 or sljghtly less than or greater than 1.000 as determined

by b j o'l og'i cal vari ance .

(i i ) Bone and Pi n An I ati on:

The bone ang'le and pin angle measurements also vary with djfferent degrees of rotation lBrodin 1955, österman 7972]. To minimize this error in measuring the deformed bone axi s for the bone angl e measurement the pin angìe v'tas taken as the most accurate angle of bone deformity. The radìo-opaque p'ins that rlrere inìtially parallel could be measured, with protractors directly, or using the method of Cobb (1948) to measure the deviation (Fig. 4.4).

This gave a radiolog'ical deformity index of two parameters.

1. Bone lengthening ratio. 2. Pin angulation.

The anguìation and ratio determjned the radiologicaì evidence of a

fus i on across the epi physeal p'l ate .

(iii) C.T. for measurement of area of fusion mass:

0nce cleared of other tjssue the bones were sent for computerized tomographjc section (G.E.9800) at Flinders Medical Centre. 3 or 4 x

1.5 mm. thick transax'ial abutting cuts at 5 mm. spaced intervals were

performed commenci ng at the superi or marker pi n i n the ep'i phys'i s . Results were recorded on a floppy disc (F.D.34-9000-18613 * Datalife by Verbatim) and also hard copy x-ray of the transaxial scan of the bone at the level of the epìphyseal p'late (Fig. 4.6). The less dense physeal cartilage could be seen in relatjon to the cortical and cancellous bone. At the level of the operation the tissue reaction

53 Ffgurc .1..6r cqtF¡tÍ!ñrsED lü(redmrPmlc 8DeÎrffi @ [Hrß TrÜn^*il; PlNf8lS. Thc arça oi f,ú¡Io,n m¡a is caler¡l,atad a¡ a porcentagl of thc total lrc¡ l,¡m'diatc1y adJaccnt to the proxi.nal e¡li¡lhyci.r at ths levcl of tlm phyri.r. fhc dsn¡t ty of tho bo¡rc f,ulLs.n i.l groater tl.lan ?5 ltour¡'¡field umLt¡ and Lr,outli.nod by thr brgken l.ina in thl¡ f{grure Dy eo¡¡nrtoriccd ple'nimtry o'n'¡cctlon¡ of th€ tl"bir thÊ atrca of phyrcal dit6¡ption va¡ calculatcd a¡ a f¡lrccatlgo of thc vhole phyrcal arca. vç could be separately identifjed. The density of the tissues was recorded in Hounsfjeld Units (H.U.) by the scanner. The density of fibrous tissue ranged fron 20-75 H.U. and bone 75 H.U.

The area of a transverse sectjon of bone at the physeaì level was estimated using a pl animeter. The average of three separate measurements was taken of the adjudged area of bone brjdge damage at the physeal level. This gave an area of the defect as a percentage of the physeaì area as a quant'itative index of the extent of the perìpheral growth plate damage necessary to produce a part'ial growth arrest.

4.3.5 Examination of Spec'imens at Autopsy:

All specimens were taken at autopsy, and soft tissues from the lower ljmbs removed below the knees. Right & left limbs were separateìy marked and immed'iately placed ìn l0% neutral buffered formalin. 'length Measurements of bone and pi n di stance v'rere taken wi th ca'l'ipers (Fig. 4.7).

4.3.6 Histoloqical Quantification:

All bones had whole coronal sections of the proxima'l tibia of the operated and non-operated side prepared (Fig. 4.8).

The marker pins were used to identify the coronal level of provocation of the fusion and also the level of the sham control side.

After fixation in 10% neutral buffered formalin a band saw was used to cut coronal sections of the bone at the marker pins. Occasionalìy because of circumferentjal bone growth, it was djfficult to find the marker p'ins. To eliminate the potentiaì for sectjonal error the

-55- (p Drsmüe[ Aç tuü(msu."tü Crni¡rcn n¡¡¡urod th¡ öi¡ttñelat ¡ftor ro¡gnsr! øf thG ú@ût ttr¡¡u¡ rmd oxsr*¡ivr b@no gmonht o,var tln pirnr.

Figure {.7.2s Spceirren fine grain x-ray at autopay. this ¡ü¡or¡ evidoneo of partial' fusiori. Þ I I i,: f lÈ / {" tf1

I -- JI I # l¡ I \

l; cl2 3 4 5 t CMS. f".M.c & Fûmrc {"t¡ FilßjPA&nüIOt CsF HnÚ' it[¡ffiinG:ÀE, ãffif!CIt. : þtml ¡uroxlnel' tibi¡ ( eont¡rol' ridc ) @f trø ¡rin traèt¡ in the opù¡üryrfË tor diatd[!¡¡lo] ênrure that th,e sectio,n lraa boon takot¡ through thc apprryriate Lç.vçL (x 3)

Fûeù¡rè ,1.8.2 r Coron¡.I. aaction of thË proxi'ml tibia rith a b'and slr etrt 5 m ri.dc at the level of tha tmrker pLnc ' +

!

I ,¡- I \¡ ) |t I ì

HISTOLOGICAL SECTION

section

pin hole

operated site

57 coronal sect'ions were pl aced i n decal ci f i cat'ion sol uti on and then the K-wires removed before cutting sections to prevent microtome damage.

Decalcification was carried out us'ing l0% EDTA (ethylene' dìamine tetra-acetic acid) in 7% Hydrochloric Acid. Fine grain radiographs checked the degree of decalcification.

Sections were embedded in paraffin wax. Three x 5u thick sections at intervals of 100u were cut using a Jung motorised sledge microtome. These sections were then stained by Haemotoxyl in and Eosin and examjned by light m'icroscopy on 3 occasions by the author and Dr. M.A.M. Rozenbilds, Dept. of Patho'logy, F.M.C. At the time of i nterpretati on of the mateni al the operati on detai I s ù'rere not known to the observers . Observat i ons tr,ere made of the state of the epiphyseal ptate and the presence of bone bridges, or of bone fusion between the epiphysis and metaphysis. A fusion was determined as be'ing present or absent.

The host tissue response of the ep'iphysis, metaphysis, periosteum, and physis was determined.

The normal physis shows the relatjve proportions of the resting, proliferative, hypertrophic and mjneral'izing zones of the sheep (See Fig. 3.6.2, Chapter 3).

In these large spec'imens the angular deformìty can be seen (Fig.

4.e) .

4.4 RESULTS:

Comp'lete data was obtained from I anjmals with I sq.cm. defect' and 2 animals with 2 sq.. cm. defect, created in the physis after the techniques of Phemister (1933).

58 p'mil¡nnn Fûgn*a! l.9r l&Cn@&C@¡PnC VIEW ol¡| A $IßIAL mßXOË. D¡fo¡nùty of, thc ùo6¡t ir r¡¡parent rj.ùh vtf,lL aagtrlatLon. f pcrlprhåral eortieo-c¡noll"l,sl¡s coqrLctc brr t¡ 8èên Joi'ning th rgnphyrir and ætaph!r.1¡. (x ll óç

;"_t ,B\ 4.4.1 Radiological Results:

The results for the radio'logical deformìty jndex are ljsted jn Table

44.

There I'Jas marked vari ati on i n the degree of angul ati on deformi ty rang'ing from 4o va'lgus to 23' varus. The bone 1 ength rati o was directly comparable to the degree of varus. If the ratio was less than 0.800 after three months then comp'lete radiological fusion had occurred. This was the case in 5 of l0 animals. lrlith three animals the bone tength ratio uras greater than 0.800 but still less than

1.000, and these an'imal s radio'logically showed a partia'l fus'ion. Two animals failed to fuse and had stimulation of growth.

Fig. 4.10 shows the variabi'lity of the provocatìve procedure in producing a growth arrest.

4.4.2 C.T. Results:

The results of C.T. scan evaluation of percentage of the phys'is damaged by the creatjon of defects are shown in Table 48. The Arithmetic Mean of the serjes was 24.2%. There was a wjde range of defect sjzes from 12.5% lo 49.3% of the whole of the physis.

4.4.3 Bone Measurements:

The results of direct measurements of bone length and pin djstance at autopsy are summarized in Table 4C.

4.4.4 Hi stol o Resul ts:

The histolog'ical occurrence of fusion and its correlation wjth the radiologìcal deformity index are shown in Table 40. There was fusion

60 fùE!ùg. N.!.Os ttAD.lOil¡@GIeâf¡, VlÑilA"Iff @ü F@ttOD' ñurûoor of, Frrtl¡X. uf"üh vlrùlf qD|*rrcot a,n'ryLatio,n and rhortc,ni.ng. vidc,ning of ttue PhYrir ' (åninal f ) h,oucver angutrtion occutrrod- (åi'lnl {) ¡ lton-fu¡ion r nù¡n*fblfø,r,r d grorth. lÜo Erouth arad qrynr*nt phytcal hr mdiell,Y. ovGrgronth of thc o¡rcratod ( Àûi.mt 3 t b@,Êo. (fnimn 2, t9

fi f>

-+ TABLE 4A

PROVOCATION SERIES

DEFORMITY INDEX AT 3 ]||ONTHS

( i ) Bone Lengtheni ng Rat i o (ii) Pin angu'lation

Lenqth Pi n Angul ati on Comment An i mal Aee Pi n Dì stance Bone gone Di fference No. (weeks ) tenqtÏ-Tãtio ratio 0perated Control (A/s ratio) (at 3 months) A ---T--

I 6 0. 135 0.r42 0 950 l8 0. 139 0.201 0 692 18'varus FUS ION

NO 2 6 0. 132 0. 143 0.920 FUS I ON 18 0. 200 0.203 0.985 4' vaìgus

NO 3 6 0.139 0. 139 I 000 l8 0. 199 0. 199 I 000 I va'lgus FUS ION

PARTIAL 4 6 0. I39 0 I 39 I .000 l8 0.192 0 2 l9 0.877 l0'varus FUS ION

5 6 0.134 0. 134 t .000 l8 0.136 0.190 0. 716 13'varus FUS ION

PART I AL 6 6 0. 140 0.142 0 986 FUS ION 18 0. 193 0.218 0 885 9" varus

7 6 0 I 45 0.148 0 980 I ON l8 0 I I3 0.212 0 533 23' varus FUS

PART I AL I 6 0. 140 0. 140 I .000 l8 0. 195 0.210 0.928 6'varus FUS ION

9 6 0. 144 0. 149 0.966 l8 0. 118 0.201 0. 587 l3 " varus FUS I ON

l0 6 0. 146 0. 147 0.993 t8 0.r22 0.207 0. 589 22 " varus FUS ION

62 C.T. SCAN RESULTS

TABLE 48 ot ol ol Defect lo Defect lo Defect Io TotaTTFea TotaT Area TotaT Area

Growth Growth Plãte Area P Pl ateTFea Run I Run 2 Run 3

L4 15'3/as.z L4'e/ao.ø 17 .2 TB.? I '8/ n .g 19.0 18.4 30 07103.6 25.?¡ 2a 25.3 2 ' 29.0 roo.t 24.t '9 ¡ loe .6 22.7 12'3 10. a7rr. 12.0 e'elee. 1l .4 o. 3 / al .e 14.0 o s 12.5 G) .9 13.8¡ 13.5710. 19.9 4 t0 ¡ ur.n 17.3 uo., 21 .5 o 2l .0 re.8¡rr., 18.07rr. 5 18.7 ¡ro.t 26.4 27.3 o 24.7 26.t 58.2¡ 27 .8¡r, 6 2e'7 /sa.o 50.7 rr.u 49.0 .u 48.3 49.3 17 -2/ L8.3¡r,., 18.17ur., 25.9 25.3 7 og .g 24.6 25.4 l5'6/oo.o La.e¡rr.o 8 16'2/ ot .o 24.2 23.4 21.0 ?2.9 20 .8¡ te.2¡rr.t 9 2L'o/es.g ?4.4 ru .u 24.3 2?.9 ?3.9

10 L7'2/gz.E 18. 6 rt '6/ gt.g 18.7 re.6¡rr., 20.5 r9.3 TABLE 4C

DIRECT I,IEASUREHENTS AT SACRIFICE OF:

Bone Pin Distance Difference

versus

Bone Length Difference. (at 3 months sacrifice)

Pin Difference Comment Bone Difference Di screoancy

( Short ) (Short)

I l0 mm Fus i on 6mm 4mm

2 4 m¡n No Fusion I mm (longer) 5mm

3 0mm No Fusion 0mm 0mm

4 6mm Parti aì Fusion 5mm lmm

5 12 mm Fusion l0 run 2mm

6 5mm Partial Fusion 4mm lmm

7 l8 mm Fus i on 6mm 12 mm

I 3mm Partial Fusion 3mm 0mm

9 15 mm Fus i on l0 mm 5mm l0 13 mm Fus i on l0 mm 3mm

64 in 4 animals by histologica'l criteria and failure of fusion by radiological criteria in 6 animals.

Histologìcal'ly, fusion has a characteristic pattern. At three months from the injtial surgery a cancellous and/or cortical bar of bone extends as continuous trabeculae between the metaphysìs and the epiphysis (Fig. 4.11.1). At higher porver (x 150 magnifìcation) the bone bar shows mature cancellous bone (F'ig. 4.11.2).

The tether to the physìs shows complete lack of organizat'ion by alteration of the width of the phys'is adjacent to the fusion mass (Fig. 4.12.1). Adiacent to the degenerative zone of the physis there appears to be an jncrease in the width of the zone of the prol iferative and hypertrophic cells, a'lthough mineral jzation of the hypertrophic zone is normal (Fig. 4.12.2).

As noted by Harris, Martin & Tile (1965), the growth plate in the transferred segment may remain viable although funct'ionless in the reversed bone block segment (Fig. 4.13). þlhere the periosteum has been djsturbed the peripheral zone of Ranvier shows a disorganjzed

'l physeal cart i age .

Non-fusjon has a variable pattern. 0n ìnspectìon the bone is not apparently angulated or shortened. X-rays fajl to show deformity or a bone bar across the physis.

Hjstological'ly there is fibrous tissue in the area of the bone bar defect. This is demonstrated in Fig.4.14.1' 4.74.2.

Attempts at an incomplete bone b¡idge occur, and there may be a vari abl e degree of tether. Immature woven bone creates the appearance of caìlus repaìr (Fì9. 4.15).

65 Fisuro a.tlr HIg$oLCIGICÀn" fvlM'Dtct oF' Fuslm¡ Doffi RtsPotrsE.

¡ Mi.mn 5 ¡Me . IxDüLüËÕraX. b@,rc fualoo @,f tb Fhnnúrtöa ¡coøøwc. ' ( * 2. S l Figrure l.ll.2 ¡ Thc high p

b ¡

;rà

( t I t À a il II

'{ ñ[tü@û¡æIe*& üvIDWìtr @ jnü8['trt Flfü'EtåL Et}ffi8.

¡ bouc tcthr iredirtely rtþecot Fig¡urç 'l.l3.l Co,nti'nr¡,or¡¡ of to an ab,u¡oml -eõ¡.umt¡¡lbyria. Thcre is Ìoe¡ of organi¿atio'a tÌ¡.a c'homdrocytc and thc phyelr i¡ n¿rrmr. (x 150) 8@D' r ldJfernt to th. dogprù.çltLvè ¡rtryloll. , - . thcre ð¡¡DEDctrü to Uc aq incrGaiG ln-Yidth of tho prchlryncrtrophie anßl hyß.itno$n¡.c eotr.h. lli,n¡rcI,i¡r¡túou ic nornl (x 25@) 'l ul; ,, {x^ ,._ '+û o l-r' <_Q

\.' /

tl

I I

-:l zr

r \ .\ i J dlt 'ìÁ Fi'en¡rq 4r 13 r Í'II8@¡OGICAL ÚYf DEÑeü Olf ltrill-FU8Id r ttt DüT8ü*X¡ T'RâñAPËII@ßGÎOW. Ir.r thc rcvcn¡¡ô bonc block th ¡ñyltr ar.n bc oblrryed to bc pr.rrat.+ trt Lr dLeorgm,i.¡ld and fr¡nctl'omLc¡¡ at 3 æ,r,rth¡ frol tho i.nltial proc.d,urt. (x 1.5) 89

::ib^;;':, Ftffi. {.t1. HrIt[t@IÆtBfCåü, wtDtrcü @f ffin-il0rüÛ'ü. üi¡nrtl l.l4.l¡ In Ami,.n¡L. 2 wlmrc tb.r¡ ü¡. g¡útill fulûqrt tho vù¡otc ¡'eettoa of, tho ¡rroxincl tlbia ¡hour thc f,ibros¡ ti¡euc and al¡o tmnipbôral bor¡c ¡ùyrit prolif,rnctiæ at t,h¡ !¡vc! of, rttrqrtrd f,u¡ù@. Íihr¡ !l dt¡emti'mity ø'f, thr bõ'aru tr*MiLüI.,(x 3.5rù r In , i'nal- I tÌ¡c fibrout tl'r¡uc ¡}læl mra @n.ntX. df¡eo'ÉrtLmulty rt tùo I'ewól of th ¿rttotrt at phyri.odc¡i.r. (x 3.5) * 69

{ rì À$Ë ,',a \ {

t

,¿,.à .n.,-. ¡liltll@ü,ÃG[iGtL úVXMret O¡f m-F0Eügr. ( âúr'me Cl ( x 350 ) It¡c fi,bro,r¡¡ ti.l¡uc ¡,brw¡ l l,om,gitudi"oaL crretrr ritlt¡ tùla brirû ørl.eoùrd rLqg tM }úat¡ @f, gËwth. Iinrturc canecllonr¡ bø,ne ¡h,ora a freetura ef,f,ect at thc lc'vc.I of, tln formr ¡ûnyril vi.th di¡eoqtùnuity gf tho bo,m tr¡boeul¡c. -'6 ''-''' .>

I ¡a ...

. ó ,'. À.=(_

ll - .::r. , ,.; \

æ7.' l+:A¡- tu-'.4 *¡.¡ '" ì ql*

,lt\ hr, lálh \\

¡rrta-:.'L -: :-t

-.{sÍùÈr

'¿¡.Q - q -i !i-

rl:_ \t

qlç { --{

I r ' È-"A;. -Flr¡;¡-. .l. The fibrous tissue is longitudinalìy oriented, as are the spicules of bone of the operated segment. The phys'i s shows angu'l ati on towards the metaphysis jn the operated segment, and this edge of physis'is further disorganized. The width of the pro'l iferative zone is increased and shows lack of ìong'itudinal array. The proliferative zone cells are more cellular and also show increase of intercellular

matri x.

4.5 DISCUSSION:

The histological findìngs of fusion and radiological parameters of fusion do not demonstrate an absolute correlation (Table 4D).

TABLE 4D

CORRELATION OF RADIOLOGICAL

& HISTOLOGICAL FUSION

Animal No. Rad i ol oq i cal Hi stol ogi cal Resul t Resul t I Fusion No Fusion 2 No Fusion No Fusion 3 No Fusion No Fusion 4 Parti a'l Fus i on No Fusion 5 Fus i on Fus i on 6 Parti al FusÍon No Fusion 7 Fus i on Fusi on 8 Partial Fusion No Fusion 9 Fus i on Fus i on I 0 Fus i on Fus i on

For exampìe, the first an'imal had a radio'logìca'l fusion that was not confirmed histologicatly. Thjs exemp'l ifies the difficulty of

quant'if i cati on of the resul ts of th'i s seri es of experiments .

From Table 4A radiologically the result was a varus deformity of 18" and a decrease jn bone length ratio from 0.950 to 0.692. From Table

-71 'length 4C there u,as a l0 mm. of di screpancy between the pi ns, but only a 6 mm. overall bone length discrepancy.

The overal'l I ength d'i screpancy from physeal damage i s I ess than predì cted. Thi s i s true i n al I the experiments. The second experimental animal had one'leg being I mm. ìonger than the control. This suggests that the distal tjbìal epiphysis may contribute to a "catch-up" phenomenon of growth to the length of the whole bone as described by Heikel in 1961. Thìs particular result, 'in conjunction with the occurrences of non-fusion both radiologically and histolog'ically in other cases, confirms that there 'is a variable degree of damage to the periphery of the growth plate that can be withstood without producing deformity.

Friedenberg in 1957 commented upon this phenomenon and suggested that the abi'lity of the physis to respond to minor perÌpheral deformities induced micro fractures of the osseous brìdges. Campbel'l' Grisolia & Zanconato (1959) and Johnson and Southwick (1960) confirmed these findings. The results of the experiments described confjrm these find'ings a'lthough question the interpretation of the histologìcal result since it is more probable that a bone bridge does not ever form and hence cannot fracture. The immature fibrous tissue

I ong'itud'inaì'ly arrayed i n the defect between the epi physi s and the metaphysis represent immature callus.

The C.T. scan (Table 4B) gave a measure of the extent of PhYsea'l damage and showed this could be used to estimate the extent of undamaged physis.

The density of tissue cavjties recorded jn Hounsfjeld Units i s such that the density of fat js from 0 to (minus) - 150". In the extremities no tissue other than fat has a negative H.U. density

-72- value. Any fluid that accumulates jn bones has a positive value. Thus ad'ipose tissue can be identified jn extremities with great accuracy.

To monitor the change in the tissue cavities with fat interposed as a graft the repeated scan would thus g'ive information on the change ìn the s'ize of the growth plate defect.

There r.,as a wide variation (Table 48) (12.5-49.3%) in the percentage damage to the phys'is produced by a standard I sq.cm. defect. This b,as thought to be due to the djffÌcuìty'in est'imating bone density difference (H.U.) between normal cancellous bone and the fibrous and bone tissue of the defect site. The contour of the physeal pìate is not pìanar and so the scan will not constantly pass through the level of the normal physis. This would tend to over-estimate the damaged segment. The data suggests that with small areas of damage, for example in animal 3 where the size of defect u,as 12.5%, the phys'is can continue to function and even break down bone bridges or prevent their formatìon by a micro fracture effect. In the jntermediate range of def ic'iency of 18.2%-26,7% a fusion may or may not occur.

Thj s i s determi ned by bi o1 ogi cal vari ants such as whether the perìosteum forms a bone bar that does not fracture, or whether the peripheraì periosteal, Pluripotential cells repair the physìs suffjciently to maintain physeal function.

The C.T. estjmation of 49.3% of physeal defect in animal number 6, wjth partial radjological deformity not confjrmed by bone bridge formation, hjstolog'ically suggests that the primary problem is the dysfunction of the physìs jtself rather than a bone bridge preventing its function. Alternatively, the higher percentage could represent a gross over-estimation of the defect s'ize, and is of concern when

73 tooking at determjn'ing the extent of the defect which could be filled wi th fat.

Fat is the only t'issue on the extremities to have negative H.U. and thus represents another marker to determjne the extent of the physis resected at reversal operati ons . It al so enabl es seri al C. T. measurements to determine with time the size of the defect.

4.6 CONCLUS ION:

These experiments confirmed that the technique of Phemister uras appropriate to a sheep tibìa model to produce partia'l peripheral physeal dysfunction, but that this peripheral lesion to the physis may not cause a premature growth arrest.

Rad'iolog'ically, a deformity index of fusion can be a rel iable indjcator of the formation of a bone bridge across the physis. However the shortening and deform'ity may also be a result of the extent of the growth of the physis that has been compromised.

Animals not showing radiolog'ica'l evidence of anguìar deformity and shortening would be discarded from the reversal operatìons.

The next series of expeniments evaluate the reparative potential of the phys'is, determine the extent to which an interpositional material could prevent bone bridge formation, and show the mechanism(s) of the recovery of physeal function.

-74- ChapteT 5 FAT INTERPOSITIONAL REVERSAL

5.1 EXPERIMENTAL AIMS:

A series of experiments was undertaken to determine: (i) the fate of free fat graft 'implants (i i) the extent of peripheral physis that can be resected and rep'laced with free fat to restore normal growth (i ji) the reparatjve potent'iaì of the physis.

5.2 REVIEt,l OF THE LITERATURE:

The treatment of choice'in iniury of the physis that causes deformity has generaììy been a corrective osteotomy. As growth conti nues, however, the deformi ty recurs, and thus the osteotomy may need to be repeated. Alternativeìy, in addition to an osteotomy the rest of the phys'is can be destroyed to prevent recurrence of angulation. This strategy results 'in the loss of 'longitudinal growth of the bone, and thus in limb shorteni ng.

In other treatments to equalize leg length the oppos'ite leg may requi re shorten'i ng by ej ther epi physeal pl ate arrest (physiodesis) or by a shorten'ing operation at skeletal maturity IPhemister 1933, Eyre-Brook 1951, Green & Anderson 1955, Menelaus 1966, Bìanco 1978, llinquist 19781 . A bone lengthen'ing operation on the shortened leg js also poss'ible ICodjvilla 1905, Magnuson 1913, Puttj 1921, Abbott & Crego 1928, Compere 1936, Sofield 1939, Harmon & Knigsten 1940, Abbott & Gìl1 1942, Brockway & Fowler 1942, Phalen 1942, Allan 1948, McCarroll 1950, Anderson 1952, Blount 1954, Goff 1960, Salter & Harris

1963, Kawamura 1968, 1981, Ilizarov 1969, Wagner 1971, 1978,

-75- Stephens 1978, Ray 1978, Moseley 1978, Monticelli & Sp'inelf i

1981c, De Basti ani 19861 . There are comp'l i cati ons assoc'i ated wìth these technÌques that incl ude; pin tract infectìon' fractures and non-union of the lengthened segment, dislocation of joints [Coleman 1986, Moseley 1988].

Many investigators have performed animal experiments to prevent the progression of deformity due to physeal fusion by remov'ing a port'ion of the physis and immediately repìacing it with an interpositional material .

In 1878, Vogt vlas the f i rst to i nvestigate the effects of epi physea'l separation. In a goat he used goì d I eaf as interpositional material on the proximal tibia, and in a dog a rubber film on the distal radius. He was able to prevent the growth of capìIIaries between the ep'iphysÌs and the metaphysis.

In 1956 Ford and Key used gel foam in a study of experimental trauma to the distal femoral epiphysìs in rabbits.

Also ìn 1956, Friedenberg and Brasheer used bone wax in the distal femur of the rabbit. Further results in the same model in 1957 brere reported by Friedenberg who used bone wax and methyl methacry'l ate.

In 1958 Key and Ford used bone wax in the distal femur of the rabbi t .

In 1970 Serafin used bone wax and muscle in the d'istal femur of the rabbjt. The free musc'le transplant was most effective in preventing bone b¡idge formation across the traumatized

epi physeal p'l ate.

-76- In 1983 Lennox, Goldner, and Sussman used fat and carti'lage in a rabbit distal femur model. Their results suggested that cartilage was superior to fat as an 'interpositional material.

All these experiments have included a peripheral iniury to the physis (a type VI iniury) to create a periphera'l defect. An i nterposi ti onal materi al was used to prevent physea'l bar format'i on , and any subsequent growth I'las observed . Resul ts have been varied, but there has been sufficient success to suggest that bone bar formation can be inhibjted or prevented. tlhen no ìnterpositional material is used bone bar formation occurs consistently, [österman 1972, Lennox, Goldner & Sussman 19831 a'lthough Bìsgard (1937), noted difficulty in obtain'ing fusjon in goats. Djfficulty in obta'ining fusion was al so confirmed 'in Chapter 4.

In the clinical field there are no reports of interposition materials being used in human studies for the preventìon of bone bnidges at the time of acute traumatic physea'l iniury. The technique of fat 'interposition has been utilized by the author at the Adelaide Children's Hosp'itaì during the open reduction of cases of comminuted type IV fractures and type VI fractures. Al though fol'l ow-up has not been to skel etal maturjty, results have been encouraging, and in all cases prevent'ion of bone brjdg'ing has occurred. Another appl ication of th j s techn'ique might occur during the exc'ision of a ben'ign tumour, a process which sometimes includes loss of the physis.

The treatment of an established physeal bar by excision of the bar has an experimental basis. The presence of iniured or dead porti ons of carti'l age i n an epi physeal p'l ate has been

-77 - shovln to prevent the formati on of a physeal bar fo'l I owi ng 'injury ILangenskiö]d & Edgren 1949, Heikel 19601.

The mechanism of repa'ir of the iniured portion of the phys'is has been assumed to be from regenerat'ion of the adiacent parts of the growth plate by a process of lateral creep. Nordentoft in 1969 observed that the physeal cartìlage regenerates and fills a drjll hole transversing the ep'iphyseal plate. However' in experiments where a bony connection exj sts between the ep'iphys'is and the metaphysis no regeneration has been observed IFord and Key 1956, Frìedenberg 1956, Campbell, Grisol ia, Zanconato 19591.

An'imal experiments have also been performed in which a bone bar js created then excised. Various interposition materials have been used w'ith varyìng degrees of success.

Bone wax has been most often used [Key and Ford 1958, Campbelì' Grisolia, Zanconato 1959, Serafin 19701. In 1957 Friedenberg used bone b,ax in the distal femur in l0 rabbits, but the results brere not encouraging because of fragmentat'ion of the bone wax.

In 1969 Nordentoft used no ìnterpositjonal material in the dog proximal t'ibi a fo] ì owì ng epi physeal/metaphyseal separati on concurrent with removal of a bone bridge between the ep'iphys'is and the metaphysis. He did not achieve positive results by this method.

In I972 österman reported the most comprehens'ive series of experiments using fat or cartììage or bone wax in the distal femur of rabbits. He concluded that suitable interposjtional

-78- materials prevent the recurrence of the bone bridge, and that deformity may be corrected. He suggested that carti'lage may be the best material. Langenskiöld (1986) has also demonstrated the effectiveness of fat as an interpositional material in a pig model.

In 1974 Bright used sil'icone rubber, s'il icone adhesive, or 'isobutyl cyanoacrylate in the medial distal femur of dogs. His conclusions were that the silastjc elastomer prevented bone bridge reformatjon.

Sudmann jn 1982, used no Ìnterpositional material in the rabbit distal femur model of part'iaì closure of the physis, and reported on the deì ay of bone bridge reformation with indomethacjn. The mechanism of this is conjectural, and has been substantjated in work by Conno'lìy et a'l (1988).

The first case in a human of fat interpositjon vras reported in 1967 by Langenskìöld. The case involved a 15 year old boy with genu recurvatum, secondary to a bone bar in the anterior proximal tibia. The aetiology was unknown. The bone bar was excised in 1965 and the space fjìled with autogenous fat. During the 1.5 year follow up the angìe of genu recurvatum 'improved l0 degrees, but there v',as no documentation of ìongitudjnal growth.

The first case document'ing longìtudinal growth was reported in 1980 by Peterson. In this case a tibia grew 76.7 cms after excision of a dìstal tibial bar in 1968 in a boy who was then 5 years of age. Follow up continued for 10 years; sheet silastic and ge1 foam were used as the interposjtional materials.

-79- Mu'ltip'le series of cl inical cases have since been reported in which several different materials have been used:

(i) fat alone v,,as reported by Langenskiöld (1967, 1975, 7978, 1979, 1981, 1986, 1987) and V'isser and Nielsen (1981);

(j) fat and bone wax has been reported by Vìckers (1980);

(iij) Silastic (S'ilastic Elastomer No. 382 - Dow Corning) has been reported by Bright (1978, 1982);

( i v ) Methyl Methacryl ate (Cran'i opl ast i c, Ino bari um] manufactured by L.D. Caulk Company, Mìlford, Delau,are' U.S.A. 19963) has been reported by Mallet (1975, 1978,) Vickers (1980), Klassen and Peterson (1982).

These methods appear to be most popular. However, few cases have been fol I owed to matuni ty to permit assessment of superiority of any one interpositional material.

The use of fat has the advantage of being autogenous, and no fore'ign material is inserted. Usually, enough fat can be found in the same incision to fill the defect, ôlthough sometimes other fat must be harvested from another site of the body such as the buttock. LangenskÌö'ld (1975) recommends the use of buttock fat.

Fat, however, has the d'i sadvantage of not produci ng haemostasis in the resected cav'ity. When the tourniquet is released the fat may float out of the cavity as bleeding occurs from the bone. Bone h,ax may be used to diminish the bone bì eedi ng IV'ickers 1980] .

-80- Closjng the periosteum over the defect to contain the fat also predisposes to neu, bone formation peripheraì1y, and to the reformation of a physea'l bar. This is undesirable since it may tether growth aga'in.

The operative defect also weakens the structure of the bone and therefore may predispose to patho'logicaì fracture IVickers 1980, Visser & Njelsen l98ll. Gradual ossification of the metaphyseaì defects do seem to occur [Peterson 1980]. The fate of the fat i n the metaphysea'l cav'ity i s sti I I undocumented. Langenskiöld (1987) suggests that hypertrophy and elongation of the autogenous fat occurs with progress'ive decrease in the size of defect in the resected physeaì segment. This is reported from animal and cì inical data ILangenskiöld, Videman, &

Neval ainen 19861 .

0f the other interpositional materials, silastic is inert, non-reactive, and virtually unaffected by long term exposure within the body. tlhen mixed with a catalyst, silcone-rubber monomer vulcanizes. ldhile it is in the semipoìymerized state the sil icone rubber can be moulded and pressed into the surgical field to fill the exact form of the cavity. It must remain in djrect contact with the exposed physis to prevent bar reformation. Steril ization of both the monomer and the catalyst is effected 12 hours prior to surgery. During the operation a culture js taken of the silastic after mixing but before insertion. The ìmplant is free-floating and easi'ly removed later. Silastic is current'ly controlled in the U.S.A. by the Food and Drug Admini stration (F.D.A. ) and its use requ'ires authorjzation since it is an Investjgat'ional New Drug.

-81 Bright (1982) recommends the use of Silastic elastomer No.382

(Dow Corning).

Recommended by Kl assen and Peterson in 7982, methyì methacrylate is lìght, thermally non-conductive, transparent, and easy to mould. Both the liquid (monomer) and the powder (poìymer) are steriìe as packaged, and may be mixed in the operating room. It is unnecessary to obtain cultures. It has had wide clinical trjals in both neurosurgery and orthopaedic surgery. tlhen used as an isolated substance it has caused no rejection, infection, or neopìastic change ICabanela 19721.

Because the solid substance fills the cavity there 'is excellent haemostas i s, and al so structural ly the bone i ntegri ty i s maintained. Klassen and Peterson (1982) strongly recommend the use of methyl methacry'late without barium. "Cranioplast" has the advantage of being radÌolucent, and this enables easier identification of physeaì bridge reformation.

In 1979, Langenskiöld and österman summarÍzed the results of the first 33 patients who had undergone clinical osseous bnidge resection wjth autogenous body fat imp'lantation. In this series there were 22 males and 1l females with a mean age of 10.3 years at the time of surgery. Five pat'ients were operated upon too recently for assessment and were not evaluated. 0f the 28 patients whose results could be determined from the tabulated lìst of cases 15 appeared to have good or excellent results. This represents 54% of the cases. Five pat'ients had fair results, equivalent to lg% of cases. Eight patients or

28% had poor results. Most of the poor results were due to

82 recurrent bridgi ng, requÍ ri ng repeat osteotomy or repeat resectjon of a recurrent osseous bridge.

In 1982, Kl assen & Peterson reported at the Mayo Cl inic

(1968-1982) on 7l cases of bridge resection. "Cranioplast" was used jn 68 cases, bone brax and fat in 2 cases, and silastic sheet and gel foam in I case. In all but one case there u,as some growth after bar excjsion. The growth of the bone operated upon as a percentage of the growth of the normal bone has varjed from 0 - 200%, with a mean average of 94% lKlassen 19821. For patients followed to maturjty the average growth 'is 84% of normal. Thus, the renewed growth may diminish the anguìar deformity, and also the rate of progression of limb

ì ength i nequal i ty. Occas i onal ì y there may even be a reduction of the length inequality (the treated limb grow'ing faster than the normal limb). In the one exception there was both proximal and di stal ti bi al bar formati on foì'l owi ng prev'ious irradiation for sarcoma of the fibula, there was no growth after bar excision at each site. Although the remainìng physis appeared radiological'ly normal it may have had no potential for growth due to radiation damage.

In 1982, using Medical Elastomer #382 (renumbered as X7-2320 for human use) Bright reported on the first 100 of over 250 consecutive patients with at least a 3-year follow up from the date of surgery. 70% had good to excellent resu'lts, 12% fair results, and 18% poor results. Over one half of the poor results were children for whom the operation was performed too late. These chjldren stopped growing within six months of the operat'ive procedure, even though their suggested at least one more year of growth remaining. For this reason the

83 recommendation was that a patient should have at least 2 years of remaining growth before considening physeal resection. Bright in 7982 also reported the occurrence of post operat'ive infections that compromised a successful result.

The use of free fat transplantation is based on the observation that it remains fat tissue in its new location. In 1924 Lexer fi rst di scussed thi s phenomenon and i t was confi rmed i n experiments by österman in 1972 and Kiviluoto 'in 1976.

5.3 MATERIALS AND METHODS:

As previously described, after the method of Phemister, on the proxìmal tibia of 20 lambs at six weeks of age a I sq. cm. defect was performed. 15 lambs had 2 sq.cm. defects and l0 lambs had 3 sq. cm. defects. The contra lateral limb served as a control. It remained unoperated upon after the initial operati on to p'l ace marker p'ins i n si tu.

X-rays were taken three months I ater to measure the bone deformity index. A confjrmation of deformity $,as present in each case which was operated upon for the reversal procedure.

5 . 3 . I Bone Bri dqe Resect i on :

The bone tether to the physis was excised with a high speed dental drill until a heaìthy cartiìage growth plate could be seen. The bone fragments were removed us'ing irrigation to minimize heat damage to the physis (Fig. 5.1).

Fat was taken from the knee fat pad and placed in the defect as a free transfer after the method of Langenskiöld (1967). It

-84- oPü ,wç*uu,e @ Pülv'ffiâ,& I'truÊß M$8eÎrffi RFOß Figrurc 5.1-1r Inei,sion over ttrc ¡rroximt tibia crt¡loltGs ¡nriorter¡m aftcr divi¡ioo of, tl¡c ¡of,t ti'c;l,lG ( hel-d ulth thc fo.rco¡l ! . ¡ Division of the porioctoutrt betvean tho Or¡ea rcflcôtcd t'lre¡nc le ¿ eonttn¡¡olc¡ tCIoo bridgo avident.

;

Figure 5.1.3: Àn osteotonre remcrves the cortical bone belou the proximal marker pin.

Figure 5.1.¡l: The bone shows the defect vhere the osteotome has cut the cortical bone. â:\''.,

-ila

L\. ..'- {t rs-t-+>-I --

tl

.. .\ I kümv I o't thr eo.lrtie¡Ir bßæ rr*n¡ll tb. Llor¡rc lÞütio,a of 'tl¡a bom ùrið!e'

I fu ür¡[& ! m &nt¡U Nt @ffi!ù€cÙ rmvtl o. BLneeuilrr lÞctPot (x ¿.51 ¡r¡i¡t 1n lûçntif,ieation of r¡otul, ti¡auc. tt r.

Þ \-: ;-¡rr

'1.-

¡> - ¿:-I

È: \ ¿\ Irri.gnti,oa r,lth ¡tcri.lo r.a!üm t,hrouqË nf.¡ri¡brr roft tie¡u tranm i¡ù thc o¡rcretlvc fi.Cld. Tho debrie is rorroved vith co,ntin'r¡o'rnc controllod ¡uctiot¡. f!.mn ã. !. t ¡ Ir.,r thc crr¡rer!.o'r ff'cfd of vfll.wr th nã.ml¡L iClioh¡¡l ¡iùryaia ca¡r bc aean.Ë trhr drrf,ret i¡¡ tllc opl¡ilrylerl nata¡ürylerX, Jrrmctlo"n l¡ ¡ècr¡ bcl,ou thc aupcrior bono m.rkcr pia. ì F-lgr¡,¡'ç 5. 1. , t Shovi.nE thc rcnr¡rtrt ¡ûry*ir ctryËriorl¡t and tlrc d,ontal b,urr pro{trG.ûively rcnoving tlrc boma bridge.

Figure 5.1.10: Pollovi,ng eoqrÎ'ote rcnovcl of th bonc bridg¡r tho nornrt rcri.&¡nl ¡nhycir c¡n bc aúUlrt€i.ted, a¡ a e'olrtLnûûGrùla lina la tho n'iddlc of thû ffcLd of vl,er.Þ ¡ Figure 5-1.11: Àrthrotoury of the ipsiJ-ateral knee joint srrous a large infrapatel-la fat pad.

Figure 5.1.12: Incision of the fat pad and harvest of the autogenous fat to be pJ-aced in the defect.

r suturG ot- tlro f,¡t to the eryi.¡tr¡r"ri.¡ or P nkcr pin to prGvå,nt loae of ¡roaltiom of the frt @cG Lt vr¡ plarecd i,n thr dcf,oat.

.Exci¡io,n of thè pcri-o¡tcr¡a to ¡lrcvcnt bfiCgc rcfomtio'¡t cn$ faitutG of th¡ ¡lroco&lro. was sutured into posit'ion with 0 dexon to the ep'iphysis or epìphysea'l p'late marker pin to prevent migration.

At the reversal procedure, 'i f overgrowth of the p'in t'ips had occurred, the epi physeaì or metaphyseal marker pì ns brere re-sited (for example An'imal ll).

After excision of the periosteum at the level of the defect the wounds b,ere closed in layers. The prev'ious post-operat'ive reg i me !,Jas undertaken j ncì ud'i ng prophy'l act i c use of antibiotìcs. ldeight-beaning v,,as allowed as soon as tolerated by the animal.

Under another anaesthet'ic one week after the reversal procedure axjal tomography (C.T.) of the tibia was performed to determine the extent of the growth p'l ate removed . The proxi ma'l epiphyseal pin b,as taken as the physeal marker, and the C.T. cut was taken at 5 mm. abutting slices distal to the p'in us'ing the techn'ique previously described.

An jmal s trlere autops'ied at three or six months to examine the difference in time response to the autologous fat impìants.

5.3.2 Radi ol oqi cal 0u a nt'i f i cat i on of Resul ts :

In all x-rays the pin distance and pin ang'le were measured.

The bone I ength and bone angì e were al so measured. The deformi ty i ndex !'Jas cal cul ated for al I groups of an i mal s at three months from the provocative procedure, and then at three months and six months after the reversal operat'ion.

92 5.3.3 C.T. Quantification of Results:

At autopsy a repeat C.T. of the tibia u,as undertaken and results were compared to the jnitial slice. Us'ing computerized d i g ì tal p'l an i metry the percentage of defect area to the p'l ate area was estimated three times, and the median taken (Fig. s.2) .

Statistjcal analysìs using the unpaired Student's t test rtas used to assess difference'in C.T. areas.

5.4 HISTOLOGICAL UANTIFICATION OF RESULTS :

The hi stol ogi cal preparat'ion b,as as previ ousìy descri bed. The whole bone sections þrere reviewed and quantification of the histology determined. a. Assessment of the response of the host tissue to the

i mpl ant :

Firstly, the degree of fjbrosis around the fat implant was determined. It was graded as zero if absent, one plus (+) if present, and two p'luses (++) if there was an organìzed capsule (Fjg. 5.3).

Secondly, the remodellìng activjty of the host cancellous bone of the epiphysis and metaphysis was estjmated by the degrees of 'l osteobl asti c acti vjty. There were 3 grades of remodel i ng : zero showed no osteoblastic actjvity; one p'lus (+) indicated active bone formation wjthout increase of trabecular wjdth; two pluses (++) indjcated active bone formation wjth increased trabecular wjdth (Fig. 5.4).

-93- Ft,gurr $.2¡ e .f . @&fAûUfIfIeA?[ffi @f DmYtFÀL Nå. Figurc 5.2.1r fhc C.1. ¡lic.c tlù.t juot i,nctr"@rd tbe Proxinal ¡rin vac takon a¡ the reprcscntatlvo ¡lLce for ealer¡l¡t aO,n of arcas- On the hard eopy tho defcet arrèa to bo ealculatod v¿rr outliaod ( broto¡¡ lino ) . figurc 5.2.2r ttrrir.rg a f,lop,py då¡e a digital ¡rlrai,rtcr tô¡ u¡cd to cal.culate thc rel¿ti.vc ¡urfaco arGaa. ThG ,¡Dåteôntügrc of dofoet to totrl lr{rü ülú cateulat.d S ti.ma cnd the ngan obtainod. RlZPLßN REY 3.29 r Rol 3l ltN 29 I sD t2 9. I R 12. I ltN r8 t sD 92.56 R 6.0

PY UU 8'C UL+C28{

?C' LRiBr2 SL'0t 94 (. f

-!:

'.t \

tu Ð À' ;*

FigUTC 5.3: HTS'T.(OLCIGICAL ÁSSESSFIET{T ÛF IIOST TISSUE RESPONSE IrO THE ITTIPLÀNT - CAPSULE FORT.IATION. Figure 5-3.1: The degree of fibrosis around the viable fat can be seen in this uhole tibial section of Ànima]- 20- (x 3)

The hi yras 0. There is a the fat and the hos Figure 5-3-22 One pJ,us (+) grade aÇ filrrncic wiflr arlìlâatêft fibres and fibroblasts as seen in i"i"-i^ll --t;zs-o it- l\lo plus ( *+ ) grade of fibrosis vith fornation of an organized (x capsuì-e in ÀninraL 26- 325) ) { - .i , -rl, ¡l. I t' \ ( \

( \¡ À 'L '')- :

t I q4rygp T!4r llE8l{Md6üeAt @!' tl@sr tta"æn rt*erIfri !O IIûtr XTPLåüE - BPTPHYSËAL T@P

FLqutc 5. {. f, ¡ ftrùG d'lgrce of epn¡ùylcrX' ncv broorl fornti'øa em b? ¡ma i,n th!,1 rmefi¡ ttbià¡' *rctûo,n øú M-ûnl trl. {¡: 3l thc hi.ghor poüèr fj.eld grðdG g¡a.3 O. thore i¡ nc, tre'bocular bonc thickeqirg lútd ll.ttle o¡tcpbilr}'ti,c aeti'ryity- (r 385) J lt

I aI

rt

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'.) lril'r'; Figure 5-4.2¿ (+) Epiphyseal bone torm¿rtaon as seetl rrr the who-l-e tibial section of Ànimal 20- (x 3) The higher lrouer field (x 325).' Note the increased width of trabecuJ-ae and osteoblastic activity- ì l'r

''!: ' s ,ó

F a _l .* I tdi- t

ì'

¡

.Þ t.,.øg ,:v'ú fig¡¡re 5-¡1.3r { ++ } G¡ri¡Ëya+al bo,rlo f,etr¡tion te s{¡'rn ln tbrc rhole tiblal ¡oction of Ànirial lT- (* 3) gh hi$þGr ¡wurrrod fi,¡td (x 32$l ñotr tbo m¡rc thør d'ø¡Ë!,r rl,dth of trabccr¡X.e,c a¡ad l.oqc of intar-trabocula spa¡ec. /t

\, ¡" Thirdly, the cortical bone thjckness of the metaphyseal bone adjacent to the defect was graded as zero (same), (+) intermediate, (++) double thickness of normal cortical bone (Fjg. 5.s).

b. The fate of the fat:

Fjrstly, fat bras assessed for preservat'ion of site of the implant: whether it remained physea'l (P)' or metaphyseal (M), or epiphyseal (E) jn situ. Secondly, the viability of the fat graft and any change in sjze !'ras graded: (i) (N) normal , (i'i) H hypertrophy, (iii) P/N partial necrosÌs, (iv) C/N comp'lete necrosis. Thirdly, the extent of interstitial fibrosis was rated as absent (0), present (+), marked (++) (Fig. 5.6). c. The recurrence of bone bridqe fusion:

Any bone bridge reformatjon was identified and the site, ejther peri pheral or central determ'i ned . The bone bri dge u,as then classified as showing cancellous woven bone or cortical bone (Fig. 5.7). d. The res se of the host sis:

The morphol og'ical features of the physeal contour were assessed.

Firstly, the th'ickness of the physis immediately adiacent to the fat graft showed a medial spur of physeal tissue. Th js v',as graded as either (i) absent (0)' (ij) present (+)' or (jii) markedìy elongated (++) (Fig. 5.8).

-100- rttoúæIcru. rûffi8,ru @ ru! !Ia88 EßDr'@ll - @llIct[ rm Mil,I"nre.

flg¡urc 5"5,1¡ (@! gr.dÇ, lt¡o eortlea]l bq,Êc i-¡ th ac ti.dth. t¡h¡rc na¡r bc ûrÐ i*eüG¡ç,c in ttu e*neorrøur b@ñc oú thr rctr¡$rgrrûc. ( r l. 5l

3, (+) grldc. Àü roclr in Àøi,mL 26 rtrcqc tho c is inerccrod in vidth. (x 2)

r (++) gr?do. A¡ sêcn ia ÀnLral e9 th corti.ccl. rca¡cd in iicc at tcdst tui.c.r tllc rf,dth of th¡ otbcr eortcr. (x 4.5) 0

VIJ p¡¡r

JO FlEeor¡ 5.ór HISîOÛÆIGICA.I" A8$tr8ffiilE @ü l*E lA? AWFO@AAFf. Flgurc 5.6.1¡ llor.ml f,at graf,t in situ at thc physeal .Lcvol (P) ui,tlmtlt ap¡ræont i¡rerca¡o in cíEc. (x 3l FÍgura 5.6.2¡ fnìmf 20 fu,r¡¡tratrr¡ el"o,ngntlon of t!toe defoet at 6 renthe fron tlro inltial ravcr¡al pr'ocedurc. It slr i.ntorprctcd a¡ cv,ü,&nç¡ of h¡ryortre¡uhy of thc graft rlth graft rcmini.ng in eontret wLth thc 'nomrl cpi.pü.rycoal bo,¡r,e. (x :¡ -{ú. ::"1& .'', isi' -": l¡" ù' .'"1..'''' ' " i ';i,z-; !.

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102 løl,r¡, 2Õ ðrmottrrto¡ ¡pirtlrX" rrrocnertl thc dcfqct rt 3 æntÙ¡r ui,th {,ntor¡titill fi,b,rorå¡ ( * I rnú' cqnu!,lr üo'rHt&ar.' Thc grlft ie rcrè rmttpþytorl (tt ia loeatloo. (x 3) r À¡ninaf &!' óm ltreto¡ eqrl'ctc r¡¡croric - tth mchd f,,mücrrti,ti'rL f{bto;ûr ( ++ } -ll Thore arc additio,nal fcatr¡rc.o in thc cpiphyro¡l dnd nrtagút¡'l¡ll, bouo.thût ¡.Imr þ@nG rlrcdol'l.ing. (¡ 3) .'Ê-.{ltt,'

I

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lo3 ülI8?0ü,OGtreA¡; À$S,E8ffifm 9D æm tnlm

FiEurG 5.7.1¡ Periphoral bo.n,c bar fonrrtior¡.? xn Àairal 38 thc c¿u¡¡e,cllous bonc b,rr cr,n bo cGGn joini*g tht ePl¡ùyris amd rtqÑvtl"r. A v{abl.o frt dcflet l¡ -pr..{üt .t thG plryecaL lcvcl. (¡q 3)

Figgryé 5.2.¿r Ccntral. bo'r¡e b¿¡r fomatiom.G IE tnùnl. I7l-ffirc-Eñcrc ir a }ergc phyaeaL dcf,oct of grcatcr than 1/3rd o'f thc diar¡rcter in thia pla.nc, a coil¡tlete eaoeellot¡a bone bridgc ia dorcn¡trated. (x 3) ''A;ì-

I

\q

104 f,ignuro 5,t¡ Xlilü'lr@¡@6Í€Aü AE&ü,ffi @F HeDSf p[fYðnÄfi, m,fif(W8t. r 5. I À¡rl,ml 3t d,cre,actntca an i¡¡dole'nt phyroal rGü¡¡otΡG. i¡ no r¡'trr of aodiat ti¡c¡¡c.( x 0 ) nfgh ¡t@ñr ft c!.dl i.l,jlurùrate¡ tto lcrtøn a^rid l"¡eü of ¡rknrÛorl hlpertro,@y. (x 325) t. t J

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lt I

_) { rlr:\i* â \ I ^...}y' ccnqllctc loetùøn oü ùtc qrylrr tibi"r I'n trrtå¡ a tylDierl ptrylcr.L rcrpømr'c ot hy¡rertropùy of püryceal'ticc¡¡G gw'e t+). (x 3) úùnGr f,i;cld ðma.¡trator ineree*c iq uith'r d qû c,¡rüi.fi¡¡n-lühr irrto thc m - ; :Éñcre ll 1o¡r of the norml callulár re,glllarity of, the pùryroel cømtor¡r' (x 325) :c¡P C :.,'-.19.¡"

. ¡F .J + . rå.gg .- .' .'- ,4¿- À tò ccctlonr of tbc r¡pp.r tiþi¿ la 'art a Erade (++) ¡rhycøal rG¡¡Do6,ûG. (x 2.5) It{.f,h pourc of thr &ru¡n f{rnd Mrtr¡tcn. ¡ bûmürcli €mt@Eû to thc plryreril prol,l,fcrðtio.n. lfhrc ir Lor,ã of nor*L phyrcaL anehltcctr¡ro i.ri contact uith th fibrou¡ eagrule of thc fat gnlüt. (x fsot g

n ì

I r'? Secondly, the proliferation of physeal tissue was examined to 'l j eval uate the type of the cart'i age . Under pol ari zed I ght

f i brocart'i'l age can be seen separate from the normal physeal cartilage (Fig. 5.9).

Third'ly, additional ectopic phys'is posìtjoned in the reg'ion of the fat graft tras identjfjed as either metaphyseal or ep'iphyseal i n s'itu. The peri pheral growth pl ate response bras ana'lysed for normality or hypertrophy (Fig. 5.10).

5.5 RESULTS OF 1 SQ. CM. DEFECTS

In the I sq. cm. defect series 20 animal s !'rere entered. In a manner sjmilar to the previous expe¡iment' the 4 animals who failed to create a deformity were discarded from further analys'is. One anaesthetic death occurred durìng the transfer of the animal to a C.T. scan. Qne premature autopsy was the result of a t,ound infection that continued despite antibiotics being admini stered for three weeks post operatively. The rema.ining l4 anjmals were in two groups. one group of 7

an'imals Was killed at three months from the reversal procedure' The other group of 7 animals was killed at six months'

5 . 5 . I Rad'i ol oqy Resul ts :

For all animals jn the I sq.cm. group the serial measurements of bone 'length, pin distance, bone angìe, and pjn angle are given in Appendix 2. Animals ll-17 had measurements at 6 weeks of ôgê, l8 t¡,,eeks and at 30 vJeeks (autopsy). The six-month group (animals l8-24) had measurements at 6 weeks of ô9ê, l8

weeks, 30 weeks and al 42 weeks (autopsy).

-108- ftg$fc .5. 9 r trl[t!@û¡@6teÀü, A$til'$ffi @f rüßlt l4¡ff1ç $ilV*,*ü, RÜgP'CIÑBE . FOI.AüT8TE LTGü'IT EX,AüIMÈIIIM. ¡ &imt 26 J gr& ( ++ I ph¡rarrl' rlrúloia¡¡. ae¡ru! cal.l,nl" trtùi.øn. (x 3Í30 ThG ¡dnrâ õGetion vi.th ¡lolarizod fi$ht. øoll,trrgnur o.f the cartlX,agn mtrl'¡( l.¡ drmn¡tratcd. (x 325) -- f ì¡>. L- ; ,¡ :r lr.\t:.lå'Jt ii +í\

t

\

I

.l I

li>' ,#l^ g

,l i; -{. jî \

'+\

î nlA!t(Ðû@!er& Aü,8ü:ß!ffi! @ üm Hgö! PHYTAAI, m8ffia - PßnrPm*nAr" Fnvtlt. Figlrrc 5.10. l: om [i croaco¡ric cxaßi&tti,om À,ninàl- 37 ahov¡ ¡o inømror øf 3 to 4 tÍm,r ùo th. üiôth oG tbr pøùryhcr*1, pùyrir. (r 2.5)q> Fotari¡od light axmin¡ti.o'a provcl thi¡ il,rgrr. (x 3¿S) ) \

I I I

I

F

\,

I

! The results of the deformìty index for I sq.cm. defects for three months survi val are summari zed i n Tabl e 54. Al I 7 animals in this group produced a bone deformity index that confirmed a medial tibial growth arrest. The mean degree of varus angul at j on v',as 22' ( range 18-33 " ) and the bone I ength ratio decreased from 1.000 to 0.600.

After the reversal procedure at 18 weeks 5 of 7 animal s showed a mean of 8.4" (range 6-14") improvement of the varus angle and concurrent improvement towards 1.000 of the bone length ratio.

Two animal s, l5 and 16, showed no improvement of either parameter, indicat'ing failure of the reversal procedure.

The results of the deform'ity index for 1 sq.cm. defects in animals of six months survival are summarized in Table 58.

In every case varus deformity was produced. The mean degree of varus was of 2l' (range 15-27'). The pin angìe difference then improved a mean of 8.5'(range 6-ll") in the first three months, and al I bone I ength rati os showed concurrent improvement towards 1.000.

In the next three months the rate of correction of varus averaged 2" (range 0-4"). There was continued improvement of the bone length ratio toward 1.000.

A typical example of improvement is anìmal 24, in which radjologica'lly a 25'varus deformity'improved 6o in the first three months and then l'in the next three months (Fig. 5.ll). The initial bone length ratio of 0.992 decreased to 0.611 then improved to 0.794 at three months and fjnally lo 0.792 at six months.

- lll TABLE 5A

1S CM. OE

3 MONTH SURVIVAL

DEFORMITY INDEX AT 3 MONTHS

Anqul ation Comment An i mal Ase Pi n Di stance Bone Length Pin Di No. (weeks ) Boñe-LenqTl-mtt o ratio fference Operated Control (A/g ratio) AB

ll 6 0. 136 0.155 0.877 l8a 0.094 0.216 0.435 33'varus REVERSAL Pin t8b 0. 141 0.216 0.653 SUCCESS reposìt. 30 0.r72 0.228 0.754 6'ìmproved

t2 6 0. 148 0.1 48 I 000 t8 0.124 0.2 t2 0 s84 l8' varus REVERSAL 30 0. 182 0.2 28 0 798 ll'ìmproved SUCCESS

13 6 0. 148 0. 158 0.937 l8 0.133 0.2r0 0.633 20' varus REVERSAL 30 0.188 0.245 0.767 5'improved SUCCESS

l4 6 0.1 55 0. 146 I .062 t8 0.1 3l 0.192 0.682 l9'varus REVERSAL / 30 0.1 85 0.209 0.885 l4'improved SUCC ESS

l5 6 0. 147 0.148 0.993 NO l8 0. 128 0.209 0.612 20'varus REVERSAL 30 0.128 0.23 0. 556 +3' varus worse FAI LURE

l6 6 0.1 48 0. 149 0.993 NO I8 0.1 l6 0.2t2 0. 547 20'varus REVERSAL 30 0.1 22 0.23 0. 53 +4'varus worse FA i LURE

T7 6 0.135 0.1 37 0.992 l8 0. 103 0.2 08 0.495 28'varus RTVERSAL 30 0.148 0.2 34 0.632 6' improved SUCCESS

112 TABLE 58

lsQ . CM. DEFECT

6 MONTH SURVIVAL

DEFORMI TY INDEX AT 3 AND 6 MONTHS

Pin Angulation Comment An i mal Ase Bone Length Di fference No. (weeks ) ratio (A/s ratìo)

t8 6 0.147 0. t48 0.993 REVERSAL 18 0.123 0.201 0.612 23' varus 30 0. 184 0.242 0.760 10"'improved SUCCESS 42 0. 236 0.284 0.830 same

l9 6 0.159 0. 160 0.994 0. 138 0.199 0.693 2l' varus REVERSAL 18 'improved 30 0. 190 0.223 0.8s2 8' SUCCESS 42 0.220 0.261 0 .843 3'improved

20 6 0. 153 0.1s0 I .020 o l8 0.143 0.177 0 .807 t7 varus REVERSAL 30 0.210 0.208 I .0r0 9 ' improved succEss 42 0.235 0.250 0.940 3 ' ìmproved

2l 6 0.149 0. 149 I .000 l8 0. r4t 0. 194 0.727 l5 " varus REVERSAL 30 0. 198 0. 215 0 .921 9 'improved SUCCESS 42 0.232 0.248 0.935 4 ' improved

22 6 0. 157 0. 164 0.957 REVERSAL 18 0. 141 0.222 0.635 19'varus 30 0. 198 0.254 0.779 I l' improved SUCCESS 42 0.234 0.275 0.851 l' improved

23 6 0. 141 0. 119 l.l8 l8 0. 123 0. 118 0.654 27' varus REVIRSAL 0. 158 0. 205 0.77 7'improved SUCCESS 30 'improved 42 0. 183 0.2t7 0.843 I '

24 6 0.1 42 0.1 43 0 .992 l8 0.1 18 0.1 93 0 6ll 25 varus REVERSAL SUCC ESS 30 0.1 73 0.2 l8 0 794 6 i mproved 42 0.1 9 0.2 4 0 792 I ì mproved

1r3 nMM)fl@¡ûffi8enG 8vt@ffiD @t FEnXgl Nþ mvGroldlt @il Dtlìffitft. (Hùi,mI ¡lt the lnittrl AP x-ray ¡llou¡ 3 re¡rth¡ l¡trr tlrrc íc tho Phrni,¡tcr o,¡rorutivc r.åtc 25' varut üofocltty. Tho EF tho Lrft md-ûrl t{bi,r.D> b@út ¡,ollEtb rltiø 0.611.

3 no,nthr ¡ftcr rcvor¡al 6 rno,nth¡ rf,tcr rcwrreal vtnûa l.t¡rovm.nt á,ad boñc tbrc'Ll eomtlewd lon'gth ratÍio nor 0 .79,a. irryr'ovcm,at.

By radiological criteria the deformity index showed improvement or success in 12 of 14 cases (85.7%).

5.5.2 C.T. Results:

The results of C.T. measurement of the extent of physeal defect at the time of the reversal procedure and at autopsy are seen in Table 5C. The complete table of measurements is given in Appendix 3.

The 7 animals in the three-month autopsy group had a physeal defect at reversal of 17.5 + 2.0% (Standard Deviation). The extent of the I esi on had 'increased to 2l .3 + 4.4% at autopsy

(p=0. looT, t=2.1) .

The 7 animals in the s'ix-month autopsy group had a physeal defect at reversal of 16.9 + 2.5%. The extent of the lesion had decreased to 15.1 + 4.6 at autopsy (p=0.5034, t=0.9).

There was no statjsticaì'ly significant difference between the i nj tj al defect si ze for the three and si x-month groups (p=0.706, ú=0.4).

For 74 animals in this fjrst group the injtÍal s'ize of the defect was 17.2 + 2.2%.

5.5.3 Hjstology Results:

Tables 5D and 5E detajl the histolog'ical findings. a. Host Response:

All animals jn the three month group showed a (+) or (++) granulation tissue about the fat Ìmp'lant. A pseudo-capsule had

- ll5 - TABLE 5C

C.T. SCAN RESULTS FOR I SQ. C]'I.

AT 3 ]-'IONTH SURVIVAL

Initial C.T. C.T. at Sacrifice Comment

(%) (%) Si ze ll l7.l 26.5 I ncreased t2 t7.9 2l .5 I ncreased l3 16.6 18.6 I ncreased l4 15.6 22.4 I ncreased

15 18.3 20.6 Increased

16 21.? 26.0 I ncreased t7 15.5 13.6 Decreased

Mean 17.5 + 2.0 21.3 + 4.4 p=0.1007

C.T. SCAN RESULTS FOR I c1,l

AT 6 MONTH SURVIVAL

Initial C.T. C.T. at Sacrifice Comment

(%) (%) Si ze

t8 16. 5 17 .7 Increased

l9 20.9 18. 3 Decreased

20 17.5 16. 7 Decreased

2l 18.8 19.4 I ncreased

22 l7.l 15.6 Decreased

23 13.5 6.4 Decreased

24 14.2 tt.7 Decreased

Mean 16.9 + 2.5 l5.l + 4.6 p=0.5034

1ló TABLE 50

H I STOPATHOLOGY

I S0. Cl.l. REVERSALS

SHEEP SACRIFICED AT 3 I.IONTHS

Animal No. ll t2 l3 l4 l5 l6 t7

C. T. I (r) t7 .r% t7 9% l6 6% 15.6% I8 3% 2l 2% l5 5% 2 (F) 26.5% 2l 5% l8 6% 22.4% 20 6% 26 0% l3 6%

Fat

Site P P P P/ì4 P Vìabiìity N N N Part i al Compl ete Compl ete N Necros i s Necros i s Necrosi s Fibrosis + ++ + ++ +

Host Ti ssue

Fibrosis ++ ++ + + + 0steobl asts + + + ++ +++ +++ + Epiphyseaì Bone ++ ++ + ++ ++ ++ ++ Cort i cal ++ + ++ 0 0 0 +

Physea'l

l4edi aì Spur 0 Indol ent + + + No No ++ Hypertrophy ++ ++ + ++ 0 0 + Per i pheral No No No Yes No No No Ectopi c Yes Ye s No Yes No No No

Bone Bridge

Site Central Compì ete Compì ete No Cancel I ous Cancel I ous Cancel I ous Nat ure Incompìete Cortical Cortical

117 TABLE 5E

H I STOPATHOLOGY

I SQ. CM. REVERSALS

SHEEP SACRIFICED AT 6 I.IONTHS

Animal No. l8 l9 20 2l 22 23 24 c.T I I l6 5% 20 9% 7 5% t8.8% t7.t% 3 5% l4 ¿/ø 2 F Ù 7% l8 3% 6 7% 19.4% t5.6% 6 4% ll 7%

Fat

Si te P P P P P P/¡4 P Viabiì ity H H H H H N H Fibrosis + + + + 0 +/ ++ +

Host Ti ssue

Fibrosis ++ + + + + + ++ 0steobl asts + 0 0 + + + + Epiphyseal Bone + 0 ++ + + ++ + Cort i cal + + 0 + ++ 0

Physeal

Mediaì Spur ++ ++ ++ + + + + Hypertrophy ++ +/ ++ + +/ ++ + + + Peri pheral No Yes No No No Yes No Ectopi c Ye s No No Yes Yes No No

Bone Bridqe

Site Attempt i ng No No No No Central No Central Fi brous Nature Fi brous

1r8 formed by six months wjth more specimens showing a (+++) degree of fibrosis. Epiphyseal bone thickening v',as very marked jn 6 of 7 cases and cortjcal thicken'ing in 4 out of 5 of the three-month group. By six months the cortical bone response was either not present or minjmal in 6 of 7 specimens. Additional'ly, the epiphyseal bone response was present in only 2 of 7 cases. b. Fat Response:

In 12 of 14 experiments the fat remained at the phys'i s/metaphyseaì iunction.

0f the 12 an i mal s 'i n wh i ch the f at remai ned at the physis/metaphysis iunction the autologous fat graft remajned normal'in appearance jn 4 out of 5 three-month follow up cases. In 6 out of 7 six-month cases there was long'itudinal hypertrophy of the fat content. In these cases there was min'imal (0 or + grade) fibrosis within the fat. Initially, the fibrosjs in the fat was marked (++ grade) in 2 of 5 three-month cases. There was extensive fibrosis in animal 14 where partiaì necrosis of the fat graft was present. c. Bone Bri e Reformat'i on :

If there is a reformation of the bone bar then continued deformity may occur. In 2 of 7 of the three-month experiments, An'imals l5 and 16, there was a complete cancellous/cort'icaì bone bar between the ep'iphysis and the metaphys'is (Fig. 5.12).

- ll9 - rotm Dnrw RiBP(tm*Erot - PüûrplilnhÀL, (}atrel fél tLthor a¡ a ro¡r¡lt of fat necro¡i¡ or lor¡ of poriti'on or ml" poaitLo'n thoro i¡ no evidonêc of, fat at thc pñyreal' Lrvclr. A corrytete corticoeaoe¡llou¡ bar JoLnr tho opLphyraal u¡û mt I bo,sc rLth of thc r.vGçral procodlrno. ¡PhYlc,a failunttl ¡ T!¡o flndLngr u rG rinilar l.n Ani.mL 15. (x 5) o(, I In 3 other animals there was a central incomplete lesion: Animal 14 in the three-month group, and Animals l8 and 23 in the six-month group. d. Physeal Response:

The response of the phys'is þlas such that a medial spur of the physeal cartiìage was seen extendìng into the metaphysis in 4 out of 5 three-month cases, and in 7 out of 7, six-month cases.

Hypertrophy in the amount of physeaì cartilage med'ialìy was most marked in 3 out of 7 six-month cases and in 3 of 5 at three months. Polarized light microscopy revealed that the nature of this tissue was fjbrocartilage, and not phenotypical of physeal carti 1 age i n al I cases. The degree of disorganÍzation of the physÌs centrally could produce a bimodal appearance where the convol utjon of the physi s gives the appearance of there be'ing two comp'l ete growth p'l ates ( Fi g . 5.13).

In the two cases (An'imals l5 and l6) of complete bone bridge reformation there was evjdence of dimjnutjon of the height of the physis.

There was marked peripheral proliferation of fibrocartÍlage in 3 of 12 animals which djd not have bone bridge reformatjon.

S'ix spec'imens showed ectopì c metaphyseaì physeal carti I age.

5.6 RESULTS OF 2 SQ. C]'l. DEFECTS:

0f the l5 animals in the 2 sq. cm. series l3 anjmaìs had reversal operat'ions. There was one anaesthetic death during

- 127 Figure 5.13: IìITERSTITIÀL PHYSEAL REPÀIR. (Àninal f7) Figure 5. 13- 1: Irrnediatel- y adjacent to the fat there is a spur of physeal. cartilage that projects into the metaphysis- This was present in 11 of L2, 1 sq. cm. defects with a successfu.I. reversal outcotæ. Hl4rertrophy of the cartilage shows loss of normaJ. ceJ-J-ul.ar orientation and increased collagen content (po].arized light). Figure 5.13.2: Ànother section shovs an apparent duplication of the grorth ptate with a birnodal orientation. This represents the increased convolution of the interstitial repair proceas. (Sinilar findings in Ani¡nals 18, 19, 2O.l , ¿-

1 i

¡r\

t I I

', II

\ transfer of the animal from the C.T. to the Animal House, and one animal died of tetanus. Eleven anjmals have complete data. Seven u,ere killed at three months and four at six months.

5.6.1 Radioloqy Results: 'length, The serial measurements of bone Þjn distance, bone angle, and pjn angle are shown in Appendix 4. Animals 25-31 had measurements at 6 weeks of age, l8 weeks and at 30 weeks (autopsy). The six-month group (Animal s 32-35) had measurements at 6 weeks of age, l8 weeks, 30 weeks and at 42 weeks.

The results of the deform'ity index for 2 sq.cm. defects for three-months survi val are summari zed i n Tabl e 5F. Al I 7 animals in this group produced a bone deformìty index that confjrmed a medial growth arrest. The mean varus angulatjon !'ras 2l'(range 12' - 37') and the bone length ratjo decreased from 1.000 to the 0.600 level.

After the reversal procedure 3 of 7 showed no worsening of the varus angle. However, on'ly one showed a significant o 'improvement i n the varus deform'i ty of 5 and concurrent

ìmprovement towards 1.000 of the bone length ratio.

Anjmal s 27, 28, 29, 30 showed no 'improvement of either parameter thus indjcating failure of the reversal procedure.

The results of the deformity index for 2 sq.cm. for six-months survival are summarized in Table 5G.

-123- TABLE 5F

2 SQ. Cr,l. DEFECT

3 HONTH SURVIVAL

DEFORI.IITY INDEX AT 3 MONTHS

Angul at i on Comment An i mal Aes Pi n Dì stance Bone Length Pi n Di No. (weeks ) Bon e Lenq th Ratio ratio fference 0perated Control (A/g ratio) A ---E--

25 6 0. 164 0. 178 0.92l l8 0. l4l 0.238 0.5 92 l8'varus REVERSAL 30 0.t72 0.255 0.6 75 5' improved SUCCESS

26 6 0 164 0. 170 0.965 REVERSAL t8 0 143 0. 218 0.656 20'varus PART I AL 30 0 188 0.265 0. 709 s ame SUCC ESS

27 6 0. 158 0.1 55 I .020 NO l8 0.150 0.2 03 0.739 l6'varus REVERSAL 30 0. l6l 0.2 72 0.591 8' worse FA I LURE

28 6 0 r62 0.162 I .000 NO l8 0 137 0.228 0.601 28'varus REVIRSAL 30 0 r53 0.267 0. 573 3' worse FA I LURE

29 6 0.165 0.159 I 030 NO l8 0. 106 0. 198 0 53 5 37'varus REVIRSAL 30 0.1t5 0. 235 0 40 9 3' worse FAI LURE

30 6 0.154 0. 161 0.957 NO l8 0. l3l 0.211 0.621 l8'varus REV ERSAL 30 0. 133 0. 240 0. 554 l0'worse FA I LURE

3l 6 0. 139 0. 140 0.928 REV ERSAL l8 0. 118 0.190 0.621 l2'varus PART IAL 30 0. l3l 0. 196 0.668 same SUCCESS

124 TABLE 5G

2 SQ. CM. DEFECT

6 MONTH SURVIVAL

DEFORMITY INDEX AT 3 AND 6 MONTHS

An i mal Ase Pi n Di stance Bone Length Pi n Anqul ati on Comment No. (weeks ) Bone en tio ratio Di fference 0perated Control (A/g ratio) A B

32 6 0.144 0.150 0.960 l8 0.t24 0. 193 0.642 l7' varus REV ERSAL 30 0.169 0.233 0.725 7' 'improved succ tss 42 0. 188 0.243 0.774 2o improved

33 6 0. 146 0. 152 0.960 l8 0. 182 0.2t2 0.858 1l ' varus REVERSAL 30 0. 205 0.244 0.840 2 " 'improved SUCC ESS 52 0.?34 0.273 0 .857 3 " 'improved

34 6 0. l5l 0. l5l I .000 NO 18 0. 125 0.202 0.619 25" varus REVERSAL 30 0.175 0.246 0. 711 I' urorse PARTIAL 52 0.195 0.272 0.716 4o ìmproved SUCC ESS

35 6 0. t6l 0. l6l I .000 l8 0.132 0.224 0. 589 25" varus REVERSAL 30 0. 188 0.254 0.740 16" improved SUCC ESS 52 0. l8l 0.300 0.603 same

125 3 out of 4 six-month animals showed improvement of the indices mostly in the init'ial three months. By radiological criteria the deform'ity index showed improvement or success in 6 of ll cases (54.5%).

5.6.2 C.T. Results:

The results of the C.T. measurement of the extent of the physeal defect at the time of the reversal procedure and at sacri fi ce are seen i n Tabl e 5H. The compl ete tabl e of measurement is in Append'ix 5. For the l1 cases with reversal the initial C.T. measured 28.0 + 6.0%.

The 7 animals in the three-month autopsy group had a physeaì defect at reversal of 26.3 + 5.3%. This had decreased to 25.0

17.3% at autoPsY (P=0.723, t=0.4).

The 4 animals in the six-month autopsy group had a physea'l defect at reversal of 30.8 + 6.9%. The extent of the lesion had decreased to 22.4 + 3.7% at autopsy. This failed to reach statistical significance (p=0.1041, t=2.7). There was not a statisticalty significant difference between the three and sjx month groups (P=0.9928).

5.6.3 Histoloqical Results:

In the 2 sq. cm. seri es, 7 of the animal s v',ere autops'ied at three months and 4 at sjx months (Tables 5I & 5J). a. Host Response:

As has been noted with the I sq.cm. defect series, the host tissue response showed an initjal osteoblastic response being +

-126- TABLE 5H

c.T. scAN RESULTS FoR 2 SQ. CM.

AT 3 MONTH SURVIVAL

An i mal Initial C.T. C.T. at Sacrifice Comment No. (%) (%) Si ze

25 30.6 30.6 Same

26 27.8 23.9 I ncreased

27 29.3 24.4 Decreased

28 30. 2 36.9 Increased

29 18.1 23. 5 I ncreased

30 3l .3 22.9 Decreased

31 23.1 13.3 Decreased

Mean 26.3 + 5.3 25.0 + 7 .3 p=0. 723

C.T. SCAN RESULTS FOR 2 SQ. CM.

AT 6 MONTH SURVIVAL

An i mal Initial C.T. C.T. at Sacrifice Comment No. (%) (%)

32 29.8 2t.3 Decreased

33 23.0 19. I Decreased

34 30.7 21.6 Decreased

35 39 .8 27 .7 Decreased

Mean 30.8 1 6.9 22.4 Y 3 .7 p=0.104

127 TABLE 5

H I STOPATHOLOGY

2 SO. CM. REVERSALS

SHEEP SACRIFICED AT 3 I''IONTHS

Animal No. 25 26 27 28 29 30 3l .3% 23.l% c. T. I I 30 6% 2t.8% 29.3% 30.2% 18. l% 3l t3.3% 2 F 30 6% 23.9% 24.4% 36.9% 23.5% 22.9%

Fat

P l{as P P S ite P P P P N c/N P/N Viabììity N P/N N N Fibrosis 0 ++ 0 0 0 ++ ++

Host Ti ssue

Fibrosis + ++ + + + ++ ++ 0steobl asts + ++ + + + ++ ++ Epiphyseaì Bone ++ + + + ++ + ++ Corti cal ++ + + +/ ++ ++ + ++

Phys eal

No + Ì'led i al Spur No ++ + + + Hypertrophy + ++ + + ++ +++ +++ Peri pheral Yes Yes No Ye s No No No Ectop i c No 7 Yes (tl) No No No No

Bone Bridqe

Site No No No No No Comp'lete No Nature Cancel I ous

128 TABLE 5J

H I STOPATHOLOGY

2SQ . CM. REVERSALS

SHEEP SACRIFICED AT 6 MONTHS

Animal No. 3? 33 34 35 c.T I (r) 29.8% 23.0% 30.7% 39.8% 2 (F) 21.3% 19.lT" 2t.6% 27 .7%

Fat

Si te P P P/¡r P Vi ab'il i ty N H N N Fibrosis + 0 0 +

Host Ti ssue

Fi brosi s ++ + + + 0steobl asts 0 0 ++ 0 Epiphyseaì Bone ++ + + + Cort i cal ++ + ++ ++

Physeal

Medial Spur + ++ ++ ++ Hypertrophy No ++ ++ No Peri pheral No No No No Ectopi c No No No No

Bone Bridge

Si te Att. No No Att. Nature (At )

129 or ++ in all three-month an'ima'ls, whereas only one six-month animal had thjs osteoblastic response.

The epiphyseal bone response showed thickening more marked in the three-month animals than the six-month specimens. However, the epiphyseal bone response was a'lways (+) for these ìarger defects. A plateau fracture of Animal 29 was noted. The width of the ep'iphyseal bone was a'lways i ncreased ei ther + or ++. The cortical bone thickness was increased in all experiments, and double the cortex width in 4 of 7 three-month cases and 3 of 4 six-month cases. b. Fat Res 0nses:

In l1 of ll experiments the fat remained in the physeal/metaphyseal iunctjon. However, in one autograft there was a remnant of fat onìy at the physeal level with evidence of complete necrosis, and a signìfjcant amount of fibrous tjssue (An'ima] 30).

7 out of 7 , three-month cases had physea'l pos'iti on at the appropri ate I evel , wi th 4 show'ing no necrosi s, 2 show'ing partÍa'l necrosis, and one indicating compìete necrosis (Animal 30). tlith complete and partial necrosis there u,ere marked fibrous tissue septae within the graft.

3 out of 4 six-month cases showed normal fat, and one, Animal 33, had hypertrophy of the autograft wjth an apparent increase in size of the fat tissue. tlhere the fat was viable, there was always mjnjmal or little tjssue fibrosis.

-130- c. Bone Bridge:

Bone bridges were not seen in 6 of 7 three-month specjmens, but the spec'imen from An'imal 30 showed a compl ete cancel I ous bone bridge with failure of the reversal procedure.

None of the six-month specìmens showed a complete bone bridge formation. However, 3 of 4 showed an incomplete bone bridge or attempt at bone bridge formatjon between the metaphysÌs and the ep'iphysis. A micro-fracture effect in Animals 32, 34, and 35 could be demonstrated (Fig. 5.14). There is an incomp'lete tether to the process of growth. The immature u,oven bone shows longitudinal array of the bone along ljnes of tensjon between the ep'iphys'is and metaphys'is (F'ig. 5.15). d. Physeal Response:

The response of the physis showed a medial metaphyseal spur formation in all specimens other than in Animal 30 where there was a complete bone bridge reformation. The medial physeal response was ++ in 3 out of 4 of the sjx-month animals.

There v,,as hypertrophy of the physeaì cart'i I age wi th d'i sorgani zati on i n 4 out of 7 three-month animal s. Thi s hypertrophy was not found in 2 oul of 4 of the six-month animals.

Peripheral physea'l enlargement was seen in 3 of the ll an'imals.

Th j s u,as noted only in the three-month spec'imens. Thi s hyperplasia of the zone of Ranvier cartiìage, was also associated with the presence of ectopic sjtes of physeaì

- l3l AFMFN A1 ruDIAL f(M ¡.NIDGß MFOIIITçTOÑ Lü FOrËi. Thie nùole ¡octi.o,m of, tibia from t¡nlml 35 do¡p,n¡tratcr thc ¡t¿bl¡ fat graft in poritlo,n and a,¡Urarcnt ridcninE of thc cplphyrcrl bonc. lmcdiatc'l.y adJa'con{ to thc p,hyrcal rc¡ilair thcrc ir an attonpt of a ear¡ccllouc bar.formtion.r (x ll ztL

\ l'igturc 5-15¡ ÀFWT A? TffiÐTÀú D6M }RIÞGE NSMffiTlIfl. (Aninal 35) (x 350) & fuhttr¡ü. tp.vûtt bûüc näømr l,oqrütuflúofl[ ¡ar.y of thn bqlnc betr6.cn thr cpipht¡ti¡ ¡r¡d rrtrgÑryrLl alomg Lfalr of, tcncion ( grwthl tmsaiatcly rdJrrcont to thc pùy¡L¡. thf,r 'mlero-fmcturc' cff¡et Ì.¡¿r ¡lro bo¡¡r r¡otod 1a Àmi.mlc 32, 3a. I .A f ,Ð(

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É-_ cartilage as for exampìe in a specìmen from Animal 27, Here, metaphyseal cartilage rests v',ere seen.

5.7 RESULTS OF 3 . CM. DEFECTS:

0f the l0 animals with a 3 sq. cm. defect one anjmal did not have a reversal procedure as part of documentation of the natural h'i story study. One animal died as a resul t of 'l mi sadventure. There u,ere thus I animal s wi th the argest defects that have complete data. Five were killed at three months and 3 at six months.

5.7. I Radi ol oqi cal Resul ts:

The serial measurements of bone length, Pilì distance, bone ang'le and pìn angle are shown in Appendix 6. Animals 36-40 had measurements at 6 weeks of ôgê, 18 weeks and at 30 weeks

(autopsy) .

The six-month group (Anjmals 4l-43) had measurements at 6 weeks of age, 18 weeks, 30 weeks and at 42 weeks.

The results of the deform'ity index for 3 sq. cm. defects at three months survival are summarized in Table 5K.

Qnìy I of the 5 three-month animals showed ìmprovement of the degree of varus. 0f the other four animal s two ltJere respect'iveìy 7" and lo !,rorse. Two showed no change in the varus

ang'l e. I'lhere the degree of varus remai ned the same or .improved, the bone length ratio ìmproved towards 1.000. For example, Anjmal 39, jn which the bone length ratio at reversal was 0.550 and a 13'varus angle showed no angular change, but the ratio improved to 0.640'. Hence where there was

-r34- TABLE 5K

3 SQ. CI,I. DEFECT

3 I,IONTH SURVIVAL

DEFORMITY INDEX AT 3 MONTHS

Pi Anqul at i on Comment An i mal Aee Pi n Di stance Eone Lenqth n No (weeks ) Bone Lenqth Ratio ratio Di fference 0perated Control (A/g ratio) A B

36 6 0. 142 0 145 0.979 NO l8 0.096 0 209 0.459 28' varus REVERSAL 30 0.076 0 256 0.297 7' worse FAI LURE

37 6 0. t90 0.157 t.2L l8 0. t35 0.232 0. 581 l7'varus PARTIAL 30 0.154 0.258 0.600 same succEss

38 6 0. 143 0.1 35 0.t 35 NO l8 0. 103 0.20l 0.20l 24' varus REVERSAL 30 0. 116 0.206 0.2 06 l' worse FA I LURE

39 6 0.128 0 147 I .870 l8 0.lll 0 203 0. 550 l3' varus PART IAL 30 0. 133 0 208 0.640 same SUCCESS

40 6 0.t22 0.120 I .02 l8 0.078 0. 178 0.270 l2' varus REVERSAL 30 0.102 0. 183 0. 557 4'improved succ Ess

r35 'improvement or no change in angle, but the bone length rat'io showed that there was some resumption of longitudinal growth; these have been determined to be evidence of a part'ia'lìy successful resul t (Tabl e 5K) .

The results of the deformity'index for 3 sq.cm. defects at six months survival are summarized in Table 51. In this group of

an'ima1s, 2 of 3 showed 'improvement initially, with one of these animals then fai'ling to sustain the improvement. The other animal (An'ima1 43) showed progressive failure of radiolog'ical bone deformity indices.

The success of the operatjon of fat interposition by radiologÌcal criterja u,as 4/8 or 50%.

5 .7 .2 C. T. Resul ts :

In Table 5M the C.T. results show that the mean of the initial defect was 34.2% + 8.4% for the three-month group. Although 4 of 5 cases showed a decrease in area, there was not a statist'ica1'ly signìficant difference between the initial C.T. and the C.T. at autopsy, where the mean was 34.7% + 8.1% for

three months (p=0 . 7625, t=1 .8) .

The six-month animals had an initial defect size of 32.7 t

6,3%, The f i nal area vJas 36.2 + 9 .6%. There v''as not a statistically signifjcant d'ifference jn the defect size (p=0.6354, ú=0.5) . The mean defect si ze 'ini t'ia1 ly for the 8 animals jn the 3 sq. cm. group was 33.7 + 7.2%'

-136- TABLE 5L

3 . CI.I. DEFECT

6 MONTH SURVIVAL

DEFORMITY INDEX AT 3 AND 6 MONTHS

Pi n Angul ati on Comment An i mal Ase Pin Di stance D'if ference No. (weeks ) Bone Lenqth Rat io 0perated

41 6 0. 139 0. 139 I .000 0.126 0. 198 0.636 20" varus REVERSAL (, 18 'improved \ 30 0.172 0.243 0.708 4" succtss 42 0.177 0.242 0.736 2" improved

42 6 0.146 0.152 0.961 NO 18 0.128 0.206 0.621 14' varus REVERSAL 14" improved INITIAL 30 0. r91 0.246 0.776 o 4? 0. 194 0.250 0.776 I worse SUCCESS

43 6 0.158 0.154 t .02 1B 0. 139 0.206 0.675 30'varus NO 30 0.128 0.23 0. 556 3' vúorse REVERSAL 42 0. 148 0.247 0.599 2" v,rorse FAI LURE TABLE 5M

C.T. SCAN RESULTS FOR 3 SQ. CM.

AT 3 MONTH SURVIVAL

An i mal Initial C.T. C.T. at Sacrifice Comment No. (%) (%) Si ze

36 35.8 24.6 Decreased

37 47 .6 37.8 Decreased

38 25.2 15.8 Decreased

39 l3 .6 24.0 I ncrea s ed

40 30.9 2t .5 Decreased

Mean 34.2 t 8.4 34.7 + 8.1 P=0. 163

C.T. SCAN RESULTS FOR 3 SQ. CM.

AT 6 MONTH SURVIVAL

Initial C.T. C.T. at Sacrifice Comment

(%) (%)

4l 27 .3 30.0 I ncreased

42 3t.2 31.4 Same

43 39.7 47 .2 I ncrea s ed

Mean 32.7 + 6.3 36.2 + 9.6 P=0.635

r38 5.7.3 Histoloqical Results:

In the 3 sq. cm. ser.ies 5 of the animal s u,ere autops'ied at three months and 3 at sjx months (Tab'le 5N, 50). a. Host Response:

The host tissue showed the features as previously described in the I sq. cm. and 2 sq. cm. series. There was initjal increase 'in cortical thickness and osteoblastic activ'ity in the epìphyseal bone noted in atl cases. At the six month review there !,ras evidence of a plateau fracture in one anjmal (Animal

41) . b. Fat Response:

In all 8 experiments the fat remajned at the physea'l level and there was no necrosis of the fat. 4 of 8 animals had minimal evidence of interstitial fibrosis. c. Bone Bridge:

Bone bnidge format'ion v,ras present jn 4 of 8 specimens. There were 2 peripheral cortical bars, and one (Animal 36) additionally had a cancellous central bar (Fig. 5.16). There were 2 othelincompl ete cancel l ous central bars . One animal (Animal 39) showed a micro fracture effect of thjs bone bridge as had been prev'ious'ly noted. This anjmal was autopsied at three months (Fig. 5.17).

d. Physeal Response:

The physeal response showed a mediaì spur in all I spec'imens.

There was hypertrophy in 3 of 3, six-month cases, and 3 of 5

-139- TABLE 5N

H I STOPATHOLOGY

3 SQ. CM REVERSALS

SHEE P SACRIFICED AT 3 I,IONTHS

38 39 40 An'imal No . 36 37 c.T I (r) 35.8% 47 .6% 25 2% 31.6% 30.9% 2 (F) ?4.6% 37.9% l5 ffi ?4.0% 21.5%

Fat

Site P P P P P Viabiì'ity N N N N N + F'i bros i s 0 + 0 0

Host Ti ssue

Fibrosis 0 0 0 ++0 0steobì asts 0 0 0(+in Peri ) ++(Peri ) ++(Peri ) Epi ph. Bone ++ + ++ +++ Cort i cal ++ + N/A ++ unabl e to cìassify

Physeaì

Hedi al Spur + + + + + Hypertrophy No No + + + Peri pheraì No Yes No Yes Yes Ectop i c No No No No No

Bone Bridqe

Site C+P No Peripheraì AttemPt at No l.ledi al'ly-Can Peri pheraì Nature Peri ph. -Cort. Cort i caì Cancel ì ous

r40 TABLE 50

H I STOPATHOLOGY

3 S0. Cl4. REVERSALS

SHEEP SACRIFICED AT 6 MONTHS

Animal No. 4l 42 43 c. T. I (r) 27 .3% 3t.2% 39 .7% 2 (F) 30.0% 3t .4% 47 .2%

Fat

Si te P P P Viability N N N Fi brosi s + + 0

Host Ti ssue

Fibrosis ++ + 0 0steobl asts 0 0 0 Peiph. Bone + +++ + Cort i cal ++ ++ +

Physeaì

Medial Spur + ++ + Hypertrophy + + ++ Peri pheral No No No Ectopi c No No No

Bone Bridqe

Si te smal I CENTRAL No No attempt i ng Nature medially cancel ì ous

141 s 'Em mru8 müEmt[tr r. fûtmX. 3ó stroüc a perl¡ütcrtl t ç rtèe G. Thcrc i¡ drforni. t lide T,it ure of, tho rcvrr¡r.tr flt lûty. (* 3.st io,uo

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{*) "d a ,. t ti' v- t J l.r\ three-month animals. One of the animals without hypertrophy of the ptate had a bone bridge reformation. There were no ectopic rests of cells. 3 of 5 three-month animals (Animals 37, 39 and 40), showed a marked peripheral physeal pro'liferation.

-144- Chapter 6 DISCUSSION

By comparing the results of the 3 groups of an'imals the inc'idence of success, and the causes of failure of fat as an'interpositional graft materia'l , can be def j ned.

In the I sq.cm. defect animals the radiological deformity showed that 72 of l4 (85.7%) animal s had 'improvement of varus. The improvement of varus also correlated with an improvement of the bone length ratio towards 1.000.

This improvement was most noticeable at three months, but the effect contjnued up to six months. However, the rate of improvement decreased. Animal 22 is an excellent example of this phenomenon where the varus of 19'improved by ll'in the first three months, and then only a further lo in the subsequent three months. The bone length ratio showed improvements from 0.654 to 0.770 at three months and finally 0.843 at six months.

In the 2 sq. cm. defect group 6 of ll animals (54.5%), showed improvement of the varus deform'ity. The rate of Ìmprovement showed the same trend as in the smaller defect group, with'improvement most marked in the first three months. Animal 32 shows this effect where the initial varus of 17" improved by 7'in the first three months, 'length and on'ly 2o in the next three months. The bone ratjo also reflected a successful reversal procedure. In one animal (Animal 26), where angu'lation remajned the same' the ratjo improved from 0.656 to 0.709 jndicatjng some continued longìtudinaì growth.

-145- In the 3 sq. cm. defect group only 4 of I cases (50.0%) showed radiological improvement. In this group the degree of angulatìon improvement was much less noticeable. For example Animal 4l showed 4" improvement initially, and 2' subsequent improvement. Two anìmals showed no varus angulation 'improvement, but improvement of the bone ìength ratio towards 1.000. The degree of improvement, and also the rate of improvement, v,,as decreased wi th the ì arger growth p'l ate resecti on.

The princìpaì cause of failure in the first series of an'imals vlas determjned by either the fajlure of survival of fat or thejnabì]ity to maintain the fat in pos'ition. Because haemostasis is not jmi 'inhì b'ited guaranteed, a process s I ar to fracture repa'iris not ' and thjs results, as Ín Anjmals 15 and 16, with a bone bridge reformati on.

The initial C.T. scan area for the I sq. cm. series was 17.2 ¡ 2.2%, The initial C.T. area for the 2 sq. cm. series was 28.0 + 6.0%. The jnitial C.T. area for the 3 sq. cm. lesion was 33.7 + 7.2%. There h,as a statistically s'ign'ificant jncrease in the size of the defect between these groups of animals (p values !{ere < 0.0001). Therefore, the size of the fat graft to fill the cavity was also jncreased. This may affect the survival of the fat. In the 2 sq. cm. series two animals showed partial necrosis, and one showed complete necrosis: in this animal a cancellous bone brìdge did occur with recurrence of deformity (Animal 30).

The bone bridges may be incomplete (Animals 32, and 35) or compìete (An'imal 38). There may also be a combination of both central and peripheral recurrence (Animal 36).

-146- For all cases the medial canceìlous bone bridges show an incomplete bone trabecular formation between the metaphysjs and the epiphysis. This may be 'interpreted as a micro-fracture effect as noted in animals where the provocation operation failed to cause a cessation of growth. Johnson & Southwick (1960) and others have commented upon this. It is evidence of the abjljty of the physìs to maintajn growth. Th'i s effect i s confj rmed i n the author's seri es of expeniments.

The reasons for failure of fat as the interpositional material are thus:

(i) fat necrosis with failure of revascularization

( i i ) fat mal posi ti on (iii) fajlure of majntenance of position.

If the fat remains vjable and in posit'ion, what are the factors that determine the host bone and physeal repair processes?

From the histologÍcal analysjs the host response to the physeal defect can be exam j ned i n reg'i ons . The ep i phys'i s responds wi th jncreased width. There is also an increase in the number of bony trabeculae acting to stress shield the defect in accordance with trlolffs'law (1892). If the defect is large enough (as in Animal 4l) then a pìateau fracture may occur.

The initi al osteobl astic response seen in al I l9 three-month spec'imens becomes much less marked with maturation and remodel'ling of the surroundi ng epi physeal and metaphyseal bone. Ini ti al ly the epiphyseal bone thickening was ++ or + in 6 of 7 with I sq. cm. defects, 7 of 7 with 2 sq.cm. defects and 5 of 5 with 3 sq.cm. defects. At six months on'ly 2 of the 14 an jmal s showed ep'iphyseal

-147- trabecular thìckening, and osteoblastjc actjvity þras diminished. Cortical bone thickening l,Jas consistent with the response of bone to load in varus. It also remodelled in accordance with t'lolffs' law.

Concerning the fat graft at the bone/fat interface: the granulation ti ssue that woul d have surrounded the fat soon afterimp] antati on graduaììy showed maturation into fibrous encapsu'lation of the fat graft. The thickened fibrous tissue at three months had become more mature by sjx months. The degree of fibrosis depended upon the fat viability. Partial necrosis and septae within the fat graft gave a more marked fibrous response and this fibrous tissue may act as a tether and inhibjt normal physeal growth.

1¡ith a viable fat material, a stable cavity can be maintained within the bone. It does not heal as would a fracture. can the phys.is respond wi th an j nterst'iti al repai r process?

It has been shown that the physis may expand either in a ci rcumferenti al (di ametri c) or I ongi tudi nal fashi on. Dj ametri c expansion as described by Hert (1972) occurs by cell division and matrix expansion within the peripheral phys'is (interstitial growth).

Tonna (1961), Solomon (1966) and Shap'iro (1977') have documented that cellular addjtion at the physis periphery occurs at the zone of Ranvier. Thjs is described by these authors as appositjonal growth and may aid in the physeal repair process.

From experimental and clinical evidence Langensk'iöìd and co-workers have confjrmed that central interstitial regeneration of the growth plate occurs ILangenksiöld 1967, 1975, 1978, 1979, 1981, 1986' 1987]'

-148- Other experiments in smaìl mammals such as the rabbit demonstrate that partì al physeal regeneratÌ on fol I ows i ncomp'lete and comp'lete physeal/epiphyseat resection IHeikel 1960, 1961, österman 19721.

However, Haas in 1931, and Banks and Compere in l94l' have been unabl e to show 'in experi mental studi es on rats and rabb'its any regenerative potential of the physis. Hert in 1972, also in rabbits h,as unable to document any central physeal interstitìal repa'ir mechanism. He did characterjze the peripheral physeal interstitial repair process.

In 1962 Calandruccio found that articular and epiphyseal cartiìages of the femoral of skeletally immature dogs were capable of 'ine proì ì ferati ve regenerati on and even comp'lete repai r wi th true hyal cartilage. The observed djfferences in the repair of incomplete and comp'lete defects suggested that granu'l ati on ti ssue from the subchondral bone of the ep'iphyseal ossifjcation centre inhibited cartilage formation. If this injtial cellular inflammatory response could be prevented or minimized, (j.e. by accurate anatomical reduction), the full potential of cartilage repair might be realized.

Calandruccio noted that cart'ilage prof iferation þ,as indicated by the presence of "chondrons", which were cartiìage lacunae with multiple nuclei. Chondron formation was a slou, process, and could occur only in the absence of granulat'ion t'issue. Such granulation was neither primariìy chondrogenic nor d'isposed to form cartÍlage secondarily, except perhaps by gradual fibrocartìlagÌnous metaplasia. Calandruccio was the first to suggest a role for the cartilage cana'l vessel s in the chondro-osseous heaì ing process. hlherever the cart'il age canal vessel s vlere present, cap'il I ary pro'l 'iferati on l{as evident and formation of granulation tissue occurred.

-149- Consistently in lamb experiments there are central metaphyseal spurs of cartjlage. These represent the interstjtial repair process. In al I cases there i s I oss of col umnar ori entati on and carti I age prol 'iferat j on wi th the formati on of cart'il age I acunae wi th mul tì pl e nuclei (chondrons). There is a metap'lasia of the physeal cartilage to fjbrocartÌ'lage as proven wjth polarÌzed light examination. It protrudes jnto the metaphysÌs for a variable extent but is always present jn some degree (Fjg. 6.1).

The f i brocart'il age shows i ncreased co'l I agen f i bre patterns wi th a longitudjnal orientation towards the metaphysis. Polarjzation shows that there is an increase in the matrix/cell ratio. The orjentation of the col 1 agen i s consi stent wi th the normal method of soft tissue/chondro-osseous continuìty which allows attachments to grov', as the bone undergoes elongation. The metaphysea'l direction of the spur of f i brocart'i'l age i s consi stent wi th the concept of a metaphyseal tether of the physi s which grov',s away from the point of iniury towards the joint surface.

The metaplasja of physeal carti'lage can be readjly characterized at the edge of the growth plate defect. In Animal 17 the third deeper cut histological section of the tibial specimen shows at the edge of the defect widening and marked physeaì metaplas'ia (Fig. 6.2).

For practìcal purposes in the larger mammal, sheep, jnterstjtial regenerat'ion wi th hyal i ne cart'il age does not exi st. Th j s f i ndi ng confirm'ing the views of Haas (1931), Banks and Compere (1941) and Hert (1972).

The periphery of the phys'i s may contr j bute to the repa'ir process IKeith 1920, So]omon 1966, Shapiro 19771. In this group of experìments a markedly djsorganìzed cartilage was often noted. The

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1 Figure 6.2¡ PHYSEÀL REP IR At tho cdgo of tho defoet ln À,ni"nal L7 c dcoprr ¡¡,ction i.ndi.eato¡ the def,cct nargln and rol¡lo,nlo of f,i.bro- caÊtilaglnour. nntopla¡La of tho Itt ¡hfe1¡..* ¡ Thc hlatologLcal cvidc¡rcc i¡ that t.hi¡ rcpro.cntr ô¡-dtffcrêntfation of tho hyalinc cartLlagc ar thc ¡mchani¡a of repair. (x 5l {. -- ('

t

4

) ð,j; \\ -t, '.$? I

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r\"

T

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152 cells for this repa'ir may originate from the Ranvjer Zone. Review of the histological work of österman (1972) and Langenskiöìd, Videman,

Neval ajnen (1986), suggest that the main growth p'l ate "repair" observed ìn the rabbit model occurred as a result of the abil'ity of these p'luripotential cells to grow interstitially. In fact comment is made on an unexp'lained step-cut peripheral lesion. In these sheep expeniments to prevent the formation of a peripheral bone bridge the periosteum ltlas excised, and these cells were not available to be part of the repair process.

An additional series of experiments IFoster' 1984, 19861 was undertaken to investigate the effect of physeal distraction on the process of repa'ir.

Rìng (1958), Hert (1969), Ilizarov (1969), Sìedge & Noble (1978)' Montjcelli & Spinelli (l98la), De Bastiani (1986), De Pablos (1986)' and others had suggested that at a slow rate of distraction physeal repl 'i cati on can occur i n accordance wi th Heuter-Vol kmann ( 1862) pri nci p'les .

In 14 animals there u,as a 20 kg. djstraction force. A further 7 animals compìeted low tension djstractjon of 5 kgs. In both series elongation of the bone averaged ll.0 mm. and majntenance of fat at the defect !'Jas cons i stentl y noted . Physeal st i mul at j on was noted onìy in the low tension group with a fracture being produced in the h'igh tension group. These animals had a fracture at different levels withjn the growth plate which would have led to premature physeal closure. All of the 7 low tension animals showed an'increased width of growth plate contour with a stretch of the physis up to twice its width. The tension process djd not however increase the interstitjal

-153- physeal regeneration to repair the fat interpositjonal space. Further work on an ideal tension for distraction js required.

The remainder of the physis maintajns longitudinal growth prov'iding

e i ther:

(i) a unilateral tether can be overcome. That is if a bone bridge does not form or a fracture of the bone bridge occurs (Fig. 6.3).

or

( i 'i ) the remai nì ng physi s has suf f i ci ent potenti a'l to mai ntai n growth.

In support of the last statement is the evjdence that the'larger the defects, the less likely is the possibility of reversal. The C.T. scans show a statìsticalìy significant increase in the defect size from group I to group 3 (p < 0.0001).

The chance of reversal 'in the I sq. cm. defect group is 100% if the 'interpositional material survives. 12 of l2 animals show ìmprovement

of radì ol ogi ca'l val ues. In the 2 sq. cm. group, where there þ,as complete or incomplete necrosis in three animals, reversal occurred ìn only 6 of 1l animals. In the 3 sq.cm. group, where 100% of the t fat survived, the reversal of deformity occurred onìy jn 4 of 8 cases and the varus angulation improvement occurred in only 2 of those

I cases. There was no reversal of the angular deform'ity as great as ( I noted in the I sq. cm. serjes. ó Statistically the C.T. data proves a significant djfference between group one and group three. i.e. between 17.2% + 2.7% and 33.6% +

6.7%, (p < 0.0001) and between group one and group two i.e. 17.2% +

- 154 - ->-

'fE¡a [:.= Ã -ìtl+ ;it: ,¡ - 'í'f

I \

r55

È \:: 2.1% and 28.0% 7 6.0% (p < 0.0001). This suggests that, where the defect is < 28% of the pìate, if a stable environment for the i nterposì ti on can be ma'inta'ined, there wi I I be resumpti on of 'long'itudinal growth and correctjon of deformity. Larger defects may j enabl e resumpt'ion of growth w thout correcti on of angul ation. t¡lhether at skel etal maturity a premature cessati on of physeal act i vi ty occurs has not been exam j ned .

It is proven that correction of ìength and angulation will occur in

100% of cases for the defect of 17.2%.

The C.T. scan data suggested that'if a bone brìdge can be prevented then a non-union effect wilì enable normal growth to proceed. If the jnitiat size of the defect is too bìg the remnant growth p'late cannot majntain growth sufficiently to prevent progressjon of a deform'ity.

Bright (1982) reported on a series of expe¡iments on 130 pupp'ies using Medical Elastomer #382 (X7 - 2320). He attempted to define the percentage of the physeal area that may be resected yet maìntain longitudinal growth. He confirmed that the lateral distal femoral growth mechanism t'tas more sensitive than the med'ial and that central defects would re-bridge if there þ,as loss of contact of the interposition with the epìphys'is. The mechanism for the resumpt'ion of longitudinal growth was not mentioned.

In attempt'ing to document a decrease in size of the physeal defect the author's study did not quantìtate that a decrease in size of the growth plate defect occurred wjth t'ime, as has been proposed by

Langensk.iöld (1986), Langenskiöld, österman & valle (1987).

The c.T. at the time of reversal did show a change jn the size of the defects (Tabìes 5C, 5H). In the I sq. cm. serjes 17.5 + 2.0%

- 156 - increased lo 21.3 + 4.4% al three months, 16.9 + 2.5% decreased to 15.1 + 4.6 at six months. In the 2 sq. cm. series 26.3 t 5.3% decreased to 25.0 + 7.3% at three months and 30.8 + 6.9% decreased to 22.4 + 3.7% at six months. In the 3 sq. cm. series 34.2% + 8.4% increased to 34.7 + 8.1% at three months and 32.7 + 6.3% increased to 36.2 + 9.6% al six months. Us'ing the unpaÌred Student's t test there u,as not a statistically s'ign'ificant alteration jn size between groups. However, if the defect does decrease in size it would seem more consistent that it is a result of relative diminution of the defect size compared to an increase in total physea'l diameter with dìametrjc growth rather than an jnterstitial repair process.

The long'itudinaì size of the defect was noted to be increased in 6 of 7 I sq. cm. defects by six months. This noticeable increase in size of the defect is probably due to the growth potential jn this group. The fat survives and maintains a pos'ition close to the epiphysis and as longitudinal growth occurs the 'lipocytes may pro'liferate to mai ntai n a space wj thi n the stabi I i zi ng bone cavÌ ty. In other experiments it u,as noted that repìacement of cultured chondrocyte impìants occurred with haemopoeìtÍc bone marrol,, fat by six months [Foster 1988]. The stable bone defect allows growth to proceed if the critical area of physeaì resection is not exceeded.

- 157 - Chapter Z

7.1 CONCLUS IONS :

I Interpositional physio'lysis with a free fat graft is confjrmed as an appropriate strategy for prevention of deformity foììowing peri pheral bone bridge resecti on. It i s suggested that' provid'ing the defect is less than 17.2% (t 2.1%l of the total physea'l area, success can be achjeved in 100% of cases.

2 Fat as an i nterpos'i ti onal materi al has the advantage of

avai I abi I i ty and bi ocompat'ibi I 'ity but the di sadvantage of :

( i ) potenti al I oss of posi tì on due to ei ther faj 1 ure of haemostasis or loss of attachment to the epiphysìs/physea'l iunction'

(ii) fat necrosis with bone bridge reformation.

3 Results of Computerized Tomograph'ic scanning have not shown a statist'icaìly significant diminution in the size of defect from three to six months. This questions the abjljty of the physis to repair with normal cartiìage. The mechanjsm of the success of these'interpositional procedures may relate to the abil'ity of the physis to maìntain its function against a physioìog'ical "non-union" produced by the interposìtional material at the physeal level.

4. The repa'ir of the central physeal cartilage is with phenotypi cal ly d'if ferent f i brocarti I age rather than spec'ial i zed

hyal i ne carti'l age. Thi s i s characteri zed by metap'l as'ia and irregularity of clones of chondrocytes.

-158- 5 Fai I ure of i nterpos i t i onal procedures I eads to bone bri dge reformation across the physis. Histolog'ically, the bone brìdges

may be corti cal or cancel ì ous, and may be I ocated ej ther centrally or peripherally, or both.

6 Provision of intact functioning physeal cartilage grafts may enabl e ì arger defects to be repl aced wi th bi o'l og'i cal I y act i ve interpositional materi al s.

7.2 FUTURE DIRECTIONS:

Recent experÌmental ìnvestigatjons suggest that successful repair of articular cartilage defects may be possible [McKibbin 797L, M'itchell

& Shepard 1976, Radin, Ehrlich & Chernack et al 1978, McKibbin &

Râli: L978, Speer 1979, Salter 1980, 1982, Mank'in 1982, 0'Drìscoll & Salter 1984, Miller, Râli5 & l'lcKibbin 1985, 0'Driscoll et al 1986,

Zarnett 19871.

The requi rements for such regenerati on of arti cul ar cartì I age i ncl ude:

(i) a population of cells that can be placed into or will

mi grate i nto a cart i I age defect , where they will prol iferate and djfferentiate into chondrocytes,

(ii) a synthetic or b'io'log'ica'l matrix into whjch cells can penetrate and eventual ly repl ace or remodel i nto a cartilage framework; or cells capable of synthesizing such a matrix,

(ijj) mechanical stimuli to enhance the cell proìiferation and di fferenti ation of chondrocYtes,

(jv) protection of the repa'ir cartjlage from excessive loads,

-159- and

(v) maintenance or restoration of the normal shape and

conformat i on of the cart'i ì age .

These crjteria are similar for physeal cartilage regeneration.

Bentley (1978) has shown that physeal chondrocytes in culture can majntajn phenotype for six weeks. Transplantation of these cells to articular defects may repa'ir articular surface defects for up to one year [Aston & Bentley 1986]. Itay in 1987 successfu'lly used cul tured embryoni c chj ck ep'i physeal chondrocytes as grafts for defects in chjck articular cartjlage.

Trippel (1980), Amadio & Ehrlich (1983), have reported a h'igh density cul ture techn'i que and subsequent transfer to the phys i s of physea'l chondrocytes [Kawabe, Ehrl ich & Mankin 1987]. Despite culture techniques, and the immunological privÌlege of physea'l cartilage alì future experiments with al I ograft materi al wi I I requ'i re hìstocompatibil ity barriers to be overcome. Additjonally' the difficulty of ma'intaining phenotypical expression will need to be cons i dered .

0ther factors that stimulate chondrocyte repa'ir may also have the potential to initiate cartilage differentiation. The mechanism of action may be by stimulating the mesenchymeal cell differentiatjon. Syftestad and Caplan (1986), report promising results wjth a 3l-kD bone matrix proteìn. Another such agent may be the newly isolated osteogenin. Th'is protein, which has an apparent molecular weight of 22-kD, in conjunction wjth insoluble collagenous matrix injtiates cart j'l age formati on ISampath 1987] .

-160- 0ther approaches include the transforming growth factor beta, ISporn 1986, Seyedin 19861 a 25-kD multifunctional growth regulator that induces cartilage proteoglycan synthesis in the mesenchyma'l cells of rat muscle [Cheung 1978]. Platelet-derived growth factor [Huang 19821, fibroblast grourth factor IZapf 1986], and insuljn-like growth factor [Hauschka 1986] may all be potentially important ìn promoting physeal cartjlage repair. The demonstration of site-specific somatomedi n receptors on physeaì chondrocytes may enabl e the stimulation of skeletal growth ITrippel 1988].

To provide a biologically active 'interpositional material experiments wj th physeal chondrocyte cul ture and aì'l ograft transfer are proceed'ing IFoster 1987, 1988, Hansen 1989 (in press)].

- 16l Appendix l.

Initial operat i on control x- ray operat i on x-rays side slre at side 3 months

I Pin D'istance 23 mm 24 mm Pi n Di stance 27 nn 39.5 mm Pin Ang'le 0 5' va'lgus Pin Angìe l4'varus l0'val gus Eone Length l7l mm 169 mm Bone Length 194 mm 197 mm Bone Ang'le 4' valgus l' va'lgus Bone Angì e 6'varus 8'vaìgus

mm 42 mm 2 Pin Distance 23 mm 25 mm Pi n Di stance 42 Pin Angìe 3 . 5' vaì gus I' vaì gus Pin Angìe 9'valgus 2o varus Bone Length 174 mm 173.5 mm Bone Length 209.5 mm 207 mm gu Bone Angì e 2' vaìgus 3'vaìgus Bone Ang'le 4'vaìgus 5'vaì s

mm 3 Pin Distance 23 mm 24 nn Pin Distance 39 mm 40 gus P'in Angì e 3' valgus 3'valgus P'in Angì e 5'vaìgus 4'val 196 mm 201 mm Bone Len Ith 166 mm 172 mm Bone Length Bone Ang l e l' va'lgus 5'vaìgus Bone Angl e 0.5'va'lgus 5' va'lgus

mm 4 Pi n D'i stance 22 nn 22.5 mm Pin Distance 38 mm 47 gu Pi n Ang'l e 3' vaìgus 2' varus P'in Angl e 2'vaìgus 9' va'l s mm Bone Length 158 mm 163 mm Bone Length 198 mm 215 Bone Angì e 0 0 Bone Angì e 4' varus 8' va'lgus

mm 39.5 mm 5 Pi n Di stance 23 mm 23 mm Pin Dist ance 25.5 gus Pi n Ang'le 4'valgus 6' va'lgus P'in Angì e I l' varus 4' va'l mm Bone Length 172 mm 172 mm Bone Len gth 187.5 mm 208 Bone Angle 2' va'lgus 2'valgus Bone Ang le 19'varus 3' valgus

mm 45 mm 6 P'in D'i stance 24 mm 24 mm Pi n Di stance 39 Pin Angìe 6'vaìgus l' vaì gus Pìn Angìe 3o varus l3' vaì gus mm 206 mm Bone Length l7l mm 169 mm Bone Length 2102 val gu s Bone Angì e 4" vaìgus 0 Bone Angle 2'vaìgus 3'

mm 43 mm 7 P'in Dì stance 24 mm 24 mm Pin Distance 21 P'in Angl e 5' vaìgus 5'valgus Pin Angìe l7'varus 5'vaìgus g mm 202.5 mm Eone Length 166 mm 161.5 mm Bone Len th 186 Bone Angì e 4' vaìgus 5'vaìgus Bone Ang I e l5'varus 7'vaìgus

mm 42 mm Pi n D'istance 23.5 mm 23.5 mm Pin Distance 39 I gu Pin Angìe 3.5" vaìgus 5. 5'vaì gu: P'in Angì e 3'vaìgus l2 'va'l s g mm 200 mm Bone Length 169 mm 167.5 mm Bone Len th 199.5 gus vaìgus Bone Angl e 3' vaìgus l' val gus Bone Ang l e 3'vaì 2'

23 mm 40 mm 9 Pin Distance 24 rm 25 mm P'in Di stance 'vaì gus Pin Angìe 3'va'lgus 0 Pin Angìe l2'varus I mm 199 mm Bone Length 167 mm 167 mm Bone Length 195 va'lgus Bone Angl e I' val gus l' vaì gus Bone Angìe l2'varus 4'

Di stance 22 mm 40 mm 10 Pin Distance 24 mm 24 mm Pin va ru s 9"va'lgus Pin Angle 0 5' vaì gus Pin Angle l8' 193 mm Bone Length 164 mm 163 mm Bone Length l8l varu s 5'val s Bone Ang'le 6 . 5' va'l gus 5'vaìgus Bone Ang'le l3'

162 Appendix 2

sheep x-rav at 6 months l No. 3 mõnTñi-õõ!î-iõiõrsa ) operation control operati on control operat I on -¡ftecontrol side side side -l¡e side

1l Pin Distance 21 m 24 17m 41 mm 3l mm 43m Pin Angle 0 0 24'varus 9'val g u s l2'valgus 8'valgus Bone Length 154 m 155 179 rm 189 mm 180 mm 188 m Bone Angle l'valgus 2' val gus I9.5'varus 4'valgus l0'varus 0

Pin repositioned Pln Dlstance 24 Ím 39 mm Pin Angle 3'valgus 5'valgus Bone Length 170 m 180 mm Bone Angle 15'varus 2'valgus

44 mm 12 Pln Distance 23 mm 23m 22 Írî 39 mm 34 mm Pin Angle 2'varus 2'valgus l2'varus l0'valgus 6'varus 9'valgus rvn 155 m 177 mm 184 mm 187 mm 193 Bone Length 155 llfr 6'valgus Bone Angl e l'varus l' varus 15'varus 6'valgus 4'varus

l3 Pin Dlstance 21 m 23m 2lm 35 mm 33 nm 46m Pin Angle 3'varus 2'valgus l7'varus 8'valgus 13'varus 7'velgus 188 rTm Eone Length 142 m 416 ÍÍr 158 m 167 mm 176 mm Bone Angle 2'varus 1'varus 14'varus 5' val gus 3' varus 5'valgus 41.5 m l4 Pin Dlstance 24m 23 rm 22m 34 mm 34 mm Pin Angle 3'valgus 2'valgus l5'varus 3'valgus 4'varus 0 196 nn Bone Length 155 m 157 m 168 m 177 mm I84 mm varus Bone Angìe l' varus l'valgus l3' varus l' val gus 7'varus l'

mm 45m Pln Distance 23.5 mm 23.5 m 23m 39.5 mm 24 0 3'valgus l8'varus 5'valgus 2l'varus 4.5'valgus Pin Angle m Bonq Length 158 m 159 m 179 m 189 mm 187.5 mm 195.5 Bone Angle 4'valgus 5'valgus l7'varus l'valgus 22'varus 2'va'ìgus 45m 16 Pin Distance 23m 23m 2lm 39 mm 23 mm Pin Angle 2'valgus 5'valgus l9'varus 4'valgus 22'varus 5'valgus Bone Length 155 m 154 m 180 m 184 mm 188 mm 195 Ím Bone Angle l' val gus 5'valgus 18'varus 5' val gus l8'varus 3'valgus

nm 51 m 17 Pin Distance 2l mr 22 Ím 20m 42 3l mm gus Pin Angìe I'vaìgus t' va 9Us 22' varus 6' val 17' varus 6'valgus Bone Length 156 m 164 193 Ím 202 mm 209 Ím 218 m Bone Angle 0 3' val gus 23' varus 3'valgus l5'varus 3'valgus

3 Sheep Inltial (3 (6 reversaì ) No. ooeretl on control on control operati on control ooeratl on control operat'i sìoe JÏd-r side side side side side -i¡e

38 mm 5lm 5lm 64m 23 mm 23m 21 m 36 mm 18 Pin Distance 7' varus 7'valgus 4'varus 4'valgus Angle l' va] gus 2'valgus 20'varus 4" va'lgus Pin 206 rm 2ll mm 216 m 225 m 156 nm 155 mm l7l mn 179 mm Bone Length I' varus l'varus l'valgus 2.5'valgus Sone Angle 0 0 l6'varus 5'valgus

31 mm 39m 40m 48m 22 nm 22ffi 21 31 mm I9 Pin Di stance varus 4'valgus 1l' varus t' v aì gus 0 2'valgus t7' varus 6' valgus 11' Pin Angle mn 170 m lg2 m 184 m 137 m 152 mÍt 156 mm 163 Bone Length 138 1'valgus 2'varus 5'varus 2'varus Bone Angle 2', vaìgus 0 t2' varu s 3'valgus 47m 5l rm 22 rîrî 22m 28 mm 34 mm 36m 20 Pin Distance 22m 3'varus 3'valgus 3'varus 0 Angle 0 2' va]gus l0'varus 5' val gus Pin 165 mm 173 Írî 200 ml 204 m Length 144 m 147 fiî 154 m 158 mm Bone 3'varus 0 3'varus l' varus Bone Angle 2'valgus 1' val gus l2'varus 2'valgus

34 mm 37 nm 47m 50 Distance 22 Ífi' 22m 22m 31 mm 2l Pin gus 2' varus l' val 9us 0 1' varus Angle 3'valgus 0 ll' varus l' val Pin mm 173 m 203 m 205 m 148 m 148 m 156 m 160 mm t72 Bone Length 1.5 'varus 2'varus 2'varus l'valgus Eone Angle 0 l' varus aru s 2'valgus

33 mm 43m 46 mn 55.5 m Distance 22 nn 23 mm 22 nn 36 mn 22 Pin gus 8'varus 0 7'varus 0 Pin Angle 0 0 18'varus l' val 166 mm 169 m 197 rn 199.5 m mm 156 nm 162 nm Bone Length 140 nm 140 6.5'varus 3'valgus 6'varus 1'valgus Bone Angle 2'varus l' varus l4'varus 4'va]gus 3I.5 mm 41.5 mm 38m 47 ñr 22.5 nn 19 mm 23 mn 36.5 mm 23 Pin Distance 7'varus 9' vaì gus 7'varus 8'va'ì9us Pin Angle 0 4'varus l5'varus 8'valgus 199 mír 202 mm 207.5 m 216 m mm 159 m 186 nm 194.5 nm Bone Length 159 l' v al gus varus 5. 5'va l gus 4'varus 6' val9us 5'varus Bone Angl e l'valgus l' valgus t2'

mm 42 nm 39 mm 495nm nîl 2l mm 36 mm 31.5 24 Pin Di stance 22 nn 22.5 8'varus 6'valgus 0 l8'varus 9" valgus l0'varus 7'valgus Pin Angle 2'varus mm 206 mm mm I82 mm 192 mm 205 154.4 mm 157 m 178.5 m 187 Bone Length 7'varus 0 5'varus 5'valgus Bone Anqle 0 0.5'va'lgus 14'varus 2'valgus

ró 3 Appendìx 3

I S0. Cltl. c.T. RESULTS

3 I.IONTH SURVIVAL

Arear Total Arear An i mal c.T Total Arear G. P, Total G. P. G. P. Ave No. G. P. Area G.P. Area G.P. Area

54.07¡n.r5 t7 l1 Initial s4.2¡n.r, 17.l 53 .7s/ s .24 17 .2 l7.l .l 6t.9¡ 62.5/ß.0 28.8 26.5 Fi nal 6s.1/ 15.7 22.7 r, .t 27 .9

60 .09 70'4/ 70.09 5 t7 .9 t2 Ini ti al ¡ ,, .ru t7 .7 tz.ss 17.5 ¡ rr.nn 18. 64 .2¡ 64 .37rr .6 21.5 Fi nal 68.87r0.. 21.5 rt ., 21.5 . , 21

67 .tr 67.8r¡rr.ro 17 16 .6 t3 Ini ti al 68 .2r ¡ ,, .r, 16.5 ¡ ,o .r, 16.03 .3 65 .67r 62.3 6l.47rt.o 17.9 I8. 6 Final t ., v.7 ¡ r,., 20.1

69. s37to. 69.0s7to.rt 68.337, 15.6 l4 Initial Ut 15.2 15.5 t . Ot l6.l Final 56.5¡ r,.o 22.0 5s.2¡ rt ., 23.9 58.7 ¡ ,r.t 2l .3 22.4

85.07tU.t l5 Ini ti al 84.67tt. t l8.l 84 .2¡ ,U.t 18.2 18.6 18.3 74.6¡rU.u 20.6 Fi nal 71.t¡r0., l9 .3 76.t¡rU.o 2t.6 20.9

91.6¡ 9l .07tr 21.4 2t.2 l6 Initiaì 9r.7¡rn.o 2t.2 rt., 21.0 . t 96 .2¡ Final 100.37rU. t 25.4 98.4 ¡ rU., 26.1 rU.o 26.4 26.0

107 .07tr., t7 Ini ti al 107 .07rr.3 15.2 107 .7 ¡t ., 16. 0 l5 .2 15.5 I 15. 17tO. 116.2¡ trz.r 14. 5 l3 .6 Fi nal n l2 .5 rr., l3 .9 ¡ ru.,

I SQ. CIII. C.T. RESULTS

6 I'IONTH SURVIVAL

Total Area/ Total Area/ Ave An i mal c.T. Total Area¡ G. P. + G. P. G. P. No. G.P. Area I G.P. Area % G.P. Area %

63 .4s7ro. l8 Ini ti al 66.087rt.rt 17.0 65. l07to. rt 16. 5 tt 16.0 16. 5 80.37r, t7 .2 76.7 18.3 75.s7tr.t 17.6 t7 .7 Flnal . t ¡ rn .o

s0.057r0. 48. 957, s0.397t0. t9 Initl al u ?t.0 . ,n 20.3 rt 2t.3 20.9

5l . 57n. 49.7 Fi nal 52.6¡t.t 18.8 o 17.5 ¡n., 18.7 18.3

s2.77¡t.o 53. 187r. 53.24¡t.rU t7 .4 17.5 20 Initial 17. I U l7 .9

59.4¡n.s 60.67t0., 7 Ff nal 60.2¡, .s 16. 5 16.7 l7 .0 t6.

ss.357ro. 56.0/ll.l7 s3 .3s7r. l7 .9 l8 .8 2l Initial rt 18.6 20.0 UU Finaì 64.4¡ rr.t 19. l 68.2¡ ,t ., 19.8 66.4 ¡ ,, .t 19.3 r9.4

62.527t0.tt 6r.70¡ro.ro 22 Initi al 62.857rr.r6 l7 .8 t6.2 17 .4 l7.l 80.s/n.a 80.87r 18.4 16.6 15.6 Fi nal 15.8 r . t 14.5 ¡ ,, .o

7s.7 79.t¡r,.o 80.07rt.t l5.l 13.5 23 Ini ti al ¡t.t n.7 l3 .8 2'92/ 2'8/ 2's/n.q 5.9 6.4 Final cs .o 6.8 qs .s 6.5

70 .2¡ 68.6¡n.n 7t.6¡t.t l3 .8 t4.2 24 Initìal r0.7 15. 2 13.7 6.6/sg.g Final 6'3lss.a ll.4 6'3/ss.z I1.5 l2 .3 r1.7

164 Appendix 4

ât SheeD (3 r) No.

on control oDe rat i on controì oDerat i on contro'ì operatl -slTe side g s ide side g

22 Ím 37m 28 rn 45 ßI 25 Pin Dlstance 22ffi 24m 5'vaì9us Pin Angle 0 3'valgus l8'varus 3'valgus 1l'varus mì 176 rn 134 mn 135 iln 156 mr 155 mm 162 Eone Length varus Bone Angle 5'varus 0.5'varus l0'varus 3'valgus 13'varus 3'

mm 44 mm 26 Ptn Distance 2lm 22 Ín 20m 32.5 mm 3l Angle 4'valgus 2'valgus l3'varus 5'v¡lgus l3'varus 6'valgus Pìn 169 ilî Bone Length 128 m 129 nn 139 m 147.5 m 165 m Bone Angìe 2'varus 3'varus l9'varus 5'valgus l5'varus 5'valgus 50 ín 21 23 riln 22m 24m 34 mm 29m Pin Dlstance 9'va gus Pi n Ang'ì e 2'valgus 7'varus l5'varus 6'varus 20'varus 142 m 160 m 170 mm 180 rn 184 rÍr Bone Length 145 fln 4'valgus Bone Angle 5'varus l2'varus l7'varus l'valgus 20'v¡rus

38 mm 28.5 mr 50.5 m Pin Distance 22 Ín 22 mm 22 nm 8'valgus Pln Angle 3'valgus 5' vaì gus 23'varus 7'valgus l0'vrrus 183 nn 187 m Bone LFngth 136 mn 135 [n l6l m 167 mm 0 Bone Ahgìe l'valgus 6' varus 22.5' varus 2'vaìgus 20'varus

42 mm 2? trnt 2l rm 16m 30.5 mn 20 ilr 29 Pln Distance l' varus Pln Angle 0 0 38'varus l'varus 40'varus 174 m 179 mm Bone Length 133 rn 132 m l5l m 151 mn 18.5'varus 0 Bone Angle l' varus l'varus 26'varus 5'varus

22m 36 mm 25m 46m 30 Pin Dlstance 22.5 m 23.5 m l0'valgus Pin Angìe 7'valgus 6'valgus 9'varus 8'vaìgus 18.5'v 146 rn 168 m l7l mn 188 m 192 ttrn Eone Length 146.5 m l4'varus 5'vaìgus Bone Angle 4'valgus 9'valgus l7'varus l'valgus 28 íÍr 42 fln nm 23m 23m 39 mm 31 Pin Dlstance 22 l' varus Angle 2'valgus l'vaì9us l5'varus l'valgus l2'varus Pin rm 214 mr 165 riln 163 ilI 194 mr 205 m 214 Bone Length varus 7'varus Bone Angìe 4'v¡l9us 2'vaìgus 19'varus l'vàlgus l5'

9 months Ini r-rav at Sheep (3 (6 rnolifil-posT re.versal ) l{o. cont¡ol oDeration controì ooeration control oDe¡atlon control ooeratlon -mle- side -mle- slde -¡itõ- sloe sfile side

34 fln 32 rn 45.5 ß¡ 37.5 m 50. 5m 32 Pln Dlstance 22.5 rn 23,5 mr 2l mr ¡l'valgus 6.5'varus 3' vaìgus l'valgus l5'varus 0 8'varus Pin Angle 2'valgus 200 m 207 mn 156 m 157 m 169 ûn 176 mm 189 m 195 r,n Eone Length l'velgus ¡l' varus t' valgus Bone Angle l4 valgus l'vaì9us l4'varus 2'vaì9us l2'varus

59.5 m 32m 38 mn 40m 47. llfn 50m 33 Pin Distance 22 ñi 23 fin 3'valgus 0 4'varus 5'valgus 2'varus 5' vaìgus l'varus Pln Angle 2'valgus 195 tffl 213 rril 218 m l5l ñÍ l5l m¡ 176 m 179 mm 195 fin Bone Length 2'varus 3.5 vaì gus 0 1.5' valgus Bone Angle 3' v¡rus 2.5'verus 5'v¡rus 2'varus

ßr 36 rn 33 mî 49m 4lm 60m 34 Pln Dlstance 23 mr 23m 2l 3'varus 2'vaìgus l7'varus 4'vaì9us l8'varus 4' vðìgus 2l'varus Pfn Angle 6' valgus 199.5 ¡rn 210 m 220 m 152 m 168 ml 178 nm 188 rÍr Bone Length 152 nn l5'varus 5' vaì9us l7'varus 2'vaì9us Bone Angìe 0 4'vaìgus l8' varus 2'valgus

mt 37 rm 64m 20 lltî 35 rn 36 fln 49.5 35 Pln Dlstance 22m 22 nnt 4'varus 20' verus 9'valgus 4'vaìgus l'v¡lgus l7' varus 5'valgus l0'varus P1n Angle 192 ñî 197 mm 204 m 212.5 m 137 m 137 iln l5l mt 156 mm Bone Length 4'vaìgus l0'varus l'valgus 22.5'varus 5'valgus Eone Angle 3'varus 0 20' varus

tó5 Append i x 5 2 SQ. CM. C.T. RESULTS

3 I'IONTH SURVIVAL

Area An i mal c.T Total Area G. P. Total G. P. G. P. Ave No. G.P. Area % G. P. Area %

63 63.37tr.t 6a.5¡ro., 25 Initia'l .37rr. t 30. 5 30. I 3I.2 30.6

67.9¡r.-.t 67 .8¡ 28. 5 Fi naì 67 .9/ 2s.7 30. 5 32 .8 r, .t 30.6

57 .6¡ 57 .8¡ 26 Initial 58.57rr.t 21.8 rr.o 21.5 rr., 22.0 2l .8 59.2¡ 58.07tr.t Finaì 6l .97r0., 24.0 ro ., 24.2 23.6 23.9

49.7 49.a¡ro.t 27 Initial 50.37r0.n 28.6 ¡ ro .o 29.1 30. 2 29.3 82.5¡,, Fi nal 83.7¡ro., 24.7 86.6¡r, .o 2q.3 ., 24.1 24.4 74.3¡rr., 28 Initial 74.2¡rr., 29.2 t5,3¡rt., 30.6 30 .8 30.2 Final 73.9¡rr., 39. I 73.0¡ru., 35.8 68.7 ¡ro.u 35.8 36.9

6s.a¡ 68.57rr.u 29 Initial 70 .3¡ rr., 18. I rt.o 19.3 16.8 l8.l 72.e¡r,., Final 70 .2¡ ru., 23.9 65.8¡rr.o 25.8 20.9 23.5

82.0¡rr.t 82.2¡ru., 3l .3 30 Initial 83 .2¡ rr., 30.9 3l .5 31.4 7 .6/ss.z Fi nal 7 .7 /gz.t 24.1 7 '3/gg.o 2L.7 22.9 22.9

trz.9/ I I0.07ru. 23.0 tr0 .7 22.5 23. I 3l Initial 27 .O 23 .9 t ¡ ro .t 9'oloe. 8's 13.4 13.3 Final 9 'o / ao.o 13.5 o 13. I / ae.+

6 ]'IONTH SURVTVAL

G. P. Ave An i mal c T G. P. G. P. lo % -E % lo

63.67tr., 6r,3¡r, 28.9 29.8 32 Initial 64 .78¡ rt .0, 30.0 30.4 ., e'r Final 9'5/ qs.q 2l .0 9.9/ cs.t 22.7 / qs.z 20.2 2l .3

62.r 59.77 59.87tn. 23 .8 23.0 33 Initial ¡ ro .o 22.6 ¡ t .u 22.6 t 8'9 8'3/qs. t8.2 19. r Final 8'8/qo. oe I9.0 / qq .a 20.0 a

52.6¡rr., 5r.6¡tr., 29. 5 30.7 34 Initial 52.5¡ru., 30.0 32.5 9'3/ 9'7 qs.z 2l 4 2l .6 F i na'l 9.e/qq.t 22.5 qq.s 20.8 /

58.5¡ 57 .3¡ 57.7¡rt.o 39.9 39 .8 35 lnitial rr.g 39.0 rt .t 40.6 I I .0700. r0.2¡ 25.2 27 .7 Final t2.t¡ oo., 30.5 t 27 .5 oo.t

16 6 at x-rav at 6 months Sheep itial 3 rnoñTfis posT-iwersal ) No. Appendix 6 operati on control oDerati on control operat i on control side -lTlle side sl¡õ- side =ìîe

14.5 mm 53.5 mm 17 mm 39 mm 36 Pin Distance 22 rn 23 mm 33'varus 5'valgus 3'val gus 23' varus 8'vaìgus Pin Angle 0 mm 207 mm nm 186.5 mm 192 Length 155 mm 158 mm 178 Bone varus 2'valgus 31" varus 4'valgus Bone Angle 0 2'valgus l9' 26 mm 46 mm 20.5 mm 38 mm 37 Pin 0istance 24.5 nrn 2l mm varus 3.5'valgus 3' valgus l4' varus 5'valgus 14' Pin Angle l' valgus 169 mn 178 mm 129 rm 134 mm 150 mm 163.5 mm Bone Length 8' valgus l8' varus 8' valgus Bone Ang'le 4'valgus 4' valgus l6'varus 45. 5mm 41.5 nm 24.5 mm 24 mm 23.5 rm 2l rm gus 38 Pin Distance 19'varus 6'valgus 17'varus 4.5 ' val Angle 2'valgus 3'valgus mm Pin 206.5 tnn 210 mm ?21 167.5 run 174 nm 203 rm Bone Length 1' varus l6' varus t' vaì gu s Bone Angle 5' vaìgus 0 l5'varus 25.5 mm 41.5 mm 25m 2l mm 39.5 rm 39 Pin Distance 21.5 rm 5'vaìgus 7' varus 6' valgus Pin Angle l' valgus 5' vaìgus 11.5'varus 170 run 190 mn 194 mnt Bone Length 168.5 nr¡ 4'valgus l2'varus l0' varus Bone Angle 4' valgus l'valgus 10.5'varus ¡¡r¡ 4l mm r¡n 16 mm 37.5 mm 22 40 Pin Di stance 22 Ín 2l 2'vaìgus 10.5'varus l'valgus 5'varus 3'valgus o. Pin Angle 2'valgus 215 mm 2?4 ¡m 176 run 204 rm 210.5 mm \ Bone Length 181 mn 4'valgus 20' varus 3'varus Bone Angle 0 5'valgus l3'varus

x-rav at 9 months Ini r at x-rav at 6 months Sheep (3 monTñT post reversal ) (6 rnonTillõsT reversaì ) No. on contro l operati on contro l on control operati on control operati operati side side side side side si¡e- side =I¡e

mm 36.5 mm 52.5 rm 23.5 mm 39.5 mn 35 mm 5l 4l Pin Di stance 24 rm 24 mn 9'varus 3'valgus valgus l5'varus 6'valgus ll'varus 3'valgus Pin Angle 0 l' 217 mm 198.5 ¡m 204 mm 210 mm 206 nm Bone Length 173 mn 173 rm 187 rrn valgus 0 10'varus 0 l' Bone Angle 3'valgus 3'valgus 14'varus mm 38 mm 5l mm 40 53 25.5 nm 23 nn 39 mm 42 Pin Di stance 24 m¡n 0 l' va1 gus l'valgus l'valgus 7'varus 7' valgus 0 0 Pin Angle 198 mm 207 mm 206 mm ?12 nn 164 nm 165 ¡m 179.5 nrn 189 mm Bone Length l'valgus 7'varus 0 3' aru 2'valgus Bone Angle 5' varus l' varus ll'varus mm 4l rm 26 ¡m 46.5 mm rn 22 ¡rn 2l r¡n 32.5 nn 2l 43 Pin 0istance 22.5 2l' varus 20' valgus 23' varus 26'vaìgus 0 3'varus 18" varus 9'vaìgus Pin Angle 164 mm 178 mm +75mm + 88 m¡n rm 143 rm l5l nm 158 rm Bone Length 142.5 l'varus 18' varus 2'valgus ll'varus 0 Bone Angle 2'varus 2' varus 9.5'varus Appendix 7

3 SQ. Cl.l. C. T. RESULTS

3 }IONTH SURVIVAL

An'imal c. T. G. P. G. P. G. P. Ave .T ol ot No. tb lo % lo

83 . 57t0. 83.2¡rr., 36 Ini ti aì 7e ,s ¡ rt., 35.9 t 36.3 35. I 35 .8 r0.2¡ 10. a7or. Fi nal t0.6¡ or., 25.1 nr., 24.7 t 24.1 24.6

68. 67to 68.3¡rr., 47 6e.37rt., 46.0 47 .6 37 Init'ial . o 49.6 .t re .0¡ 18.870, Fi naì 18.470r. t 37.7 r0., 37.9 ., 37 .8 37.8

s2.8¡rr.u e3.6¡ 90.2¡rr.t 25.4 25.2 38 I n I t 1 aI 23.2 r, ., 26.9 8'8/sg 8'4/sz.e 15.9 8'o/sz.g 15. I l s.8 F ì na I . o 16.5

tr6.2¡ rr2.r¡ 14.5 13.6 39 I n I t I aI ll5.17to.n 12.5 tr., l3 .9 ru., 12.4¡ rr.3¡ 23.2 tr.7 23.6 24.0 F i naI ot.t 2s.2 nt., ¡ on.,

tr?.5¡tu., rt?.2¡tt.n 30.2 30.9 40 I n 'i t l aì ttl.87rn., 3l .2 3l .3 6a.37rr Final 68.87r0. t 2l .5 6a.2¡ rr.t 21.5 ., 2t.6 2t.5

6 I.IONTH SURVIVAL

Total Are T tal Area G. P. Ave c.T Total Area G. P. G. P. T ol d lo lo G. P. Area lo G;P. Area lo G. P. Area =-

ß0.4¡ru.t ee .3¡ 27.9 27.3 4l Initial ee.r¡ r, ., 27 .4 26.7 r, ., re .5¡ 18. lTut 29.4 18. lTrt 29.2 30.0 Fi naì ur., 3l .4 . t .,

8e.4¡r, 88.6¡rt.o 3l .6 3t.2 42 I n I t i aI 88.3¡rt.o 3l .7 ., 30.4 20.r¡uo.t 33.2 t8.3¡ 29.5 3l .4 F i na 1 re.4¡ur., 3l .6 ur.,

7a.3¡rt., 38.0 73.8¡rr.n 38. 5 39.7 43 I n i t I a 1 73.0¡tr., 42.7 25.r 25.2¡rt.n 46.7 25.8¡ 46. I 47 .2 F i na I ¡r, .n 48.8 r, .n

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