UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The effect of immobilization on ligamentous healing and strength of the medial collateral ligament of… Pisesky, Wayne Anthony 1982

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1982_A6_7 P57.pdf [ 17.88MB ]
Metadata
JSON: 831-1.0095502.json
JSON-LD: 831-1.0095502-ld.json
RDF/XML (Pretty): 831-1.0095502-rdf.xml
RDF/JSON: 831-1.0095502-rdf.json
Turtle: 831-1.0095502-turtle.txt
N-Triples: 831-1.0095502-rdf-ntriples.txt
Original Record: 831-1.0095502-source.json
Full Text
831-1.0095502-fulltext.txt
Citation
831-1.0095502.ris

Full Text

THE EFFECT OF IMMOBILIZATION ON LIGAMENTOUS HEALING AND STRENGTH OF THE MEDIAL COLLATERAL LIGAMENT OF THE RAT KNEE by WAYNE ANTHONY PISESKY B . S c , The U n i v e r s i t y o f A l b e r t a , 197^ M.D., The U n i v e r s i t y o f A l b e r t a , 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department o f Anatomy F a c u l t y of Medicine We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1982 £c) Wayne Anthony P i s e s k y , 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 DE-6 (2/79) i i ABSTRACT The purpose of t h i s study was to determine the e f f e c t s of varying periods of immobilization on ligamentous healing and strength i n a r a t experi-mental model. Sixty-one mature male Wistar r a t s were used. The l e f t knee medial c o l l a t e r a l ligament was s u r g i c a l l y exposed, divided, and repaired. The r a t s were randomly placed into one of four groups: Group A, no immobiliza-t i o n , Group B, 2 weeks' immobilization, Group C, 6 weeks' immobilization, and Group D, 10 weeks' immobilization of the operated limb. The r i g h t knee served as a c o n t r o l . The ligaments were studied h i s t o l o g i c a l l y and biomechanically at 2 weeks, 6 weeks, 10 weeks and 20 weeks post-operatively. H i s t o l o g i c samples were o b j e c t i v e l y evaluated with the l i g h t microscope using a Maturity Index Score and Scale that were devised based on the numbers and o r i e n t a t i o n of the f i b r o b l a s t s and the amount and o r i e n t a t i o n of the collagen f i b r e s . Ligament-bone preparations were studied using an Instron material t e s t i n g machine to determine the biomechanical properties of the ligament u n t i l f a i l u r e . U t i l i z i n g the Maturity Index Score and Scale, i t was shown that Group A, with no immobilization, matured more r a p i d l y than the other groups, and achieved f u l l maturity at 20 weeks post-operatively. The other groups a l l showed a retarded rate of healing while immobilized. The electron microscopic study supported t h i s data by demonstrating the l e v e l of metabolic a c t i v i t y of the f i b r o b l a s t s which decreased with increasing maturity and by demonstrating that the s i z e , amount and o r i e n t a t i o n of the collagen f i b e r s increased with mobilization. i i i The biomechanical t e s t i n g showed that at 2 weeks post-operative, Group A had achieved a strength which was 46% of controls while Group B was only 29% of controls (p = 0.055). At 6 weeks Group A was 65% of controls, Group B was 56% of controls and Group C was 39% of controls (p = 0.0004). At 20 weeks Group A was 83% of controls, Group B was 71% of controls, Group C was 66% of controls and Group D was 48% of controls (p = 0.0005). Group A was 71% stronger than Group D at t h i s time, i n d i c a t i n g that the healing medial c o l l a -t e r a l ligament attained a greater strength and h i s t o l o g i c a l l y matured more rapi d l y i f mobilization i s begun immediately. i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS i x INTRODUCTION 1 A. Current Management F o l l o w i n g Knee Ligament I n j u r y or Reconstruction 1 B. The E f f e c t s o f Immobilization 1 C. Normal Ligament 4 D. Cytology o f the F i b r o b l a s t 4 E. Ligament Healing 5 F. T e n s i l e P r o p e r t i e s 6 G. The Role o f Function i n Ligament Healing 7 PURPOSE OF STUDY 8 MATERIALS AND METHODS 9 A. Population and Procedure 9 B. Immobilization Procedure 10 C. H i s t o l o g i c Test Procedure 11 D. H i s t o l o g i c E v a l u a t i o n 12 1. L i g h t Microscopy 12 2. E l e c t r o n Microscopy 13 E. Biomechanical Test Procedure lH V Table o f Contents (Cont.) Page MATERIALS AND METHODS (Cont.) F. Biomechanical E v a l u a t i o n 14 RESULTS 16 A. General 16 B. His t o l o g y - L i g h t Microscopy 16 1. C o n t r o l s 16 2. Two Weeks 17 3. S i x Weeks 17 4. Ten Weeks 18 5. Twenty Weeks 19 C. H i s t o l o g y - E l e c t r o n Microscopy 20 1. C o n t r o l 20 2. Two Weeks 21 3. S i x Weeks 21 4. Ten Weeks 22 5. Twenty Weeks 23 D. Biomechanical Te s t i n g 24 1. Maximum Load at F a i l u r e 24 2. Separation Force Ratio (S.F.R.) 24 3. Elongation 26 4. Compliance 26 5. S i t e o f Ligament F a i l u r e 27 DISCUSSION 28 A. The Experimental Model 28 B. Histology 28 v i Table o f Contents (Cont.) Page DISCUSSION (Cont.) C. Biomechanical T e s t i n g 30 D. C l i n i c a l Relevance 32 SUMMARY AND CONCLUSION 35 TABLES 36 FIGURES 44 BIBLIOGRAPHY 118 v i i LIST OF TABLES Table Page I M a t u r i t y Index Score 36 I I M a t u r i t y Index Scale 37 I I I Range o f Motion 38 IV M a t u r i t y Index Score 39 V Biomechanical T e s t i n g o f Medial C o l l a t e r a l Ligament 40 VI Repaired Ligament / Control Ligament 41 VI I S i t e o f F a i l u r e 42 V I I I S i t e o f Ligament F a i l u r e 43 v i i i LIST OF FIGURES Figures Pages 1 Rat Skeleton 44 2 Ligament Bone Holder 45 3 Instron Materials Testing Machine 46 4- 5 Light Micrographs of Control Ligaments 47- 48 6-30 Light Micrographs of Repaired Ligaments 49- 74 31 Maturity Index Score Graph 75- 76 32-34 Electron Micrographs of Control Ligaments 77- 80 35-60 Electron Micrographs of Repaired Ligaments 81-110 61 Separation Force Ratio Graph 111 62 Separation Force Ratio Bar Graph 112 63 Separation Force Ratio Graph 113 64 Ligament Elongation Graph 114 65 Ligament Elongation of Repaired vs Control Graph 115 66 Ligament Compliance Graph 116 67 Ligament Compliance of Repaired vs Control Graph 117 ACKNOWLEDGEMENTS I would l i k e to extend my si n c e r e g r a t i t u d e to my sponsor Dr. B. Day f o r h i s support and i n t e r e s t i n t h i s p r o j e c t . I would a l s o l i k e to thank Dr. K.S. Morton who allowed me to spend a year i n the Department o f Anatomy as part o f my orthopaedic t r a i n i n g . Thanks are a l s o i n order to V i r g i n i a L a i f o r her help i n the prepara-t i o n o f the h i s t o l o g i c s e c t i o n s and t o Dr. F. Weinberg i n the Department o f Metullurgy f o r h i s a s s i s t a n c e i n the biomechanical t e s t i n g . F i n a l l y , I would l i k e to thank Dr. S.M. Friedman and Dr. C.E. Slonecker and a l l the other members and s t a f f o f the Department o f Anatomy f o r t h e i r support. INTRODUCTION A. Current Management Following Knee Ligament Injury or Reconstruction. Since the time of Hippocrates, physicians have been advocating immobil-i z a t i o n of damaged ligaments and j o i n t s with various forms of plaster casts, s p l i n t s and bandages. However, the optimal period of immobilization following N • 4 . • ^ ^ • -, 5,9,14,26,30 a knee ligament repair or reconstruction i s c o n t r o v e r s i a l Various authors (Palmer, O'Donoghue, Noyes) have advocated periods of immobil-5 24 i z a t i o n of 6-12 weeks a f t e r knee ligament repair or reconstruction ' This i s followed by an extensive period of mobilization and physiotherapy. 5 14 9 30 Other authors (Aim , Haggmark , Dehne and Perkins ) have advocated more con t r o v e r s i a l methods of post-operative, or post-injury management. 5 Aim f e e l s that four weeks of p l a s t e r immobilization i s s u f f i c i e n t while 14 Haggmark believes that immediate cast brace applications i s the preferred 9 30 method of management. Both Dehne and Perkins believe that no immobili-zation i s necessary. Yet there i s no published well designed experiment studying the e f f e c t s of varying periods of immobilization on the healing l i g a -ment. Authors, such as Clayton and Weir, have stated that "attempts were made to determine the e f f e c t s of immobilization on the rate of healing, but, since i t was d i f f i c u l t to maintain s a t i s f a c t o r y immobilization by plaster, the 8 r e s u l t s were discarded" . B. The E f f e c t s of Immobilization. The claims of r e s t have been advocated, i n the past, by such notable 30 surgeons as H.O. Thomas, John H i l t o n , and S i r Reginald Watson-Jones . They regarded r e s t as a matter of noninterference as i n the case of John Hi l t o n or as a p o s i t i v e treatment as i n the teaching of H.O. Thomas. "He believed and 30 preached that rest had magical properties" . However the harmful e f f e c t s of prolonged rest and immobilization have been overlooked by these great men. George Perkins has pointed out the damage to the knee j o i n t when immobilizing 30 a lower extremity i n the treatment of a tuberculous hip . Numerous inves t i g a t o r s have demonstrated the deleterious e f f e c t s o f immobility on bone, 1-3 9 10 14 19 20 j o i n t s and soft t i s s u e structures i n both animals and man > > > » » » 22,24,26,30,32,33,35,36,38^ 2 6 Noyes has demonstrated a decrease i n bone density of immobilized bones. He has also demonstrated subperiosteal bone resorption at s i t e s where ligaments of the knee i n s e r t d i r e c t l y into bone through periosteum leading to a higher incidence of ligament f a i l u r e at these s i t e s following immobili-zat i o n . This applies to the t i b i a l i n s e r t i o n s of the medial and l a t e r a l c o l l a t e r a l ligaments of the knee. The Femoral attachments of these ligaments and the attachments of the cruciate ligaments are through a zone of f i b r o -c a r t i l a g e which did not appear to be s i g n i f i c a n t l y a f f e c t e d by the immobiliza-t i o n process. He also found a s i g n i f i c a n t decrease i n the strength of the normal anterior cruciate ligament to 61% of controls a f t e r being immobilized f o r 8 weeks. The strength of these ligaments was s t i l l only 91% of controls a f t e r 12 months of reconditioning. Evans"*"^ studied immobilization and remobilization of rat knee j o i n t s and found that a f t e r prolonged immobilization ( a f t e r 30 days) contractures of the muscles of the l e g and of the capsule of the knee j o i n t lead to a r e s t r i c -tion of motion. He found a p r o l i f e r a t i o n of intracapsular connective t i s s u e and the formation of a large number of i n t r a - a r t i c u l a r adhesions. He also documented major i r r e v e r s i b l e c a r t i l a g e a l t e r a t i o n s including matrix f i b r i l l a -t i o n , c l e f t formation and u l c e r a t i o n with adjacent subchondral l e s i o n s . These 3 c a r t i l a g e changes were present at as early as 30 days of immobilization and were at areas of t i b i a l - f e m o r a l contact and patello-femoral contact. 1 2 3 36 Using histochemical techniques Akeson ' ' and Woo have docu-mented the response o f the connective t i s s u e s around the knee to varying periods of immobilization. They demonstrated a s i g n i f i c a n t loss o f chondroitin-4 and -6-sulphate, hyaluronic a c i d , and water i n p e r i a r t i c u l a r connective tissues of immobilized knees. They found that the t o t a l amount of collagen remained constant but that there was an accelerated rate of turnover of t h i s collagen and that immature collagen was being formed. Their con-clusions were that the l u b r i c a t i n g properties of hyaluronic acid probably con-t r i b u t e to the ease of motion between adjacent f a s c i a l , capsular and l i g a -mentous structures. They postulate that "motion of connective tissue would appear to i n h i b i t the contracture process through several mechanisms: a) stimulation o f proteoglycan synthesis, thereby l u b r i c a t i n g and maintaining a c r i t i c a l distance between e x i s t i n g f i b e r s ; b) ordering (rather than ran-domizing) the deposition o f new collagen f i b e r s so as to r e s i s t t e n s i l e stresses; c) prevention of anomolous c r o s s l i n k s i n the matrix by preventing a stationary f i b e r - f i b e r a t t i t u d e at intercept points" Other authors have demonstrated the b e n e f i c i a l e f f e c t s which motion has • . v . . , . . * ^ 1. « . * . * . • «. 9,10,14,20,22,24,26,30,35,38 on the i n t e g r i t y and strength of ligaments ' ' ' ' ' ' i-> t-> t-> n 20 Laros found that at ligament-bone junctions i n dogs e x i s t i n g at various l e v e l s of physical a c t i v i t y , strength diminished as physical a c t i v i t y dimini-35 shed and t h i s decrease was prevented by exercise. Tipton also found that immobilization of a normal r a t knee j o i n t lead to a decrease i n ligament strength and that forced a c t i v e mobilization lead to an increase i n t h i s l i g a -mentous strength. Zuckerman compared ligament strength o f r a t s subject to various a c t i v i t y l e v e l s and found that i n the forced exercise group (swimming if or running) there was a s i g n i f i c a n t increase i n the ligament strength. 26 Noyes has also documented the e f f e c t s of reconditioning of immo-b i l i z e d ligaments showing an increase i n strength with reconditioning but with only p a r t i a l recovery a f t e r 8 weeks of immobilization followed by 20 weeks of reconditioning. He also believes that mechanical forces have a favorable i n -fluence on healing properties by a f f e c t i n g the alignment and or i e n t a t i o n of collagen f i b e r s as well as stimulating the maturation phase of the healing process. He states that the s p e c i f i c d e t a i l s as to the timing, magnitude and mode of a p p l i c a t i o n of mechanical forces to achieve such e f f e c t s are not known. C. Normal Ligament. When examined h i s t o l o g i c a l l y , ligament can be seen to consist mainly of p a r a l l e l collagen f i b e r s with small numbers of c e l l s interspersed between the collagen f i b e r s . U t i l i z i n g the electron microscope these collagen f i b e r s can be seen to have a c h a r a c t e r i s t i c regular crossbanding with a p e r i o d i c i t y of . o 640A. This crossbanding i s the r e s u l t of the overlapping of the three basic units of tropocollagen which are composed of amino acid chains and twisted into a left-handed superhelix with a pitc h of 28.6A"*"^ ' The tropocollagen filaments have a high tendency to f i b r i l formation which occurs extra-c e l l u l a r l y and i n a manner where they are arranged p a r a l l e l to each other. Situated between the collagen f i b e r s are the c e l l s of the ligament, the immature f i b r o b l a s t s and the mature f i b r o c y t e s , as well as occasional c a p i l l a r i e s . The f i b r o b l a s t i s responsible f o r the production of the tropo-collagen filaments as well as for the production of the small amount of ground 15 substance seen within the ligament . This consists of the protein poly-saccharides, hyaluronic acid and chondroitin sulphate. D. Cytology of the Fi b r o b l a s t . The f i b r o b l a s t i s an act i v e c e l l engaged i n the production of collagen 5 and connective t i s s u e matrix. I t s dormant phase i s the f i b r o c y t e . Histo-l o g i c a l l y i t can be seen that the f i b r o b l a s t contains a l l of the i n t r a c e l l u l a r organelles associated with a metabolically a c t i v e c e l l . The most d i s t i n c t i v e feature of the f i b r o b l a s t i s the highly developed rough endoplasmic reticulum 31 ( r . e . r . ) . This consists of f l a t or d i l a t e d sacs or channels that occupy approximately 35% of the volume of the c e l l . I t i s ribosome studded and i s responsible for protein synthesis. The r . e . r . i s associated with a well developed, randomly dispersed g o l g i apparatus which i s a secretory complex responsible for carbohydrate synthesis and the packaging of proteins and enzymes. The f i b r o b l a s t also contains numerous large mitochondria for energy production. Other features include a large ovoid nucleus with one or more prominent n u c l e o l i as well as v e s i c l e s , l i p i d droplets and cytoplasmic filaments. E. Ligament Healing. L i k e most other t i s s u e s i n the body, ligament healing can be divided into three overlapping stages of r e p a i r : the inflammatory phase, the p r o l i -15 22 31 f e r a t i v e phase and the remodelling phase ' ' . The f i r s t phase i s the inflammatory phase which l a s t s four to seven days"^'^. I n i t i a l l y there i s bleeding, exudation and the formation of a f i b r i n c l o t which w i l l act as a s c a f f o l d for early r e p a i r . The ends of the ligament become edematous and there i s an invasion of inflammatory c e l l s into the wound. These consist of polymorphonuclear leucocytes followed by mono-cytes and macrophages. These c e l l s are responsible for cleaning up necrotic material and c e l l u l a r debris. As early as three or four days, undifferen-t i a t e d mesenchymal c e l l s and f i b r o b l a s t s can be seen migrating into t h i s area. This signals the beginning of the p r o l i f e r a t i v e phase which overlaps with the inflammatory phase. The invasion of large numbers of f i b r o b l a s t s i s 6 accompanied by an ingrowth of c a p i l l a r y buds and the beginning of collagen synthesis. Collagen synthesis accelerates and a ligament callous i s 22 formed . This phase of healing i s reported to l a s t 20 to 45 days i n tendon 22 healing The t h i r d phase of healing i s the reorganization or remodelling phase. Here the ligament callous i s reorganized into a structure resembling normal ligament i n terms of collagen amount and organization as well as c e l l popu-l a t i o n . The f i b r o b l a s t s become l e s s active metabolically and are fewer i n number. They gradually a l i g n themselves along the l o n g i t u d i n a l axis of the ligament or tendon i n between the collagen f i b e r s . The collagen f i b e r s also become aligned along the l o n g i t u d i n a l ligament axis and are larger i n 12 22 31 s i z e ' ' . The length of t h i s phase has been reported to be between 60 22 and 360 days i n length i n tendons of d i f f e r e n t species of animals F. T e n s i l e Properties. The t e n s i l e properties of tendon healing have been documented by Mason 22 and A l l e n . They found three phases of t e n s i l e strength: 1) A phase of rapid diminution of t e n s i l e strength corresponding to the phase of exudation and fibrous union and l a s t i n g approximately f i v e days. 2) A phase of increas-ing t e n s i l e strength to reach a plateau at about the sixteenth day. This corresponds to the phase of f i b r o p l a s i a or the p r o l i f e r a t i v e phase. 3) A second phase of increasing t e n s i l e strength which corresponds to the phase of maturation and remodelling. This phase begins between the nineteenth and twentyfirst days and continues u n t i l maturity. The structure responsible f o r the t e n s i l e strength of ligaments and tendon i s the collagen. I t has been reported that a collagen f i b e r 1mm. i n 12 diameter requires a load of from 10-40 kg. to disrupt i t . I t i s f e l t that the c r o s s - l i n k s between the p a r a l l e l collagen f i b e r s are the key factor i n 7 t h i s s t r e n g t h . G. The Role of Function i n Ligament Healing. The r o l e that f u n c t i o n plays i n ligament h e a l i n g remains contro-2 6 v e r s i a l . Noyes b e l i e v e s t h a t mechanical fo r c e s have a favourable i n -fluence on the h e a l i n g p r o p e r t i e s of ligament by a f f e c t i n g the alignment and o r i e n t a t i o n o f the c o l l a g e n f i b e r s . However, he does not support t h i s w i t h s c i e n t i f i c data. Moreover, he a l s o b e l i e v e s i n i m m o b i l i z i n g ligaments 15 p o s t - o p e r a t i v e l y f o r at l e a s t a s i x week p e r i o d . H i r s c h s t a t e s t h a t " p a r t i a l i m m o b i l i z a t i o n a f t e r tendon surgery i s mandatory or suture i n -22 s u f f i c i e n c y w i l l ensue". Mason and A l l e n summarized published experiment-a l data on the r o l e o f f u n c t i o n i n tendon h e a l i n g and found that tendons healed and regenerated i n t o normal appearing tendons "when subjected to 22 t e n s i o n " 8 PURPOSE OF STUDY The purpose of t h i s study was to determine the e f f e c t s of immobiliza-t i o n on ligament healing of a r a t medial c o l l a t e r a l ligament. These e f f e c t s were to be evaluated h i s t o l o g i c a l l y and biomechanically. For h i s t o l o g i c evaluation, an objective scoring system was devised to be used with the l i g h t microscope. The electron microscope was used f o r further evaluation and for v e r i f i c a t i o n of the objective scoring of the ligament healing. For bio-mechanical evaluation, the ligament was tested u n t i l f a i l u r e and the r e s u l t i n g load-deformation curves analayzed. The r a t was chosen as an experimental model because i t s knee c l o s e l y resembles the human knee anatomically. The only s i g n i f i c a n t differences are the presence of o s s i f i e d menisci i n the adult r a t and the ha b i t u a l l y flexed p o s i t i o n of the knee of the r a t . 9 MATERIALS AND METHODS A. Population and Procedure. Sixty-one mature, male Wistar r a t s were used i n t h i s study providing 122 knee preparations. The average weight of the animals at the time of operation was 405 grams. The operative procedure for a l l animals was i d e n t i c a l . The rat was anesthetized with an i n t r a p e r i t o n e a l i n j e c t i o n of Sodium Pentobarbitol at a dose of approximately 50 mg/kg body weight. The l e f t hind limb was shaved and then cleaned with an alcohol s o l u t i o n . The operative procedure was performed under clean but not s t e r i l e conditions. The rat was placed on an operating board with the knee held i n 90° of f l e x i o n and exposing the medial surface. A l o n g i t u d i n a l i n c i s i o n 1.5 to 2.0 cm i n length was made over the medial aspect of the knee j o i n t . Using a d i s s e c t i n g microscope, the medial c o l l a t e r a l ligament was exposed and then completely divided at the l e v e l of the knee j o i n t . The ligament was then repaired with a hor i z o n t a l mattress suture of 7-0 "Ticron"* on an atraumatic needle. The ligament edges were opposed but not placed under excess tension. A s i n g l e mattress suture appeared to adequately oppose the t i s s u e s without compromising the ligament i n t e g r i t y . Tissues were kept moist at a l l times with an i s o t o n i c s a l i n e s o l u t i o n . The wound was then closed i n laye r s , the skin being closed with 4-0 Dexon*. Following surgery the r a t was randomly placed into one of four groups with varying periods of immobilization: Group A (17 rats) had no immobiliza-* Trademark Davis and Geek Company 10 ti o n of the operated limb. Group B (16 rats) had two weeks immobilization of the operated limb. Group C (17 rats) had 6 weeks immobilization of the operated limb. Group D (8 rats) had 10 weeks immobilization of the operated limb. In addition there were 3 r a t s (Group Z) that were studied bio-mechanically only at the time of surgery (time zero). These served as an i n d i c a t i o n of the strength of the r e p a i r i t s e l f . In a l l cases, the r i g h t knee served as a control and had no surgery or immobilization. A l l r a t s were kept i n i n d i v i d u a l cages 18 cm x 18 cm x 25 cm and were fed on a standard d i e t of r a t chow and water ad l i b i t u m . The rats were studied at time periods of 0,2,6,10 and 20 weeks post-operative using h i s t o -l o g i c a l and biomechanical techniques. B. Immobilization Procedure. Because of the anatomy of the r a t and t h e i r constant p o s i t i o n of knee f l e x i o n , immobilization with a plaster of paris cast alone i s d i f f i c u l t . Therefore, following the operative procedure, two .035 inch diameter s t a i n l e s s s t e e l Kirschner wires were inserted i n the limb, one i n the mid femur and one i n the mid t i b i a . The Kirschner wires penetrated both sides of the leg ( F i g . 1). A plaster of paris cast was then applied incorporating the Kirschner wires for f i x a t i o n to the l e g . The knee was immobilized at an angle of approximately 90 degrees of f l e x i o n and the foot was l e f t out of the cast to allow the r a t to have r e l a t i v e l y unhindered a c t i v i t y . Ninty degrees of knee f l e x i o n was chosen as t h i s i s the physiologic r e s t i n g angle of the knee i n the 34 r a t as reported by Tipton While s t i l l wet, the p l a s t e r of paris was sprayed with t i n c t u r e of Benzoin to give the cast a b i t t e r taste, discouraging the rats from eating t h e i r casts as they are known to do. This method proved very e f f e c t i v e . Casts were changed at two weeks and at s i x weeks post-operative to ensure 11 a good f i t on the atrophied l e g minimizing any motion. Casts were a l s o r e i n -f o r ced or changed more f r e q u e n t l y i f necessary because of excess wasting of the l e g and p a r t i a l d e s t r u c t i o n of the c a s t . The average weight of the cast and K i r s c h n e r wires was 25 grams or approximately 6% of body weight. Casts were removed by soaking them o f f i n warm water with the r a t under pento-b a r b i t a l a n esthesia. F o l l o w i n g cast removal, the r a t s were e i t h e r studied or allowed normal caged a c t i v i t y u n t i l the time of study. C. H i s t o l o g i c Test Procedure. The r a t was anesthetized and placed on an o p e r a t i n g board, f o l l o w i n g cast removal i f a p p l i c a b l e . A s k i n i n c i s i o n was made on the medial aspect o f the knee, on the l e f t u t i l i z i n g the o l d i n c i s i o n . Using the d i s s e c t i n g micro-scope the medial c o l l a t e r a l ligament was exposed, m o b i l i z e d and then freed from i t s femoral and t i b i a l i n s e r t i o n s using sharp d i s s e c t i o n . The r e p a i r s i t e of the ligament was not subject to s t r e s s during the d i s s e c t i o n and a l l t i s s u e s were kept moist with an i s o t o n i c s a l i n e s o l u t i o n . The ligament was immediately placed i n Karnovsky's f i x a t i v e a t room temperature f o r 30 minutes. The procedure was repeated f o r the r i g h t knee and then the r a t was s a c r i f i c e d with an overdose of Sodium P e n t o b a r b i t o l . A f t e r t h i r t y minutes of f i x a t i o n , the ligament was trimmed i n t o smaller segments f o r study. The area bounded by the sutures were used f o r study and a corresponding area of the r i g h t medial c o l l a t e r a l ligament was l i k e w i s e trimmed. The ligaments were then f i x e d f o r a f u r t h e r two hours, washed i n sodium cocadylate b u f f e r and then placed i n t h i s b u f f e r u n t i l they were embedded i n pure epon moulds. From these epon moulds, 1.5 micron t h i n s e c t i o n s were cut and s t a i n e d w i t h T o l u i d i n e blue f o r l i g h t microscopy. A f t e r e v a l u a t i o n under the l i g h t . o microscope, the ligament was trimmed f u r t h e r and 600-900A t h i c k s e c t i o n s were cut from appropriate areas. These were s t a i n e d w i t h saturated u r a n y l acetate 12 and lead c i t r a t e for electron microscopy. D. H i s t o l o g i c Evaluation. 1. Light Microscopy In order to o b j e c t i v e l y evaluate the maturity of a healing ligament, a Maturity Index Score and a Maturity Index Scale were devised. These are based on an evaluation of the r e p a i r i n g area of the ligament using the l i g h t micro-scope and looking at four v a r i a b l e s : a) The numbers of f i b r o b l a s t s per high power f i e l d ( h . p . f . ) . Three separate representative areas on two separate sections of the area of ligament r e p a i r were looked at, at 400X magnification and the numbers of f i b r o b l a s t n u c l e i were counted. This number was averaged and a range was determined. b) Orientation of the f i b r o b l a s t s i n r e l a t i o n to the lo n g i t u d i n a l ligament a x i s . That i s whether they were p a r a l l e l to t h i s axis or whether they were angled to i t or whether there was any organization at a l l to t h e i r alignment. c) Orientation of the collagen f i b e r bundles to the l o n g i t u d i n a l ligament a x i s . Branching was defined as f i b e r s which angled from the normal axis i n t o a second plane. Transverse f i b e r s and f i b e r s seen i n cross-section were also noted. d) Amount of collagen present. This was graded on whether the collagen was t i g h t l y packed or loosely packed or mixed. The numerical assignment f o r each v a r i a b l e i s l i s t e d i n Table I with the lowest numbers i n d i c a t i n g f u l l maturity. These variables are then totaled and applied to the Maturity Index Scale (Table II) which represents phases of ligament healing following the inflammatory phase and continuing to f u l l maturity. The remodelling phase, which l a s t s the longest period of time, has been divided into three stages: 13 early, raid and l a t e . This scale then enables investigators to numerically compare ligament healing throughout i t s t o t a l process and i n fac t should apply to a l l species. 2. Electron Microscopy Factors studied with the electron microscope included the numbers and orie n t a t i o n of the f i b r o b l a s t s as well as the s i z e of the c e l l s and the maturity or l e v e l of metabolic a c t i v i t y of the f i b r o b l a s t s . The amounts of rough endoplasmic reticulum, g o l g i complexes and mitochondria were taken as an i n d i c a t i o n of t h i s a c t i v i t y with the most immature f i b r o b l a s t s having the greatest amounts of these i n t r a c e l l u l a r organelles as well as being the lar g e s t i n s i z e . The collagen f i b e r s were also looked at i n terms of t h e i r organization, s i z e and amount present. E. Biomechanical Test Procedure. The r a t was anesthetized and k i l l e d with an overdose of Sodium Pento-b a r b i t o l . Both hind limbs were then removed and dissected free of a l l s o f t t i s s u e except f o r the ligaments and capsule of the knee j o i n t . Tissues were kept moist at a l l times with an i s o t o n i c s a l i n e s o l u t i o n . The limbs were then placed i n s p e c i a l l y designed holders and pre-set i n rapid s e t t i n g epoxy with the knee at 90 degrees of f l e x i o n ( F i g . 2). This angle was chosen based on a 34 previous study by Tipton where he r a d i o l o g i c a l l y studied the physiologic angle of knee f l e x i o n i n the r a t and found i t to be 87.7 ± 4.7 degrees. This holder had a removable bar on i t to s t a b i l i z e the preparation thereby protecting the ligament from stresses u n t i l the time of t e s t . Once the epoxy had set and j u s t p r i o r to t e s t i n g , the knee was then further dissected using a dis s e c t i n g microscope. A l l capsular attachments and ligaments were removed except for the medial c o l l a t e r a l ligament. This provided a functional unit of a femur-medial c o l l a t e r a l ligament-tibia preparation. The holders were then 14 placed i n an Instron materials t e s t i n g machine ( F i g . 3), the s t a b i l i z e r bar was removed, and the ligament was tested u n t i l f a i l u r e with the force elongation curve being recorded on an o s c i l l o g r a p h . The rate of extension chosen was 0.02 mm/second. This i s a slower rate than that causing most i n -34 j u r i e s , however, i n a previous study by Tipton , he found that there was no s i g n i f i c a n t d i f f e r e n c e i n t h i s rate versus a f a s t e r physiologic rate of loading when applied to the medial c o l l a t e r a l ligament of the r a t . 34 A l l ligaments were studied within three hours of s a c r i f i c e . Tipton studied the influence of delay i n t e s t i n g on the maximum load at f a i l u r e and he found that there was no s i g n i f i c a n t difference between immediate t e s t i n g and a delay of eight hours. Longer than eight hours seemed to produce a difference i n values. F. Biomechanical Evaluation. The o s c i l l o g r a p h recordings or force elongation curves were analyzed for the following v a r i a b l e s : 1. Maximum load i n kilogram force at the time of f a i l u r e . 2. Separation Force Ratio (S.F.R.). This i s the r a t i o of the maximum load at f a i l u r e i n kilograms to the body weight of the animal i n kilograms. 34 Tipton has shown that the maximum load at f a i l u r e of the r a t medial c o l l a t e r a l ligament, varies with the body weight of the rat and that by d i v i d i n g the force by the body weight, a constant S.F.R. i s obtained. 20 This has also been found to be a constant r a t i o i n other species 3. Elongation of the ligament i n millimetres p r i o r to rupture. This was determined by c a l c u l a t i n g the time u n t i l f a i l u r e o f f of the graph and then multip l y i n g by the rate of extension. As a l l of our r a t s i n t h e i r respective groups were of s i m i l a r weights and s i z e and we did not have a way of measuring the t o t a l length of the ligament accurately to very small 15 differences, for comparison purposes, the elongation was considered to be an i n d i c a t i o n of the s t r a i n of the ligament (change i n length divided by the o r i g i n a l length). The o r i g i n a l r e s t i n g length measured by our methods was 9.7 to 9.9 milimeters i n a l l of the r a t s studied. The amount of elongation of the bone and holders was taken to be i n s i g n i f i c a n t . 4. Compliance i s the r a t i o of the elongation i n milimeters to the load at f a i l u r e or separation force i n kilograms. This i s e s s e n t i a l l y the inverse of Youngs Module of E l a s t i c i t y . T e n s i l e strength or s t r e s s (force per u n i t area) could not be c a l -culated as we did not have an accurate method of measuring the cross-sectional area of a ligament the s i z e of a rat medial c o l l a t e r a l ligament. S t a t i s t i c a l analyses of the biomechanical data were performed with a computor using, where appropriate, students t - t e s t and paired t - t e s t , analysis of variance and multiple range t e s t . Values were defined to be s t a t i s t i c a l l y s i g n i f i c a n t at P values l e s s than 0.05. In addition to t h i s evaluation of the force elongation curve, the s i t e of ligament f a i l u r e was also noted and the range of motion of the knee was measured at the time of t e s t i n g . 16 RESULTS A. General. A l l wounds healed primarily with no evidence of i n f e c t i o n . Any r a t s developing complications which consisted of pin t r a c t i n f e c t i o n s , were d i s -carded from the study. A t o t a l of 61 r a t s were studied, 39 biomechanically and 22 h i s t o l o g i c a l l y . A l l ra t s continued to gain weight although while a r a t had one limb immobilized i t s rate of weight gain was decreased. A l l limbs immobilized, demonstrated gross muscle atrophy which appeared to increase as the length of time immobilized increased although t h i s was not quantitated. A normal r a t knee has approximately 115 degrees of motion subscribing an arc of between 45 and 160 degrees. When compared to the control knee, a l l immobilized knees showed a decrease i n t h i s range of motion (See Table I I I ) . In Group B (2 weeks immobilization), a decrease to 85 to 95 degrees of motion was seen. This had been regained from the s i x t h week test onwards. In Group C (6 weeks immobilization), a decrease to 20 to 40 degrees of motion was seen. At twenty weeks, t h i s group s t i l l lacked 20 to 30 degrees of motion. In Group D (10 weeks immobilization) a decrease to only 20 to 30 degrees of motion was seen. At twenty weeks post-operative, t h i s group s t i l l lacked 40 to 60 degrees o f motion. Group A had a normal range of motion a t a l l t e s t periods. B. Histology - Light Microscopy. 1. Controls (Figs. 4 and 5). Control ligaments displayed a r e g u l a r i t y of collagen f i b e r bundles within the ligament which were l o n g i t u d i n a l l y oriented and densely packed. 17 Fibr o b l a s t s were small and very elongated i n appearance with small darkly s t a i n i n g n u c l e i . The number of f i b r o b l a s t s per high power f i e l d was 3-6. The Maturity Index Score was four. 2. Two Weeks. A large amount of callous was seen which appeared greater i n Group A. Microscopically the s i t e of laceration was e a s i l y i d e n t i f i e d i n both groups although grossly they appeared healed. Occasional inflammatory c e l l s were seen i n both groups. In Group A (no immobilization), (Figs. 6 and 7), there was a marked h y p e r c e l l u l a r i t y of over 100 f i b r o b l a s t s per high power f i e l d . These f i b r o b l a s t s were larger i n siz e and had large pale s t a i n i n g n u c l e i . The f i b r o b l a s t s displayed some lon g i t u d i n a l o r i e n t a t i o n along the ligament a x i s . There was early collagen f i b e r formation i n a l o n g i t u d i n a l d i r e c t i o n . In Group B (2 weeks immobilization), ( F i g s . 8 and 9 ) , there was also an increase i n the number of f i b r o b l a s t s to 30-45 per high power f i e l d . These f i b r o b l a s t s were also larger i n s i z e and had large pale s t a i n i n g n u c l e i a l -though t h i s difference was not as marked as i n Group A. Early collagen f i b e r formation was present but t h i s displayed no organization. The Maturity Index Score for Group A was 13 and for Group B i t was 14. 3. Six Weeks. A l l ligaments appeared grossly healed. There were no inflammatory c e l l s seen. A l l ligaments displayed some call o u s although t h i s was l e s s than at two weeks. Group A ( F i g s . 10 and 11), showed a decrease i n the numbers of f i b r o -b l asts to 32-43 per high power f i e l d . The f i b r o b l a s t s were smaller i n s i z e and had smaller darkly s t a i n i n g n u c l e i . These f i b r o b l a s t s were arranged l o n g i t u d i n a l l y , i n alignment with the ligament a x i s . The collagen f i b e r s were l o n g i t u d i n a l l y oriented completely bridging the lacerated area and were as 18 densely packed as i n c o n t r o l s . The M a t u r i t y Index Score was E i g h t . Group B ( F i g s . 12 and 13), showed a smaller decrease i n the numbers o f f i b r o b l a s t s to 18-31 per high power f i e l d . These f i b r o b l a s t s were a l s o s m a l l e r w i t h smaller darker s t a i n i n g n u c l e i . They were now mainly l o n g i -t u d i n a l i n o r i e n t a t i o n with the ligament a x i s . Collagen f i b e r s were now present i n increased amounts and these appeared to be more l o n g i t u d i n a l and be t t e r organized than at two weeks. However they s t i l l d i s played branching and were not as densely packed or as w e l l organized as i n Group A. The Ma t u r i t y Index Score was eleven. In Group C ( F i g s . 14 and 15), there was an increase i n the f i b r o b l a s t s to 52-64 per high power f i e l d . The f i b r o b l a s t s were l a r g e r than i n Groups A and B w i t h l a r g e d a r k l y s t a i n i n g n u c l e i and w i t h l a r g e numbers of i n t r a -c e l l u l a r i n c l u s i o n s . Their arrangement tended to be i r r e g u l a r and not i n alignment w i t h the ligament a x i s . The c o l l a g e n f i b e r bundles tended to be i r r e g u l a r , w i t h branching and f i b e r s at r i g h t angles to each other i n d i c a t i n g l i t t l e o r g a n i z a t i o n . They were a l s o l e s s densely packed. The M a t u r i t y Index Score was fourteen. 4. Ten Weeks. A l l ligaments appeared g r o s s l y healed and c a l l o u s formation was minimal. In Group A ( F i g s . 16 and 17), there was a f u r t h e r decrease i n the numbers of f i b r o b l a s t s to 18-28 per high power f i e l d . These f i b r o b l a s t s were small with small darkly s t a i n i n g n u c l e i . The f i b r o b l a s t s were a l l o r i e n t e d l o n g i t u d i n a l l y . Collagen f i b e r bundles were l o n g i t u d i n a l l y o r i e n t e d , densely packed and w e l l organized appearing the same as c o n t r o l s . The M a t u r i t y Index Score was f i v e . Group B ( F i g s . 18 and 19), showed a decrease i n the numbers of f i b r o -b l a s t s to 15-25 per high power f i e l d . The f i b r o b l a s t s were small with small 19 darkly s t a i n i n g n u c l e i . Occasional more immature looking c e l l s were seen. The collagen f i b e r bundles were l o n g i t u d i n a l l y oriented, regular and densely packed. The Maturity Index Score was 5. In Group C ( F i g s . 20 and 21), the numbers of f i b r o b l a s t s had decreased to 16-28 per high power f i e l d and the f i b r o b l a s t s were more l o n g i t u d i n a l l y oriented although some disarray was s t i l l present. The f i b r o b l a s t s were also smaller i n siz e and had smaller n u c l e i . The collagen f i b e r bundles were more l o n g i t u d i n a l l y oriented although there was s t i l l a large amount of branching from the ligament a x i s . The collagen f i b e r s were not as densely packed as i n Groups A and B. The Maturity Index Score was nine. In Group D (Figs. 22 and 23), the numbers of f i b r o b l a s t s present was 45-52 per high power f i e l d . These f i b r o b l a s t s were larger than i n the other groups and displayed an i r r e g u l a r organization which was i n places at r i g h t angles to the ligament a x i s . The collagen f i b e r bundles were also present i n a disorganized fashion with branching and f i b e r s at r i g h t angles to the l i g a -ment a x i s . They were also not as densely packed as i n the other groups. The Maturity Index Score was twelve. 5. Twenty Weeks. A l l groups were grossly healed with only minimal callous present i n Group D. In Group A ( F i g . 24), the numbers of f i b r o b l a s t s had decreased to 4-8 per high power f i e l d . The f i b r o b l a s t s were the same as controls i n terms of c e l l u l a r and nuclear s i z e as well as l o n g i t u d i n a l organization. The collagen f i b e r bundles were also the same as controls with' a densely packed, regular, l o n g i t u d i n a l arrangement. The Maturity Index Score was four. In Group B (F i g s . 25 and 26), the numbers of f i b r o b l a s t s had decreased to 10-15 per high power f i e l d . The f i b r o b l a s t s appeared the same as controls 20 i n terms of c e l l u l a r and nuclear s i z e with a l o n g i t u d i n a l o r i e n t a t i o n . The collagen f i b e r bundles were l o n g i t u d i n a l l y oriented and densely packed, simi-l a r to co n t r o l s . The Maturity Index Score was f i v e . In Group C (Figs. 27 and 28), the number of f i b r o b l a s t s was s t i l l 14-22 per high power f i e l d but they were now l o n g i t u d i n a l l y oriented with only a small number being angled to the ligament a x i s . The f i b r o b l a s t s had de-creased i n s i z e . The collagen f i b e r s were densely packed and regular with mainly a l o n g i t u d i n a l o r i e n t a t i o n but with some branching. The Maturity Index Score was seven. In Group D (F i g s . 24 and 30), the numbers of f i b r o b l a s t s had decreased to 20-30 per high power f i e l d and these f i b r o b l a s t s had decreased i n s i z e to that of the controls. The f i b r o b l a s t s were mainly l o n g i t u d i n a l i n o r i e n t a t i o n with some angled to the ligament a x i s . The collagen f i b e r bundles were densely packed and were arranged i n a l o n g i t u d i n a l fashion with some branching present. The Maturity Index Score was seven. The r e s u l t s of Maturity Index Score applied to the Maturing Index Scale, are l i s t e d i n Table IV and depicted g r a p h i c a l l y i n Figure 31. This i l l u s t r a t e s the rapid maturation of Group A (no immobilization) which achieved f u l l maturity at twenty weeks post-operative. The other groups a l l displayed a retarded rate of maturation while the limb was immobilized. This increased once mobilization commenced but none of these groups had achieved f u l l maturity at twenty weeks post-operative and appeared to be plateauing i n the l a t e remodelling phase. C. Histology - Electron Microscopy. 1. Control ( F i g s . 32,33,34). The f i b r o b l a s t s displayed evidence of maturity and minimal metabolic a c t i v i t y with only small numbers of mitochondria, rough endoplasmic reticulum 21 ., o and g o l g i complexes. The c o l l a g e n f i b e r s demonstrated the r e g u l a r 640A band-i n g c h a r a c t e r i s t i c o f c o l l a g e n and were densely packed and o r i e n t e d i n a l o n g i t u d i n a l f ashion along with the f i b r o b l a s t s . 2. Two Weeks. In Group A ( F i g s . 35 and 36 ) , the f i b r o b l a s t s were markedly increased i n numbers. The f i b r o b l a s t s were l a r g e and demonstrated a high l e v e l o f c e l l u l a r metabolic a c t i v i t y . There were l a r g e numbers of mitochondria, abund-ant rough endoplasmic r e t i c u l u m , l a r g e g o l g i complexes and a la r g e number o f o v e s i c l e s . The c o l l a g e n f i b e r s demonstrated the r e g u l a r 640A p e r i o d i c i t y o f banding and di s p l a y e d some l o n g i t u d i n a l o r g a n i z a t i o n . These f i b e r s were small i n diameter and l o o s e l y packed. A s m a l l amount o f e x t r a c e l l u l a r d e b r i s was present. The f i b r o b l a s t s i n Group B ( F i g s . 37 and 38 ) , were not as l a r g e , nor were they as a c t i v e m e t a b o l i c a l l y . They had increased numbers of mitochondria and rough endoplasmic r e t i c u l u m although not as much as i n Group A. There was no r e g u l a r i t y to the o r i e n t a t i o n o f the f i b r o b l a s t . The c o l l a g e n f i b e r s were smaller i n diameter, very l o o s e l y arrayed and d i s p l a y e d no o r g a n i z a t i o n w i t h f i b e r s seen running i n a l l three planes. The maturity and metabolic a c t i v i t y were not as w e l l developed as i n Group A, and there was a l a r g e amount of e x t r a c e l l u l a r d e b r i s s t i l l present. 3. S i x Weeks. In Group A ( F i g s . 39 and 40), the f i b r o b l a s t s had decreased i n s i z e , numbers," and l e v e l of metabolic a c t i v i t y . Increased numbers of mitochondria and rough endoplasmic r e t i c u l u m were s t i l l present, but t h i s was decreased from at two weeks. The c o l l a g e n f i b e r s were l a r g e r i n s i z e , densely packed and organized mainly l o n g i t u d i n a l l y . Group B ( F i g s . 41 and 42) f i b r o b l a s t s were s i m i l a r i n s i z e , numbers and 22 l e v e l of metabolic a c t i v i t y to Group A and had attained some l o n g i t u d i n a l o organization. The collagen f i b e r s displayed the c h a r a c t e r i s t i c 640A banding and had increased i n s i z e although a large degree of v a r i a b i l i t y was present. The o r i e n t a t i o n of the collagen was l o n g i t u d i n a l with branching and transverse f i b e r s but displaying some organization. E x t r a c e l l u l a r debris was s t i l l present but only i n small amounts. Group C (F i g s . 43 and 44) f i b r o b l a s t s were larger i n s i z e than Groups A or B and were very active metabolically with abundant mitochondria, rough endoplasmic reticulum and g o l g i complexes. The collagen f i b e r s were smaller in s i z e , loosely packed i n some areas and were poorly organized with f i b e r s seen running i n three d i f f e r e n t planes. There were large amounts of extra-c e l l u l a r debris. 4. Ten Weeks. The f i b r o b l a s t s of Group A (F i g s . 45 and 46), showed a further decrease i n numbers, siz e and l e v e l of metabolic a c t i v i t y . The amount of mitochondria, rough endoplasmic reticulum and g o l g i complexes was s t i l l greater than i n controls. The collagen f i b e r s were larger i n diameter, densely packed and mainly l o n g i t u d i n a l i n organization. Group B (Figs. 47 and 48) f i b r o b l a s t s were larger than i n Group A but had also decreased both i n s i z e i n the l e v e l of metabolic a c t i v i t y . The collagen f i b e r s were regular i n s i z e , were densely packed and were lo n g i -t u d i n a l l y oriented but with some branching. Group C (F i g s . 49 and 50) f i b r o b l a s t s were larger than i n Groups A or B and displayed greater metabolic a c t i v i t y than these groups with increased amounts of rough endoplasmic reticulum, mitochondria and g o l g i complexes. The collagen f i b e r s were more densely packed than at s i x weeks and were better organized. There s t i l l were f i b e r s running i n a l l three planes i n close prox-23 i m i t y and e x t r a c e l l u l a r debris was s t i l l present. Group D ( F i g s . 51 and 52) f i b r o b l a s t s were not as l a r g e as i n Group C nor were they as m e t a b o l i c a l l y a c t i v e with fewer numbers of mitochondria, rough endoplasmic r e t i c u l u m , and g o l g i complexes. The c e l l s d i splayed no d e f i n i t e l o n g i t u d i n a l o r g a n i z a t i o n . The c o l l a g e n f i b e r s were not as densely packed as i n the other groups and they were smaller i n diameter. The c o l l a g e n displayed l i t t l e o r g a n i z a t i o n with f i b e r s running i n a l l three planes. E x t r a -c e l l u l a r d e b r i s was present i n increased amounts. 5. Twenty Weeks. Group A ( F i g s . 53 and 54) f i b r o b l a s t s were i d e n t i c a l to c o n t r o l s i n s i z e and l e v e l o f metabolic a c t i v i t y . The co l l a g e n f i b e r s were l o n g i t u d i n a l l y o r i e n t e d and as densely packed as i n c o n t r o l s . The c o l l a g e n f i b e r diameter was s i m i l a r to c o n t r o l s although some smaller f i b e r s were s t i l l seen. Group B ( F i g s . 55 and 56) f i b r o b l a s t s were a l s o s i m i l a r to c o n t r o l s i n both s i z e and metabolic a c t i v i t y with decreased amounts of rough endoplasmic r e t i c u l u m and g o l g i complexes from at ten weeks. The c o l l a g e n f i b e r s were l o n g i t u d i n a l l y o r i e n t e d and densely packed although not as dense as i n con-t r o l s . Occasional branching was seen and the c o l l a g e n f i b e r diameter was l e s s than t h a t o f Group A or c o n t r o l s . Group C ( F i g s . 57 and 58) f i b r o b l a s t s were s t i l l more numerous, l a r g e r and more m e t a b o l i c a l l y a c t i v e than c o n t r o l s although the amount of rough endo-plasmic r e t i c u l u m and g o l g i complexes had decreased from at ten weeks. The o r g a n i z a t i o n of the c o l l a g e n f i b e r s had improved although branching was s t i l l q u i t e extensive and o c c a s i o n a l groups of f i b e r s were seen i n a cross s e c t i o n beside l o n g i t u d i n a l ones. The col l a g e n f i b e r s were more densely packed than a t ten weeks but s t i l l l e s s than c o n t r o l s and the diameter o f the f i b e r s was a l s o s t i l l s maller than i n c o n t r o l s . 24 Group D ( F i g s . 59 and 60) f i b r o b l a s t s were more numerous than i n con-t r o l s . Their l e v e l of metabolic a c t i v i t y was l e s s than that of Group C but not the same as controls. The numbers of mitochondria and rough endoplasmic reticulum had decreased. The collagen f i b e r s displayed minimal organization with an o v e r a l l l o n g i t u d i n a l array but with a large degree of branching f i b e r s at r i g h t angles and f i b e r s i n cross-section. They were also not as densely packed or as large i n diameter as i n Group A or i n c o n t r o l s . D. Biomechanical Testing. Just p r i o r to t e s t i n g , the ligaments were inspected grossly with the d i s s e c t i n g microscope (40X) and the findings agreed with those reported i n the previous section on Histology i n terms o f appearance of healing and c a l l o u s formation. 1. Maximum Load at F a i l u r e . See Table V where the r e s u l t s are l i s t e d . This w i l l not be discussed as r e s u l t s c o r r e l a t e with the Separation Force Ratio r e s u l t s which w i l l be discussed i n d e t a i l . 2. Separation Force Ratio (S.F.R.). 34 As shown by Tipton , the S.F.R., which i s a r a t i o of the separation force or maximum load at f a i l u r e of the ligament to the animal body weight, i s an accurate method of comparing r e s u l t s within an animal experimental model. Table V l i s t s the S.F.R. of a l l of the ligaments tested and these are displayed g r a p h i c a l l y i n Figure 61. A sharp r i s e i s seen i n the f i r s t two weeks i n Groups A and B with Group A showing the greatest increase. Applying the t - t e s t to these r e s u l t s , p = 0.056, a value not quite s i g n i f i c a n t , but one that probably would have been i f our population i n these groups was l a r g e r . At s i x weeks, Groups A and B were s i g n i f i c a n t l y d i f f e r e n t from Group C (P < 0.0005). At ten weeks, 25 Groups A and B are showing a f a l l o f f i n t h e i r rate of increase while Group C, now out of i t s cast f o r four weeks, showed a sharp increase. At twenty weeks, Groups A, B and C are l e v e l l i n g o f f while Group D has shown more of an i n -crease now that they were out of t h e i r casts. At t h i s time there i s a s i g -n i f i c a n t difference between these groups (p = 0.0005). The S.F.R. of the repaired ligament was compared to the S.F.R. of the control ligament as shown i n Table VI and i l l u s t r a t e d g r a p h i c a l l y i n Figures 62 and 63. These r e s u l t s p a r a l l e l those discussed above. At two weeks post-operative, Group A was 37$ stronger than Group B and had achieved a strength which was 46% of control strength while Group B was only 29% of con-t r o l strength. Applying the t - t e s t p = 0.055, a value not quite s i g n i f i c a n t but one that probably would have been i f our populations i n these groups were l a r g e r . At s i x weeks post-operative, Group A was 65% of controls, Group B was 56% of controls and Group C was 39% of controls. These differences were s i g -n i f i c a n t (p = 0.0004). Group A was 96% stronger than Group C at t h i s time. At ten weeks post-operative, Group A was 87% of co n t r o l s , Group B was 64% of controls, Group C was 51% of controls and Group D was 37% of co n t r o l s . The population of these groups tested at t h i s time period, was too small to apply any meaningful s t a t i s t i c a l a n a l y s i s . At twenty weeks post-operative, Group A was now 83% of controls, Group B was 71% of controls, Group C was 66% of con-t r o l s and Group D was 48% of controls. These differences were s i g n i f i c a n t (p = 0.0005). At t h i s time, Group A was 11% stronger than Group B, 32% stronger than Group C and 71% stronger than Group D. A l l groups displayed a s i g -n i f i c a n t change over the time studied between t h e i r values at two weeks, s i x weeks and twenty weeks when studied within the i n d i v i d u a l groups (p < 0.01). This a n a l y s i s showed that Group A (no immobilization) had a s i g -n i f i c a n t l y higher S.F.R. than the other groups as compared to controls and 26 regained i t s functional unit strength at a fa s t e r rate than the other groups. Group B (two weeks immobilization) also displayed a s i g n i f i c a n t l y higher S.F.R. than Group C at s i x weeks but t h i s difference diminished over the next fourteen weeks. I t was also seen that once the casts were removed and mobili-zation commenced, the rate of gain of strength increased. 3. Elongation. See Table V where the r e s u l t s are l i s t e d and Figure 64 where they are displayed g r a p h i c a l l y . The r e s u l t s do not indicate any s p e c i f i c pattern nor are the differences within groups s i g n i f i c a n t . The elongation of the repaired ligament was compared to the elongation of the control ligament as l i s t e d i n Table VI and i l l u s t r a t e d i n Figure 65. This shows that a l l groups follow a si m i l a r pattern with the maximum elongation occuring at 6 weeks (98% - 117%) then s t e a d i l y decreasing to 20 weeks where values were 61% - 72% of con t r o l s . When s t a t i s t i c a l analysis was applied, the differences between the groups were not s i g n i f i c a n t nor were the differences within the i n d i v i d u a l groups at the various time i n t e r v a l s s i g n i f i c a n t (p = 0.087 - 0.34). 4. Compliance. Compliance i s the r a t i o of the amount of elongation of the ligament to the unit load at f a i l u r e . The r e s u l t s are l i s t e d i n Table V and i l l u s t r a t e d g r a p h i c a l l y i n Figure 66. These r e s u l t s show a trend towards an improvement i n the compliance i n a l l groups over time without a s i g n i f i c a n t difference between the groups at the various time periods. The compliance of the re-paired ligament was compared with the control ligament as l i s t e d i n Table VI and i l l u s t r a t e d i n Figure 67. This shows an improvement i n compliance over time with no s t a t i s t i c a l differences i n the groups at the various time periods. When looking at i n d i v i d u a l groups over time, both Groups A and B showed a s i g n i f i c a n t improvement over time (p < 0.01). However the d i f f e r -27 ences within Groups C and D were not s i g n i f i c a n t . At 20 weeks, a l l groups had attained a compliance which was not s t a t i s t i c a l l y d i f f e r e n t from controls whereas when compared to controls a l l groups had previously been s t a t i s t i c a l l y d i f f e r e n t (p < 0.05). 5. S i t e of Ligament F a i l u r e . This data i s l i s t e d i n Tables VII and VIII and shows that from s i x weeks onwards, a l l f a i l u r e s occurred at a s i t e other than at the repair s i t e i n d i c a t i n g that by t h i s time the strength of the r e p a i r was greater than that of the ligament i t s e l f or of the ligament-bone i n s e r t i o n . At two weeks, one of the specimens from Group A f a i l e d at the t i b i a l i n s e r t i o n , i n d i c a t i n g that i t had already achieved a strength of repair greater than the ligament-bone i n s e r t i o n . As can be seen, 83% of the f a i l u r e s occurred at the t i b i a l i n -s e r t i o n . This concurs with the r e s u l t s of Tipton and Zuckerman and 26 supported by Noyes . Noyes showed that the t i b i a l i n s e r t i o n of the medial c o l l a t e r a l ligament was d i r e c t l y into bone and periostium and that with im-m o b i l i z a t i o n o f 8 weeks, t h i s area underwent subperiosteal resorption a c t u a l l y d i s r u p t i n g the attachment of the ligament i n places, leaving i t attached only to periostium. This also explains why t h i s appears to be the weakest point i n 20 the normal medial c o l l a t e r a l ligament. Laros and associates have shown that the t i b i a l i n s e r t i o n of the medial c o l l a t e r a l ligament i s subject to resorptive changes even i n non-immobilized limbs where the animal has been caged and t h i s l e d to an area of weakness definable h i s t o l o g i c a l l y . 28 DISCUSSION A. The Experimental Model. Ligament and tendon healing has been studied i n various animal experi-. , .7,11,15,17,18,19,21,22,27,28,29 . mental models including the r a t ' ' ' ' ' ' ' ' ' ' and the manner of healing i s i d e n t i c a l i n a l l of these models, as has been outlined. These studies have been h i s t o l o g i c , biochemical, and or, biomechanical but up u n t i l now, an objective method of h i s t o l o g i c a l l y assessing and comparing l i g a -ment healing has not been reported. The e f f e c t s of immobilization on normal ligaments have also been - A- A • • • i A •, 4,10,20,23-26,34-36,38 „ . studied i n various animal models . However the e f f e c t s of varying periods of immobilization on ligament healing have not been pre-v i o u s l y studied. The immobilization method u t i l i z e d i n t h i s study combined * u A A ' • 4 - u *. A- 7,10,18,20,26-29,35 . . . methods used i n other studies and proved to be very s a t i s f a c t o r y with minimal complications. Using the r a t as the model had advantages which included 1) the r a t s used were a pure inbred s t r a i n of s i m i l a r s i z e and maturity which decreases the variables i n the experimental model and provides far greater reproduc-i b i l i t y of the experiments, 2) the rats were easy to work with and perform the experiments on as well as being an inexpensive animal to maintain, 3) the anatomy of the rat knee i s s i m i l a r to other animals and humans with the only s i g n i f i c a n t differences being the presence of o s s i f i e d menisci i n the adult rat and the h a b i t u a l l y flexed p o s i t i o n of the rat knee. B. Histology. The Maturity Index Score and Maturity Index Scale form a simple and 29 e a s i l y applied method of o b j e c t i v e l y assessing the stage of maturity of a r e -p a i r i n g ligament allowing numerical comparison betwen various groups. I t takes into account the factors which appear to be the most important i n assessing the healing process of a ligament. That i s , the numbers and orien-t a t i o n of the f i b r o b l a s t s and the amount and or i e n t a t i o n of the collagen f i b e r bundles. This data has shown that the maturation of the ligament progressed more r a p i d l y i n the group with no immobilization, and at twenty weeks i t had attained the maturity of control ligaments. It also shows that the maturation process i s slow while the limb i s immobilized and then progresses at a more rap i d rate once the cast i s removed and the l e g mobilized. A l l immobilized groups displayed t h i s trend and none of the immobilized groups had f u l l y matured at twenty weeks post-operative. This scoring system has been supported by the electron microscopic work which has demonstrated the metabolic a c t i v i t y of the f i b r o b l a s t s at the various stages of healing showing that the metabolic a c t i v i t y decreasd with increasing maturity. Also shown was that the greatest l e v e l of metabolic a c t i v i t y was present at cast removal and with increasing periods of mobiliza-t i o n , t h i s i n i t i a l l e v e l of metabolic a c t i v i t y decreased. The f i b r o b l a s t s that displayed the greatest l e v e l of metabolic a c t i v i t y were those seen at two weeks i n the group which had no immobilization. Also demonstrated, was the maturation of the collagen f i b e r s which progressed more r a p i d l y i n the group with no immobilization and i n the other groups once mobilization commenced. At two weeks, organized collagen formation was already seen i n Group A. The collagen maturation progressed with increased deposition of collagen f i b e r s which gradually increased i n s i z e and lo n g i t u d i n a l p a r a l l e l organization with mobilization. With immobilization, the collagen appeared to be l a i d down i n a randomized fashion and with mobilization, t h i s collagen appeared to become or-30 ganized and mature. The other i n t e r e s t i n g factor, was the amount of e x t r a c e l l u l a r debris present. In Group A, t h i s was minimal at two weeks and nonexistant a f t e r t h i s . In the other groups e x t r a c e l l u l a r debris was present f o r quite a long period following t h i s . In Group D, there was s t i l l debris present at 10 weeks post-operative. It appeared that t h i s debris was r a p i d l y cleared once mobilization commenced. Therefore, with the a p p l i c a t i o n of physiologic stresses and mobiliza-t i o n of the extremity, the healing ligament h i s t o l o g i c a l l y matured more rapid-l y with: 1) e a r l i e r maturation of f i b r o b l a s t s which had been more ac t i v e metabolically, 2) e a r l i e r alignment of the f i b r o b l a s t s along the lo n g i t u d i n a l ligament a x i s , 3) e a r l i e r alignment of the collagen f i b e r s along the longitud-i n a l ligament axis 4) increased deposition of larger more mature collagen 5) increased c l e a r i n g of e x t r a c e l l u l a r debris. These factors were shown to be i n progress at as early as two weeks post-operative i n the group i n which mobilization had begun immediately. C. Biomechanical Testing. The r e s u l t s of the biomechanical t e s t i n g correlated with those derived h i s t o l o g i c a l l y . I t showed that there was a more rapid and s i g n i f i c a n t l y greater increase i n the strength of the ligament-bone unit with no immobiliza-t i o n . At two weeks post-operative, one of the ligaments from Group A, had already attained a strength of re p a i r which was greater than the strength of the t i b i a l i n s e r t i o n , i n d i c a t i n g a more advanced state of maturation. A f t e r s i x weeks post-operative, our r e s u l t s p a r a l l e l e d those i n the l i t e r a t u r e which shows that with increased immobilization of a limb, the 26 27 strength of the ligament and of the ligament-bone unit decreases ' . With increased immobilization, there i s also a longer period before the ligament 31 26 strength returns to normal . This data also indicates that once the heal-ing ligament has achieved a certain level of maturation, the weakest point in the medial collateral ligament is its tibial insertion and not the repair site. Since a l l groups tested at this time were at different stages of matur-ation (Maturity Index Scale: Group A-3, Group B-4, Group C-5), this supports the concept that mobilization should begin early as there was greater strength, even in the most immature, in the repair site than in the tibial insertion. The compliance of the healing ligament was significantly increased at the time of cast removal and with mobilization, this rapidly decreased to that of the control groups. This indicates that with mobilization, the ligament experienced a more rapid maturation and stiffening of the repair such that the application of a load led to less elongation or stretch of the ligament. We had no way of accurately judging the stability of the knee joints of the rats to varus or valgus stresses, except for gross palpation of the knee joint when subjected to these stresses. With this gross method of testing, no instability was detected. This is an important factor to consider when eval-uating functional properties of a healing ligament and should probably be included in future studies of this nature. It is unlikely, in our model, that any significant laxity of the healing ligament occurred as the rats did not take part in any stressfull activities and tended to splint the operated limb until they were comfortable. They were seen in a l l cases, to be easily moving about their cages with full weight bearing in no apparent discomfort one to three days post-operative. Knee motion was present in the group with no immo-bilization from the first post-operative day onwards. Therefore, we feel that only physiologic stresses were applied to this healing ligament. The leg wasting and decreased range of motion seen in a l l of our rats 32 10 26 ^6 that were immobilized p a r a l l e l s that reported in other seri e s ' ' . I t shows the dramatic decrease i n the range of motion of the knee that occurs a f t e r prolonged immobilization and the length of time i t takes to regain t h i s motion. At twenty weeks post-operative neither Group C (6 weeks immobiliza-tion) or Group D (10 weeks immobilization) had regained t h e i r f u l l range of motion. D. C l i n i c a l Relevance. 30 George Perkins stated: " I f orthodox views are correct, they w i l l p r e v a i l against any opposition, i f they are f a l s e , the sooner they are d i s -carded the better. The proposition I am putting forward, i s that movement i s often better treatment than r e s t " . He f e l t that inflammation should be treated by r e s t as long as one interpreted inflammation c o r r e c t l y as being a time when the body reacts to a harmful i n s u l t . Once a ligament has been re-paired t h i s i n s u l t then ceases and so does inflammation although the inflamma-tory response slowly subsides. Following t h i s , i s the repair stage where the debris i s removed and the repair t i s s u e l a i d down. According to Mason and 22 30 A l l e n , the inflammatory phase ends on the t h i r d to f i f t h day. Perkins then f e e l s that the repair phase should "be treated by a c t i v i t y , namely the resumption of function and movement". 9 Ernst Dehne and Richard Torp f e e l that j o i n t i n j u r i e s including ligamentous i n j u r y , should be treated by immediate mobilization. This i s based upon the "Spinal adaption concept", where they f e e l that repairs are "an adaptive process s e n s i t i v e to adverse external s t i m u l i and responsive to r e -gulated feed-back exposure". 32 33 S a l t e r ' has shown, i n h i s experiments on continuous passive motion used following i n t r a - a r t i c u l a r fractures i n rabbit knees which were s u r g i c a l l y repaired, that passive motion begun immediately, was b e n e f i c i a l to 33 the h e a l i n g process of the a r t i c u l a r c a r t i l a g e . He has shown that continuous passive motion i s w e l l t o l e r a t e d i n the r a b b i t and when a p p l i e d to i n t r a a r t i c u l a r f r a c t u r e of the knee j o i n t , there was: " 1) No adhesion formation 2) s i g n i f i c a n t l y f a s t e r r e c o n s t i t u t i o n of the subchondral bone p l a t e 3) s i g n i f i c a n t l y more frequent h e a l i n g of the a r t i c u l a r c a r t i l a g e defects 4) s i g n i f i c a n t l y l e s s development of degenerative a r t h r i t i s post-op." 14 Haggmark and E r i k s o n presented a prospective randomized t r i a l o f s i x t e e n p a t i e n t s where a standard c y l i n d e r cast was compared with a mobile c a s t brace with f o r t y degrees of motion, both a p p l i e d seven days post opera-t i v e f o l l o w i n g a n t e r i o r c r u c i a t e ligament r e c o n s t r u c t i o n . They evaluated muscle atrophy u s i n g muscle b i o p s i e s and biochemical and h i s t o l o g i c methods. Results were a l s o assessed c l i n i c a l l y up to twelve months pos t - o p e r a t i v e . They found no d i f f e r e n c e i n the f u n c t i o n a l and c l i n i c a l end r e s u l t s of the two groups while the group with the standard cast showed atrophy of type I (slow t w i t c h ) f i b e r s , not seen i n the cast-brace group. They a l s o noted a more r a p i d r e t u r n of a normal range of motion ( e i g h t weeks versus s i x t e e n weeks) and a more r a p i d r e t u r n t o s p o r t s i n the cast-brace group. They f e e l that the cast-brace protected the reconstructed ligament from valgus, varus and r o t a -t i o n a l s t r e s s e s while a l l o w i n g a l i m i t e d range of motion. This a l l o w s physio-l o g i c s t r e s s e s to be a p p l i e d to the h e a l i n g ligament and provides the i n t r a - a r t i c u l a r b e n e f i t s o f j o i n t motion. I t a l s o a l l o w s f o r increased c i r -c u l a t i o n to the extremity and minimizes muscle wasting. Our r e s u l t s are i n agreement with the views j u s t presented. We f e e l 34 that early mobilization of the j o i n t and extremity with the a p p l i c a t i o n of physiologic stresses to the healing ligament i s b e n e f i c i a l to the healing process and maturation and to the function of the extremity as a whole. The 14 method of Haggmark and Erikson i s probably the method of choice for c l i n i -c a l use as i t provides some protection from abnormal stresses but allows for j o i n t motion and the a p p l i c a t i o n of physiologic stresses. I t also i s applied early as soon as swelling and pain w i l l allow. The use of no cast immobilization following knee ligament surgery i s an i n t e r e s t i n g concept to consider for c l i n i c a l use. Experimental work being done with continuous passive motion may support t h i s concept, but for now, most investigators believe that some form of post-operative protection i s r e -. .4,5,14,26-29 quired ' ' ' 35 SUMMARY AND CONCLUSION The e f f e c t s of varying periods of immobilization on the maturation and strength of the healing process of the rat medial c o l l a t e r a l ligament were studied. A Maturity Index Score and a Maturity Index Scale were devised based on: 1) the numbers of f i b r o b l a s t s present 2) the o r i e n t a t i o n of the f i b r o b l a s t s 3) the o r i e n t a t i o n of the collagen f i b e r bundles 4) the amount of collagen present U t i l i z i n g t h i s scale, supported by the electron microscopic study, i t was shown that a healing ligament matures more r a p i d l y when exposed to physio-l o g i c stresses and mobilization of the extremity, and that immobilization delays t h i s maturation. This has been correlated with biomechanical t e s t i n g which showed a more rapid return of the strength of the ligament and ligament-bone unit with mobi-l i z a t i o n of the extremity without d i s p l a y i n g any detrimental e f f e c t s to the elongation or compliance of the ligament. The detrimental e f f e c t s of immobi-l i z a t i o n on ligament-bone i n s e r t i o n strength were shown as well as the decrease i n the range of motion of the knee. We f e e l that c l i n i c a l studies and further animal studies are warranted to document the c l i n i c a l a p p l i c a t i o n of immediate or early mobilization o f extremeties following acute ligament rep a i r s of the knee. 36 M a t u r i t y Index Score Score 1. Numbers of F i b r o b l a s t s per high power f i e l d Normal ligament <10 1 10-30 2 30-50 3 50-100 4 Immature and A c t i v e i n Repair >100 5 2. O r i e n t a t i o n of the F i b r o b l a s t s . L o n g i t u d i n a l i n alignment w i t h the ligament a x i s 1 l o n g i t u d i n a l plus angled to the ligament a x i s 2 random order i n c l u d i n g transverse to the a x i s 3 3. O r i e n t a t i o n of the c o l l a g e n f i b e r bundles L o n g i t u d i n a l i n alignment w i t h the ligament a x i s 1 mainly l o n g i t u d i n a l w i t h some f i b e r s angled (Branching) 2 l o n g i t u d i n a l w i t h extensive branching 3 minimal o r g a n i z a t i o n w i t h some transverse f i b e r s 4 random o r g a n i z a t i o n i n a l l 3 planes 5 4. Amount of Collagen Present. t i g h t l y packed 1 some areas t i g h t , some loose 2 l o o s e l y packed 3 TABLE I 37 M a t u r i t y Index Scale M a t u r i t y Index Group Score 1 Normal - mature 4 2 Late remodelling phase 5 - 7 3 Mid remodelling phase 8 - 1 0 4 E a r l y remodelling phase 11 - 13 5 Immature - i n p r o l i f e r a t i v e phase 14 - 16 TABLE I I 38 Range of Motion Group T o t a l motion C o n t r o l 115° 45--160° arc A 115° at 2 wks and 20 wks B 85°-95° 115° at at 2 wks 6 wks and 20 wks C 20°-40° 85°-95° at .-\at 6 wks 20 wks D 20°-30° 55°-75° at at 10 wks 20 wks Table I I I 39 M a t u r i t y Index Score Time Nos. of O r i e n t a t i o n O r i e n t a t i o n Amount T o t a l M a t u r i t y i n F i b r o b l a s t s of of of Index Group Wks per h.p.f. F i b r o b l a s t s Collagen Collagen Scale A 2 5 2 3 3 13 4 B 2 3 3 5 3 14 5 A 6 3 2 2 2 9 3 B 6 2 2 4 3 11 4 C 6 4 3 5 2 14 5 A 10 2 1 1 1 5 2 B 10 2 1 1 1 5 2 C 10 2 2 3 2 9 3 D 10 3 3 4 2 12 4 A 20 1 1 1 1 4 1 B 20 2 1 1 1 5 2 C 20 2 2 2 1 7. 2 D 20 2 2 2 1 7 2 Con t r o l 1 1 1 1 4 1 See t e x t f o r d e s c r i p t i o n of M a t u r i t y Index Score and M a t u r i t y Index Scale. TABLE IV Biomechanical Testing of Medial Col l a t e r a l Llf lament Maximum Load Elonj j a t i o n Compliance Time kg S.F .R. mm mm/kg Group Wks N L e f t Right L e f t Right L e f t Right L e f t Right A 2 2 1.30 2.80 2.73 5.90 4.32 3.94 3.34 1.41 + .11 + .02 +.19 + .05 + .26 +.13 +.10 + .06 B 2 2 0.93 3.25 2.00 6.92 3.30 3.94 3.54 1.23 +.02 +.48 + .13 +.72 +.51 +.38 +.47 +.07 A 6 4 2.38 3.67 4.70 7.27 3.49 3.11 1.47 0.84 +.36 +.30 + .68 +.59 +.58 + .79 +.18 +.15 B 6 3 1.93 3.47 4.13 7.45 3.30 3.38 1.75 1.00 +.27 +.50 +.55 +1.00 +.36 +. 52 +.36 +.24 C 6 6 1.09 2.78 2.40 6.13 3.28 3.40 3.26 1.21 +.21 +.30 +.45 +.61 +.59 +.70 +.15 +.17 A 10 1 2.75 3.14 5.15 5.87 3.56 3.81 1.30 1.21 B 10 1 2.45 3.86 4.82 7.59 2.54 3.30 1.04 0.86 C 10 1 2.14 4.18 4.19 8.18 2.03 2.29 0.95 0.55 D 10 2 0.99 2.73 2.12 5.80 2.29 2.80 2.48 1.02 +.04 + .14 +.50 +.06 +.26 +.26 +.78 +.06 A 20 3 3.29 3.97 5.76 6.93 2.88 4.06 0.89 1.02 +.11 +.06 +.32 +.19 +.48 +.55 +.20 +.13 B 20 3 2.96 4.18 5.21 7.34 3.30 5.50 1.13 1.32 +.15 +.02 +.52 +.31 +.62 +.43 +.25 +.14 C 20 4 2.56 3.89 4.36 6.62 3.43 4.89 1.35 1.27 +.14 +.14 +.25 +.23 +.56 +1.10 +.25 +.30 D 20 4 1.98 4.16 3.37 7.09 2.61 3.75 1.38 0.90 +.26 +.14 +.41 +.33 +.60 +.45 +.46 +.11 Z 0 3 0.16 2.82 0.32 5.83 3.13 3.94 27.0 1.40 +.02 +.50 +.05 +1.01 +.54 +.52 +5.2 +0.05 — — — — — — — Group A=No Immobilization; Group B=2 wks Immobilization; Group C=6 wks Immo b i l i z a t i o n Group D=10 wks Immobilization; Group Z=test at time of surgery. Table l i s t s mean values i n the L e f t (repaired) and the Right ( c o n t r o l ) Legs. Table V Repaired Ligament/Control Ligament Group Time Wks N Maximum Load S.F.R. E l o n g a t i on Compliance A A 2 2 0.46 0.46 1.10 2.37 B 2 2 0.29 0.29 0.86 2.88 A 6 4 0.65 0.65 1.17 1.82 B 6 3 0.56 0.56 0.98 1.77 C 6 6 0.39 0.39 1.05 2.69 A 10 1 0.88 0.88 0.93 1.07 B 10 1 0.64 0.64 0.77 1.21 C 10 1 0.51 0.51 0.89 1.73 D 10 2 0.36 0.36 0.82 3.02 A 20 3 0.83 0.83 0.72 0.88 B 20 3 0.71 0.71 0.61 0.88 C 20 4 0.66 0.66 0.75 1.15 D 20 4 0.48 0.48 0.72 1.60 Z 0 3 0.06 0.06 0.79 19.14 Group A=No Immobilization; Group B=2 wks Immobilization; Group C= 6 wks. Immobilization; Group D=10 wks Immobilization; Group Z=test at time of surgery. Table l i s t s mean values of the r a t i o of the L e f t (repaired) Ligament to the Right ( c o n t r o l ) Ligament. TABLE VI L e f t S i t e of F a i l u r e Right Midsubstance Midsubstance Midsubstance Midsubstance Time T i b i a l Femoral tear tear not T i b i a l Femoral te a r tear not Group wks N a v u l s i o n a v u l s i o n at sutures at-sutures a v u l s i o n a v u l s i o n at sutures at sutures N z 0 3 3 3 3 A 2 2 1 1 2 2 B 2 2 2 2 2 A 6 4 4 4 4 B 6 3 3 2 1 3 C 6 6 4 1 1 6 6 A 10 1 1 1 1 B 10 1 1 1 1 C 10 1 1 1 1 D 10 2 2 2 2 A 20 3 3 3 3 B 20 3 2 1 3 3 C 20 4 3 1 4 4 D 20 4 2 2 4 4 T o t a l 39 27 3 6 3 38 0 0 1 39 TABLE V I I 43 S i t e of Ligament F a i l u r e Number Percentage T i b i a l A v u l s i o n 65 83 Femoral A v u l s i o n 3 4 Midsubstance te a r at sutures 6 8 Midsubstance te a r not at sutures 4 5 T o t a l 78" 100 TABLE V I I I 44 Figure 1: A drawing of the caudal one h a l f of the appendicular skeleton o f a r a t , i n d i c a t i n g the p o s i t i o n of placement of the two Kirschner wires which penetrated both medial and l a t e r a l sides of the l e g . 45 F i g u r e 2: S p e c i a l a l u m i n u m h o l d e r s d e s i g n e d f o r t e s t i n g o f l i g a m e n t - b o n e p r e p a r a t i o n i n t h e I n s t r o n m a t e r i a l t e s t i n g m a c h i n e . F e m u r -m e d i a l c o l l a t e r a l l i g a m e n t - t i b i a p r e p a r a t i o n h a s b e e n s e t i n e p o x y i n t h e s e h o l d e r s . N o t e t h e r e m o v a l b a r b r i d g i n g t h e t w o p o r t i o n s o f t h e h o l d e r t o s t a b i l i z e t h e p r e p a r a t i o n p r i o r t o t e s t . 46 Figure 3: I n s t r o n m a t e r i a l s t e s t i n g machine. 47 Figure 4: Light micrograph of a control ligament. Arrow indicates d i r e c t i o n of l o n g i t u d i n a l ligament a x i s . Collagen f i b e r s are densely packed and p a r a l l e l to ligament a x i s . X 720. Figure 5: Light micrograph of a control ligament. F i b r o b l a s t nuclei (n) are arranged p a r a l l e l to collagen f i b e r s and are few i n number. X 180. 48 49 Figure 6: Light micrograph of a r e p a i r i n g area of a ligament i n Group A at 2 weeks post-operative. Arrow indicates ligament axis. There i s a marked h y p e r c e l l u l a r i t y of p a r a l l e l f i b r o b l a s t n u c l e i (n). Collagen f i b e r formation i s present. X 720. Figure 7: Light micrograph of a r e p a i r i n g area of a ligament i n Group A at 2 weeks post-operative. There i s a marked h y p e r c e l l u l a r i t y of mainly p a r a l l e l f i b r o b l a s t s . Collagen f i b e r formation i s present which has some attempt at p a r a l l e l organization. X 720. s o 51 Figure 8: Figure 9: Low power l i g h t micrograph of a r e p a i r i n g area of a ligament i n Group B at 2 weeks post-operative. There i s a h y p e r c e l l u l a r i t y of f i b r o b l a s t s with no organization. Early collagen f i b e r formation i s present with no organization. X 285. Higher power l i g h t micrograph of the same area i n Figure 8. Fi b r o b l a s t s are large with dark s t a i n i n g n u c l e i and have no organization. Collagen i s present, but i n small amounts and without any organization. X 720. 5L Group B 2wks. 53 Figure 10: Light micrograph of the r e p a i r i n g area of a ligament i n Group A at 6 weeks post-operative. The number of f i b r o b l a s t s has decreased and they are p a r a l l e l to the ligament axis as indicated by the arrow. The collagen i s densely packed and also p a r a l l e l to the ligament a x i s . X 720. 11: High power l i g h t micrograph of the r e p a i r i n g area of a ligament i n Group A at 6 weeks post-operative. F i b r o b l a s t n u c l e i (n) are seen i n smaller numbers and p a r a l l e l to the ligament a x i s . Collagen i s dense and regular. X 1,800. 54 55 Figure 12: Light micrograph of a r e p a i r i n g area of a ligament i n Group B at 6 weeks post-operative. The f i b r o b l a s t s have decreased i n numbers and are now p a r a l l e l to the ligament axis as indicated by the arrow. The collagen has increased i n amount and i s now mainly p a r a l l e l to the ligament a x i s . X 720. Figure 13: Light micrograph of a r e p a i r i n g area of a ligament in Group B at 6 weeks post-operative. The f i b r o b l a s t n uclei (n) have decreased i n numbers and are p a r a l l e l to the ligament a x i s . The collagen f i b e r s have some organization although not as much as i n Group A. The collagen i s not as densely packed as i n Group A. X 1,800. 5b Group B 6wks. 57 Figure 14: Light micrograph of a r e p a i r i n g area of a ligament i n Group C at 6 weeks post-operative. F i b r o b l a s t n u c l e i are large, darkly s t a i n i n g and have some organization i n r e l a t i o n to the ligament a x i s as indicated by the arrow. Collagen f i b e r s are loosely packed and also have some organization although f i b e r branching from the ligament axis i s seen. X 720. Figure 15: Light micrograph of a r e p a i r i n g area of a ligament i n Group C at 6 weeks post-operative. Numerous large f i b r o b l a s t s are seen with large darkly s t a i n i n g n u c l e i and abundant intracytoplasmic v e s i c l e s . Collagen f i b e r s are loosely packed. X 1,800. 5S Group C 6 w k s . 59 Figure 16: Light micrograph of a r e p a i r i n g area of a ligament i n Group A at 10 weeks post-operative. Fibroblast n u c l e i have decreased i n number and are a l l p a r a l l e l . Collagen f i b e r s are densely packed and regular. X 285. Figure 17: Light micrograph of a r e p a i r i n g area of a ligament i n Group A at 10 weeks post-operative. F i b r o b l a s t n u c l e i (n) are small i n number and p a r a l l e l to the ligament axis as indicated by the arrow. Collagen f i b e r s are densely packed, regular and well organized. X 720. 60 Group A 10wks. 61 Figure 18: L i g h t micrograph of a r e p a i r i n g area o f a ligament i n Group B at 10 weeks post-operative. F i b r o b l a s t n u c l e i are few i n number and p a r a l l e l . Collagen f i b e r s are densely packed and w e l l organized. X 720. Figure 19: L i g h t micrograph of a r e p a i r i n g area o f a ligament i n Group B at 10 weeks post-operative. F i b r o b l a s t n u c l e i are p a r a l l e l t o the ligament a x i s as i n d i c a t e d by the arrow. The c o l l a g e n f i b e r s are a l s o p a r a l l e l to t h i s a x i s and are densely packed and w e l l organized. X 720. Group B 10wks. 63 Figure 20: Light micrograph of a r e p a i r i n g area of a ligament i n Group C at 10 weeks post-operative. F i b r o b l a s t n u c l e i have decreased i n numbers and are better organized. Collagen f i b e r s are packed densely and are better organized than at 6 weeks. X 720. Figure 21: Light micrograph of a r e p a i r i n g area of a ligament i n Group C at 10 weeks post-operative. F i b r o b l a s t n u c l e i have decreased i n numbers. Collagen f i b e r s are packed denser than at 6 weeks but s t i l l deviate from the ligament axis as indicated by the arrow. X 720. Group C 10wks. 65 Figure 22: Light micrograph of a r e p a i r i n g area of a ligament i n Group D at 10 weeks post-operative. F i b r o b l a s t n u c l e i are numerous, large and poorly organized. X 720. Figure 23: Light micrograph of a r e p a i r i n g area of a ligament i n Group D at 10 weeks post-operative. F i b r o b l a s t n u c l e i here show some organization but a large v a r i a b i l i t y i n s i z e . Collagen i s packed loose i n t h i s area but with some lo n g i t u d i n a l organiza-t i o n to the ligament axis as indicated by the arrow. Collagen branching from t h i s axis i s seen. X 720. bio Group D 10wks. 67 Figure 24: Light micrograph of a r e p a i r i n g area of a ligament i n Group A at 20 weeks post-operative. F i b r o b l a s t n u c l e i are few i n number and p a r a l l e l to the lon g i t u d i n a l ligament axis as indicated by the arrow. The collagen f i b e r s are densely packed, regular and p a r a l l e l to the ligament axis. The appearance i s the same as the controls. X 720. 69 Figure 25: A l i g h t micrograph of the re p a i r i n g area of a ligament i n Group B at 20 weeks post-operative. F i b r o b l a s t n u c l e i are more numerous than i n Group A and are p a r a l l e l to the ligament a x i s . Collagen f i b e r s are densely packed and regular. X 720. Figure 26: Light micrograph of a r e p a i r i n g area of a ligament i n Group B at 20 weeks post-operative. F i b r o b l a s t n u c l e i are p a r a l l e l to the ligament axis as indicated by the arrow and are more numerous than i n Group A. Collagen f i b e r s are also p a r a l l e l to the axis and well organized. X 720. 10 Group B 20wks. 71 Figure 27: Light micrograph of a r e p a i r i n g area of a ligament i n Group C at 20 weeks post-operative. F i b r o b l a s t n u c l e i remain numerous and are p a r a l l e l and l o n g i t u d i n a l . The collagen f i b e r s are densely packed and regular. X 285. Figure 28: Light micrograph of a r e p a i r i n g area of a ligament i n Group C at 20 weeks post-operative. F i b r o b l a s t n u c l e i are numerous and p a r a l l e l to the ligament axis as indicated by the arrow. The collagen f i b e r s are densely packed, regular and p a r a l l e l to the ligament a x i s . X 720. 11 7 3 Figure 29: Light micrograph of a r e p a i r i n g area of a ligament i n Group D at 20 weeks post-operative. F i b r o b l a s t n u c l e i have decreased i n number, but s t i l l ' are more numerous than i n controls, and are now mainly p a r a l l e l to the ligament a x i s . The collagen f i b e r s are densely packed and regular. X 720. Figure 3 0 : Light micrograph of a r e p a i r i n g area of a ligament i n Group D at 2 0 weeks post-operative. F i b r o b l a s t n u c l e i are numerous and p a r a l l e l to the ligament axis as indicated by the arrow. The collagen f i b e r s are densely packed and mainly p a r a l l e l to t h i s a x i s although some branching i s seen. X 7 2 0 . 7V Group D 2 0 w k s . 75 Figure 31: A graph of the results of the Maturity Index Score as applied to the Maturity Index Scale versus time (in weeks) post-operative. Group A has the most rapid maturation and achieves full maturity at 20 weeks post-operative. All of the other groups show a decreased rate of maturation which is more pronounced while their limbs are immobilized. At 20 weeks post-operative Groups B, C and D are all in the late remodel-ling phase of healing. Maturity Index Scale I I i i L i i ' I I I I 0 2 4 6 8 10 12 14 16 18 20 Time (weeks) 77 Figure 32: Electron micrograph of a control ligament. A one micron bar marker i s seen i n lower r i g h t corner. The f i b r o b l a s t s are small i n siz e and not very active metabolically. The collagen f i b e r s are densely packed, regular and p a r a l l e l to the l o n g i -tudinal ligament axis as indicated by the s o l i d arrow. X 14,300. Figure 33: Electron micrograph of a control ligament with a one micron bar marker i n the lower r i g h t corner. Rough endoplasmic reticulum (r) i s seen i n t r a c e l l u l a r l y i n the f i b r o b l a s t and tropocollagen f i b r i l formation i s seen at the c e l l periphery (open arrow). The regular 640A banding of the collagen f i b e r s i s seen (two small arrows). X 28,000. 13 Control 79 Figure 34: Electron micrograph of a control ligament. A one micron bar marker i s i n the lower r i g h t corner. The r e g u l a r i t y and dense packing of the collagen f i b e r s i s seen. The 640A banding i s indicated by the two small arrows. X 28,000. 90 81 Figure 35: Electron micrograph of a r e p a i r i n g area of a ligament i n Group A at 2 weeks post-operative. A one micron bar marker i s i n the lower r i g h t corner. The f i b r o b l a s t nucleus (N) i s large with clumps of chromatin seen. The c e l l i s very large and metabolically active with abundant mitochondria (m), rough endoplasmic reticulum (r) and g o l g i complexes (g). X 14,300. Figure 36: Electron micrograph of a r e p a i r i n g area of a ligament i n Group A at 2 weeks post-operative. A one micron bar marker i s seen i n the lower r i g h t corner. The collagen f i b e r s are seen to be forming i n an organized fashion p a r a l l e l to the ligament axis as indicated by the large s o l i d arrow. The collagen i s loosely packed and small i n diameter. X 28,000. Group A 2 w k s . 83 Figure 37: Electron micrograph of a r e p a i r i n g area of a ligament i n Group B at 2 weeks post-operative. The ligament axis i s indicated by the large s o l i d arrow. The collagen f i b e r s (c) are seen to have no organization and can even be seen i n cross-section (X). Large amounts of e x t r a c e l l u l a r debris are seen (open arrow). A one micron bar marker i s i n the lower r i g h t corner. X 14,3000. Figure 38: Electron micrograph of a r e p a i r i n g area of a ligament i n Group B at 2 weeks post-operative. A smaller f i b r o b l a s t nucleus (N) i s seen. The c e l l i s not very a c t i v e metabolically as i n d i c a t -ed by the lack of i n t r a c e l l u l a r organelles ( a s t e r i x ) . Some collagen f i b e r s (c) are seen. A one micron bar marker i s i n the lower r i g h t corner. X 14,300. 64 Group B 2wks. 85 Figure 39: Electron micrograph of a r e p a i r i n g area of a ligament i n Group A at 5 weeks post-operative. The f i b r o b l a s t s are s t i l l large and metabolically a c t i v e with abundant mitochondria (m) and rough endoplasmic reticulum ( r ) . The collagen f i b e r s are packed more densely and are p a r a l l e l to the ligament axis (large s o l i d arrow). A one micron bar marker i s i n the lower r i g h t corner. X 14,300. Figure 40: Higher power electron micrograph of the same area seen i n Figure 39. Numerous g o l g i complexes (g) are seen. The collagen f i b e r s are very regular and more densely packed than at 2 weeks. A one micron bar marker i s i n the lower r i g h t corner. X 28,000. 8b Group A 6 w k s . 87 Figure 41: Electron micrograph of a re p a i r i n g area of a ligament i n Group B at 6 weeks post-operative. The f i b r o b l a s t has increased i n si z e and i s more active metabolically than at 2 weeks with abundant mitochondria (m) and rough endoplasmic reticulum (r) . Collagen f i b e r s are seen more densely packed and with some organization when compared to the ligament axis (large arrow) although f i b e r s i n cross-section (X) are seen i n the same area. One micron bar marker i n lower r i g h t corner. X 2 8 , 0 0 0 . Figure 42: Electron micrograph of a r e p a i r i n g area of a ligament i n Group B at 6 weeks post-operative. A large f i b r o b l a s t with abundant mitochondria (m) and rough endoplasmic reticulum (r) i s seen. A one micron bar marker i s i n the lower r i g h t corner. X 2 8 , 0 0 0 . 88 Group B 6wks. 89 Figure 43: Electron micrograph of a r e p a i r i n g area of a ligament i n Group C at 6 weeks post-operative. The ligament axis i s indicated by the s o l i d arrow. The collagen has some organization although f i b e r s i n cross-section (X) are seen beside l o n g i t u d i n a l ones. A one micron bar marker i s seen i n the lower r i g h t corner. X 14,300. Figure 44: Electron micrograph of a r e p a i r i n g area of a ligament in Group C at 6 weeks post-operative. A portion of a large f i b r o b l a s t i s seen which i s very a c t i v e metabolically. There i s abundant mitochondria (m) and rough endoplasmic reticulum ( r ) . Tropocollagen filaments (open arrow) are seen at the c e l l margin. E x t r a c e l l u l a r debris (small s o l i d arrow) i s present. A one micron bar marker i s i n the lower r i g h t corner. X 28,000. 90 91 Figure 45: Electron micrograph of a r e p a i r i n g area of a ligament i n Group A at 10 weeks post-operative. The f i b r o b l a s t s are l e s s active metabolically as indicated by fewer i n t r a c e l l u l a r organelles ( a s t e r i x ) . Rough endoplasmic reticulum (r) i s present and tropocollagen filaments are seen at the c e l l boundary (open arrow). A one micron bar marker i s i n the lower r i g h t corner. X 28,000. Figure 46: Electron micrograph of a r e p a i r i n g area of a ligament i n Group A at 10 weeks post-operative. The collagen f i b e r s are densely packed, regular and l o n g i t u d i n a l l y oriented p a r a l l e l to the ligament axis (as indicated by the s o l i d arrow). A one micron bar marker i s i n the lower r i g h t corner. X 28,000. 91 93 Figure 47: Electron micrograph of a re p a i r i n g area of a ligament i n Group B at 10 weeks post-operative. A small f i b r o b l a s t nucleus i s seen (N). Collagen f i b e r s are s t i l l branching and smaller i n s i z e than Group A. A one micron bar marker i s i n the lower l e f t corner. X 14,300. Figure 48: Electron micrograph of a r e p a i r i n g area of a ligament i n Group B at 10 weeks post-operative. Two f i b r o b l a s t n u clei are seen (n) and the c e l l s are not very a c t i v e metabolically as indicated by fewer i n t r a c e l l u l a r organelles ( a s t e r i x ) . Rough endoplasmic reticulum i s seen (r) . The collagen f i b e r s have increased i n organization although they s t i l l are not as densely packed or regular as Group A. The ligament axis i s indicated by the large s o l i d arrow. A one micron bar marker i s i n the lower l e f t corner. X 28,000. 95 Figure 49: An electron micrograph of a r e p a i r i n g area of a ligament i n Group C at 10 weeks post-operative. A large f i b r o b l a s t with a large nucleus (N) i s seen. This f i b r o b l a s t i s metabolically very a c t i v e with large numbers of mitochondria (m). The collagen f i b e r s are not as well organized with l o n g i t u d i n a l f i b e r s and f i b e r s i n cross-section (X) seen. The large arrow indicates the ligament a x i s . A one micron bar marker i s in the lower r i g h t corner. X 7,500. Figure 50: An electron micrograph of a r e p a i r i n g area of a ligament i n Group C at 10 weeks post-operative. A large metabolically active f i b r o b l a s t with rough endoplasmic reticulum (r) i s seen. As seen i n Figure 49 the collagen i s not as well organized as Groups A or B and displays extensive branching (open arrow). A one micron bar marker i s i n the lower r i g h t corner. X 14,300. % 97 Figure 51: Electron micrograph of a r e p a i r i n g area of a ligament i n Group D at 10 weeks post-operative. A small f i b r o b l a s t nucleus (N) i s seen and the f i b r o b l a s t i s not very a c t i v e metabolically. Some of the collagen f i b e r s are l o n g i t u d i n a l l y oriented (large s o l i d arrow) while others are seen i n cross-section (X) and oblique (o). E x t r a c e l l u l a r debris i s s t i l l present (small s o l i d arrow). A one micron bar marker i s seen i n the lower r i g h t corner. X 14,300. Figure 52: Electron micrograph of a r e p a i r i n g area of a ligament i n Group D at 10 weeks post-operative. The metabolic a c t i v i t y of the f i b r o b l a s t i s r e f l e c t e d by the small numbers of mitochondria (m) and rough endoplasmic reticulum ( r ) . Tropocollagen filaments are seen at the c e l l membrane (open arrow). A one micron bar marker i s i n the lower r i g h t corner. X 28,000. 98 99 Figure 53' An e l e c t r o n micrograph of a r e p a i r i n g area of a ligament i n Group A at 20 weeks post-operative. A small f i b r o b l a s t i s seen with a nucleus (N) and rough endoplasmic r e t i c u l u m ( r ) . There are only small amounts o f i n t r a c e l l u l a r o r g a n e l l e s ( a s t e r i x ) as i n c o n t r o l s . The c o l l a g e n f i b e r s are densely packed and l o n g i t u d i n a l as i n d i c a t e d by the s o l i d arrow. A one micron bar marker i s i n the lower r i g h t corner. X 14,300. Figure 54: An e l e c t r o n micrograph of a r e p a i r i n g area of a ligament i n Group A at 20 weeks post-operative. As i n Figure 53 there are only small numbers of i n t r a c e l l u l a r o r g a n e l l e s ( a s t e r i x ) i n c l u d i n g rough endoplasmic r e t i c u l u m ( r ) . The c o l l a g e n i s densely packed and r e g u l a r . A one micron bar marker i s i n the lower r i g h t corner. X 28,000. I 00 54 Group A 2 0 w k s . 101 Figure 55: An electron micrograph of a r e p a i r i n g area of a ligament i n Group B at 20 weeks post-operative. The f i b r o b l a s t s are small but remain a c t i v e metabolically with abundant mitochondria. The collagen i s not as densely packed as i n Group A but i s mainly l o n g i t u d i n a l as seen by the s o l i d arrow. A one micron bar marker i s i n the lower r i g h t corner. X 14,300. Figure 56: An electron micrograph of a r e p a i r i n g area of a ligament i n Group B at 20 weeks post-operative at higher magnification. A metabolically a c t i v e f i b r o b l a s t i s seen with abundant mito-chondria (m) and rough endoplasmic reticulum ( r ) . The collagen i s not as densely packed as i n Group A or controls. A one micron bar marker i s i n the lower r i g h t corner. X 28,000. iOL 103 Figure 57: An electron micrograph of a r e p a i r i n g area of a ligament i n Group C at 20 weeks post-operative. The f i b r o b l a s t i s large with a large nucleus (N) and i s s t i l l very a c t i v e metabolically with abundant mitochondria (m) and rough endoplasmic reticulum ( r ) . Tropocollagen filaments (open arrow) are seen at the c e l l margin. The collagen i s more densely packed that at 10 weeks but s t i l l i s not the same as controls and displays some branching. A one micron bar marker i s i n the lower r i g h t corner. X 28,000. 104 Group C 20wks. 1 105 Figure 58: An electron micrograph of a r e p a i r i n g area of a ligament i n Group C at 20 weeks post-operative. A f i b r o b l a s t with a large nucleus (N) i s seen. This f i b r o b l a s t i s not as metabolically a c t i v e but tropocollagen filaments (open arrow) are seen at the c e l l margin. The collagen f i b e r s are s i m i l a r to those seen i n Figure 57 with branching seen and oblique f i b e r s (o) i n some portions. The l o n g i t u d i n a l axis i s indicated by the large s o l i d arrow. A one micron bar marker i s i n the lower r i g h t corner. X 14,300. Group C 20wks. 107 Figure 59: An electron micrograph of a r e p a i r i n g area of a ligament i n Group D at 20 weeks post-operative. A small f i b r o b l a s t i s seen and there i s l i t t l e metabolic a c t i v i t y with some rough endoplasmic reticulum (r) present. The collagen f i b e r s are i r r e g u l a r and not as densely packed. A one micron bar marker i s i n the lower r i g h t corner. X 28,000. I 08 Group D 2 0 w k s . 1 0 9 Figure 60: An electron micrograph of a r e p a i r i n g area of a ligament i n Group D at 2 0 weeks post-operative. The collagen f i b e r s mainly follow a l o n g i t u d i n a l organization as indicated by the large s o l i d arrow but i r r e g u l a r i t y and branching i s s t i l l present. E x t r a c e l l u l a r debris (small s o l i d arrow) i s s t i l l present. A micron bar marker i s i n the lower r i g h t corner. X 28,000. I / 0 I l l Figure 61: A graph of the Separation Force Ratio (see text) of the required ligament i n each of the groups versus time ( i n weeks) post-operative. Group A had a s i g n i f i c a n t l y higher separation force r a t i o regaining i t s strength at a fa s t e r rate than the other groups. 112 A bar graph of the Separation Force Ratio of the repaired ligament as compared to the control ligament versus time (in weeks) post-operative for each of the groups. Group A had a significantly higher separation force ratio and regained its strength at a faster rate than the other groups (see text). At 20 weeks post-operative Group A was 11$ stronger than Group B, 32% stronger than Group C and 71% stronger than Group D. 113 Separa t ion Force Ratio of Repa i red vs. Cont ro l Ligament 100 r-ot ' 1 1 1 1 I I i i i 2 4 6 8 10 12 14 16 18 20 Time (wks) Figure 63: A l i n e graph of the separation force r a t i o of the repaired ligament as compared to the control ligament versus time ( i n weeks) post-operative. The data i s the same as i n Figure 62. 114 Elongation of Repaired Ligament 5 r -E E 0 J I L J I I I I 0 2 4 6 8 10 12 14 16 18 20 Time (weeks) Figure 64: A graph of the elongation of the repaired ligament in m i l i -meters versus time ( i n weeks) post-operative. No s p e c i f i c pattern could be derived and the differences between the versus groups was not s i g n i f i c a n t . Group A = mmMmmmm Group B = »«:x:3k Group C = ii im D I I I Group D = 115 i Elongation of Repaired vs Control Ligament j 2 4 6 8 10 12 14 16 18 20 Time (weeks) A graph of the elongation of the repaired ligament as compared to the control ligament versus time ( i n weeks) post-operative. A l l groups showed a peak at 6 weeks and decreased fdrom then on but there was no s i g n i f i c a n t differences between the various groups. Group A - mmmkmmmm Group 6 = k-<s::v:v"* Group C = fjiiiibiin Group D = mmmm 116 i Compliance of Repaired Ligament <C I I I I I I I 1 1 1 0 2 4 6 8 10 12 14 16 18 2 0 Time (weeks) Figure 66: A graph of the compliance of the repaired ligament (see text) versus time i n weeks post-operative. A trend i n improvement i s seen i n a l l of the groups with no s i g n i f i c a n t differences between the various groups. Group A = wmmmmmm Group B = . o c s o a Group C = i i i i l i i l M Group D = 117 3 . 5 r 3.0 » 2.5 E o cn Compliance of Repaired vs. Control Ligament T3 CD o CL CD 22.0 c o cu (J c o "o. E o u 1.5 1.0 0.5 0, J I L _L J I L J '0 2 4 6 8 10 12 14 16 18 20 Time (weeks) Figure 67: A graph of the compliance of the repaired ligament as compared to the control ligament, versus time ( i n weeks) postoperative. A l l groups showed an improvement i n compliance with Groups A and B being s i g n i f i c a n t l y better than Groups C and D. Group A = M M M a a i Group B = c5K"3C5k3t3 Group C = LB ai I I I I 11 Group D = tmm:mm 118 BIBLIOGRAPHY 1. Akeson, W.H. An Experimental Study of Joint S t i f f n e s s . J . Bone. J t . Surg. 43A(7): 1022-1034. 2. Akeson, W.H. Connective Tissue Response to Immobility. C l i n . Orthop. Rel. Res. 51: 194-197, 1967.' 3. Akeson, W.H., Woo, S.L.Y., Amiel, D., et a l . The Connective Tissue Response to Immobility: Biochemical Changes i n P e r i a r t i c u l a r Connective Tissue of the Immobilized Rabbit Knee. C l i n . Orthop. Rel. Res. 9 3 : 356-362, 1973. 4. Aim, A., Ekstrom, H., Stromberg, B. Tensile Strength of the Anterior Cruciate Ligament i n the Dog. Acta. Scan. Chir. Suppl. Vol. 445: 15-189. 5. Aim, A., G i l l q u i s t , J . Reconstruction of the Anterior Curciate Ligament By Using the Medial Third of the P a t e l l a r Ligament. Acta. C h i r . Scand. 140: 289-296, 1974. 6. Aim, A., L i l j e d a h l , S.O., Stromberg, B. C l i n i c a l and Experimental Experience i n Reconstruction of the Anterior Cruciate Ligament. Orth. C l i n . N.A. 7(1): 181-189, 1976. 7. Cabaud, H.E., Rodkey, W.G., Feagin, J.A. Experimental Studies of Anterior Cruciate Ligament Injury and Repair. Am. J . Sports Med. 7(1): 18-22, 1979. 8. Clayton, M.L., Weir, G.J. Experimental Investigation of Ligamentous Healing. Am. J . Surg. 98: 3 7 3 - 3 7 8 , 1959. 9. Dehne, E., Torp, R.P. Treatment of Jo i n t I n j u r i e s by Immediate Mo b i l i z a t i o n . C l i n . Orthop. Rel. Res. 77: 218-232, 1971. 10. Evans, E.B., Eggers, G.W.N., Butler, J.K., Blumel, J. Experimental Immobilization and Remobilization of Rat Knee J o i n t s . J . Bone. J t . Surg. 42A(5): 737-758, I960. 11. Forward, A.D., Cowan, R.J. Tendon Suture to Bone. J. Bone J t . Surg. 45A(4): 807-822, 1 9 6 3 . 12. Grant, M.E., Prockop, D.J. The Biosynthesis of Collagen. New Eng. J . Med. 286: 194-199, 242-249, 291-300, 1972. 13. Greene, E.C. Anatomy of the Rat. Vol. 27, Hofner Publishing Co. N.Y. 1955, 375 pp. 119 14. Haggraark, T., Erikson, E. Cylinger or Mobile Cast Brace a f t e r Knee Ligament Surgery. Am. J. Sports Med. 7(1): 48-56, 1979. 15. Hirsch, G. Tensile Properties During Tendon Healing. Acta. Orthop. Scand. Suppl. No. 153: 8-79, 1974. 16. I p p o l i t o , E., N a t a l i , P.G., Postucchini, F., et al_. Morphological, Immunochemical and Biochemical Study of Rabbit A c h i l l e s Tendon at Various Ages. J . Bone J t . Surg. 62A(4): 583-597, 1980. 17. Jack, E.A. Experimental Rupture of the Medial C o l l a t e r a l Ligament of the Knee. J . Bone J t . Surg. 3 2 B ( 3 ) : 396-402, 1950. 18. Kappakas, G.S., Brown, T.D., Goodman, M.A., e_t _al. Delayed Surgical Repair of Ruptured Ligaments: A Comparative Biomechanical and H i s t o l o g i c a l Study. C l i n . Orthop. Rel. Res. 135: 281-286, 1978. 19. K l e i n , L., Lewis, J. Simultaneous Quantification of H-Collagen Loss and H. Collagen Replacement During Healing of Rat Tendon Grafts. J. Bone J t . Surg. 54A(1): 137-146, 1972. 20. Laros, G.S., Tipton, CM., Cooper, R.R. Influence of Physical A c t i v i t y on Ligament Insertions i n the Knees of Dogs. J . Bone J t . Surg. 53A(2): 275-286, 1971. 21. Lindsay, W.K., Thomson, H.G. D i g i t a l Flexor Tendons: An Experimental Study. Br. J . P l a s t i c Surg. 12: 289-319, I960. 22. Mason, M.L., A l l e n , H.S. The Rate of Healing of Tendons. Annals of Surgery 1 1 3 ( 3 ) : 424-459, 1941. 2 3 . Noyes, F.R., DeLucas, M.S., Torvik, P.J. Biomechanics of Anterior Cruciate Ligament F a i l u r e : An A l y s i s of Strain-Rate S e n s i t i v i t y and Mechanisms of F a i l u r e i n Primates. J. Bone J t . Surg. 56A(2): 236-253, 1974. 24. Noyes, F.R., Torvik, P.J. , Hydo, W.B. Biomechanics of Ligament F a i l u r e . J . Bone J t . Surg. 56A(7): 1406-1418, 1974. 25. Noyes, F.R., Grood, E.S. The Strength of the Anterior Cruciate Ligament in Humans and Rhesus Monkeys. J . Bone J t . Surg. 58A(8): 1074-1082, 1976. 26. Noyes, F.R. Functional Properties of Knee Ligaments and A l t e r a t i o n s Induced by Immobilization. C l . Orth. Rel. Res. 123: 210-242, 1977. 27. O'Donoghue, D.H., Rockwood, C.A., Zaricznys, B., Kenyon, R. Repair of Knee Ligaments i n Dogs. J . Bone J t . Surg. 43A(8): 1167-1178, 1961. 28. O'Donoghue, D.H., Rockwood, C.A., Frank, G.R., et a l . Repair of the Anterior Cruciate Ligament i n Dogs. J . Bone J t . Surg. 48A(3): 503-519, 1966. 120 29. O'Donoghue, D.H., Gael, R.F., Jeter, G.L., et al_. Repair and Reconstruction of the Anterior Cruciate Ligament i n Dogs. J. Bone J t . Surg. 53A(4): 710-718, 1971. 30. Perkins, G. Rest and Movement. J. Bone J t . Surg. 35B(4): 521-539, 1953. 31. Ross, R. The F i b r o b l a s t and Wound Repair. B i o l . Rev. 43: 51-96, 1968. 32. S a l t e r , R.B., F i e l d , P. The E f f e c t s of Continuous Compression on L i v i n g A r t i c u l a r C a r t i l a g e . J . Bone J t . Surg. 42A(1): 31-^9, I960. 33. S a l t e r , R.B., Simmonds, D.F., Malcolm, B.W., et_ al_. The Bi o l o g i c E f f e c t of Continuous Passive Motion on the Healing of F u l l Thickness Defects i n A r t i c u l a r C a r t i l a g e . J . Bone J t . Surg. 62A(8): 1232-1251, 1980. 34. Tipton, CM., S c h i l d , R.J., F l a t t , A.E. Measurement of Ligamentous Strength i n Rat Knee. J . Bone J t . Surg. 49A(1): 63-72, 1967. 35. Tipton, CM., S c h i l d , R.J., Tomanck, R.J. Influence of Physical A c t i v i t y on the Strength of Knee Ligaments i n Rats. J . P h y s i o l . 212(4): 783-787, 1967. 36. Woo, S.L-Y., Matthews, J.V. , Akeson, W.H., et a l . Connective Tissue Response to Immobility. A r t h r i t i s and Rheumatism 18(3): 257-264, 1975. 37. Zingg, W. Bioengineering Analysis of Healing Tissues. J . Sports Med. 3(2): 61-70, 1975. 38. Zuckerman, J. , S t u l l , G.A. E f f e c t s of Exercise on Knee Ligament Separation Force i n Rats. J . Appl. Physiol. 26(6): 716-719, 1969. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0095502/manifest

Comment

Related Items