THE INDIVIDUAL AND COMBINED EFFECTS OF EXERCISE AND COLLAGENASE ON THE RODENT ACHILLES TENDON Rachel Candace Dirks Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Anatomy and Cell Biology, Indiana University October 2013 ii Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. _____________________________________ Stuart J. Warden, Ph.D., PT, Chair _____________________________________ Matthew R. Allen, Ph.D. Doctoral Committee _____________________________________ Robyn K. Fuchs, Ph.D. August 30, 2013 _____________________________________ Alexander G. Robling, Ph.D. iii ACKNOWLEDGEMENTS I owe my deepest gratitude to my mentor, Dr. Stuart Warden for his guidance and support. I am very grateful to Dr. Warden for all he has taught me and for allowing me to learn and contribute to many studies in the area of musculoskeletal research. I am also grateful to the members of my research committee, Drs. Alex Robling, Matt Allen, and Robyn Fuchs, for their assistance and advice throughout these studies. I would also like to thank former members of our lab, Matt Galley and Jeff Richard, for all of their help with the daily work required to complete the studies of this dissertation. Also, I am grateful for all of the work done by the Histology Lab and for Paul Childress for all of his help and direction with techniques and data analysis. Much of this research was made possible by help from laboratories outside of Indiana University. I would like to thank Drs. Steve Britton and Lauren Koch at the University of Michigan for developing and providing us with the strain of rats utilized in this dissertation. Also, I am indebted to Drs. Alex Scott and Angie Fearon at the University of British Columbia for lending their expertise in the study of tendinopathy, as well as spending countless hours assisting with data analysis. I would like to thank my parents for all of their love and encouragement, which has allowed me to reach this point. Finally, I am thankful for my husband, Jeremy, who has stood by me with encouragement, patience, and support throughout all of my ups and downs. iv ABSTRACT Rachel Candace Dirks THE INDIVIDUAL AND COMBINED EFFECTS OF EXERCISE AND COLLAGENASE ON THE RODENT ACHILLES TENDON Tendinopathy is a common degenerative pathology that is characterized by activity related pain, focal tendon tenderness, intratendinous imaging changes, and typically results in changes in the histological, mechanical, and molecular properties of the tendon. Tendinopathy is difficult to study in humans, which has contributed to limited knowledge of the pathology, and thus a lack of appropriate treatment options. However, most believe that the pathology is degenerative as a result of a combination of both extrinsic and intrinsic factors. In order to gain understanding of this pathology, animal models are required. Because each tendon is naturally exposed to different conditions, a universal model is not feasible; therefore, an appropriate animal model must be established for each tendon susceptible to degenerative changes. While acceptable models have been developed for several tendons, a reliable model for the Achilles tendon remains elusive. The purpose of this dissertation was to develop an animal model of Achilles tendinopathy by investigating the individual and combined effects of an intrinsic and extrinsic factor on the rodent Achilles tendon. Rats selectively bred for high capacity running and Sprague Dawley rats underwent uphill treadmill running (an extrinsic factor) to mechanically overload the Achilles tendon or served as cage controls. Collagenase (intrinsic factor) was injected into one Achilles tendon in each animal to intrinsically break down the tendon. There were no interactions between uphill running and collagenase injection, indicating that the v influence of the two factors was independent. Uphill treadmill running alone failed to produce any pathological changes in the histological or mechanical characteristics of the Achilles tendon, but did modify molecular activity. Intratendinous collagenase injection had negative effects on the histological, mechanical, and molecular properties of the tendon. The results of this dissertation demonstrated that the combined introduction of uphill treadmill running and collagenase injection did not lead to degenerative changes consistent with human Achilles tendinopathy. Intratendiouns collagenase injection negatively influenced the tendon; however, these changes were generally transient and not influenced by mechanical overload. Future studies should consider combinations of other intrinsic and extrinsic factors in an effort to develop an animal model that replicates human Achilles tendinopathy. Stuart J. Warden, Ph.D., PT, Chair vi TABLE OF CONTENTS LIST OF TABLES x LIST OF FIGURES xi LIST OF ABBREVIATIONS xii GLOSSARY OF TERMS xiv CHAPTER ONE: INTRODUCTION 1.1 Introduction 1 1.2 Tendon anatomy and function 2 1.2.1 Macroscopic tendon structure 2 1.2.2 Microscopic tendon structure 3 1.2.3 Tendon metabolism 10 1.2.4 Tendon function and biomechanics 11 1.3 Human tendon pathology 17 1.3.1 Pathology 17 1.3.2 Etiology 26 1.3.3 Pathogenesis 31 1.3.4 Prevalence 33 1.3.5 Treatment 34 vii 1.4 Models of tendinopathy 40 1.4.1 In vitro models 41 1.4.2 Ex vivo models 42 1.4.3 In vivo models 43 1.5 The Achilles tendon 48 1.5.1 Anatomy and function 48 1.5.2 Etiology and pathology 50 1.5.3 Symptoms and diagnosis 52 1.5.4 Treatment 53 1.6 Summary and aims 56 1.6.1 Tendinopathy summary 56 1.6.2 Dissertation overview 56 1.6.3 Aims 60 CHAPTER TWO: THE EFFECT OF UPHILL RUNNING ON THE HISTOLOGICAL APPEARANCE OF THE ACHILLES TENDON IN HIGH CAPACITY RUNNING RATS 2.1 Introduction 62 2.2 Methods 64 2.2.1 Animals 64 2.2.2 Treadmill running 64 2.2.3 Histology 66 2.2.4 Statistics 67 viii 2.3 Results 68 2.4 Discussion 70 CHAPTER THREE: THE EFFECTS OF UPHILL RUNNING AND COLLAGENASE INJECTION ON THE ACHILLES TENDON IN HIGH CAPACITY RUNNING RATS 3.1 Introduction 73 3.2 Methods 75 3.2.1 Animals 75 3.2.2 Intrinsic factor 76 3.2.3 Extrinsic factor 77 3.2.4 Histopathological analysis 77 3.2.5 Mechanical testing 79 3.2.6 Gene expression 81 3.2.7 Statistics 82 3.3 Results 82 3.3.1 Histopathological appearance 86 3.3.2 Mechanical properties 86 3.3.3 Gene expression 89 3.4 Discussion 89 CHAPTER FOUR: THE EFFECTS OF UPHILL RUNNING AND COLLAGENASE INJECTION ON THE ACHILLES TENDON IN ix SPRAGUE DAWLEY RATS 4.1 Introduction 96 4.2 Methods 97 4.2.1 Animals 97 4.2.2 Treadmill acclimation 98 4.2.3 Intrinsic factor 98 4.2.4 Extrinsic factor 99 4.2.5 Histopathological analysis 99 4.3 Results 102 4.4 Discussion 106 CHAPTER FIVE: SUMMARY AND FUTURE DIRECTIONS 5.1 Dissertation summary 108 5.2 Strengths and limitations 109 5.3 Future directions 111 APPENDIX Representative photomicrographs of the graded histological characteristics 114 REFERENCES 117 CURRICULUM VITAE x LIST OF TABLES 2.1 Running protocol used for the running group of rats 65 2.2 Semiquantitative scale used to grade tendons 68 2.3 Weekly running distances by rats in the run group 69 2.4 Differences in individual histopathological categories and total histopathological score in Achilles tendons 70 3.1 Running protocol used for the running group of rats 78 3.2 Weekly running distances by rats in the 4 week run group 84 3.3 Weekly running distances by rats in the 10 week run group 85 3.4 Collagenase and treadmill running effects on histolopathological characteristics 88 4.1 Running protocol used for the running group of rats 100 4.2 Semiquantitative scale used to grade tendon ground substance 101 4.3 Weekly running distances by rats in the run group 103 4.3 Collagenase and treadmill running effects on histopathological characteristics 105 [...]... tendon 87 Collagenase and treadmill running effects on the mechanical properties of the Achilles tendon 90 Collagenase and treadmill running effects on the gene expression of the Achilles tendon 91 3.5 3.6 4.1 Cumulative distance ran on the treadmill by rats in the run group 102 4.2 Collagenase and treadmill running effects on the Achilles tendon 104 xi LIST OF ABBREVIATIONS 5-HPETE arachidonic acid... function 1.2.1 Macroscopic tendon structure Structurally, tendons have two points of attachment: the myotendionus junction (MTJ) between the tendon and muscle, and the osteotendionous junction (OTJ) between the tendon and bone These attachments are referred to as the origin at the MTJ and insertion at the OTJ At the origin, the collagen fibrils of the tendon protrude deep into the myofibroblasts, allowing... ran on the treadmill by rats in the run group 69 2.2 Representative photomicrographs of the Achilles tendon 70 3.1 Collagenase injection into the Achilles tendon 76 3.2 Set-up for the mechanical testing of rat Achilles tendons 80 3.3 Cumulative distance ran on the treadmill by rats in the run group 83 3.4 Collagenase and treadmill running effects on the histological presentation of the Achilles tendon. .. by the contractile proteins of the muscle to be transmitted to the tendon collagen fibers [1-3] This junction is the weakest point of the muscle -tendon unit [4,5] There are two types of insertions, referred to as the fibrous enthesis and fibrocartilaginous enthesis, with the former describing the tendon attaching directly to the periosteum and the latter consisting of a transitional zone [6,7] The. .. observation reveals a white color of tendons with variation in their shape, depending on the muscles and bones to which they attach Typically, the tendons of more powerful muscles are short and broad, while tendons of muscles performing more delicate movements are long and slender Tendons may also be surrounded by a combination of other structures, depending upon their location and function Many structures... development of appropriate treatment options, and thus tendon pathology, once present, often persists throughout an individual s life Because of the limited number of studies pertaining to tendons and tendinopathy, discrepancies in findings are prevalent and there is a need for research on both the normal and pathological characteristics and functions of tendons 1 1.2 Tendon anatomy and function 1.2.1... minimal cellularity, immature tendons have a much greater number of cells The tenoblasts found in newborn tendons are arranged in long, parallel chains and vary in shape and size, including long, round, and polygonal [22] These cells are responsible for the formation of the ECM and their composition enables the high metabolic activity responsible for the synthesis of the matrix This includes well developed... coordinating the response to loading of the tendon [25] 4 With aging, the number of cells decreases as the amount of matrix increases During this process, the majority of tenoblasts become tenocytes, as they elongate and undergo changes in composition, leaving them with a nucleus and very little cytoplasm The elongation is believed to allow for continued contact between the cells and matrix as the number of. .. prevents quick repair of any damage done to the tendon, which may be further aggravated by activity prior to full recovery 1.2.4 Tendon function and biomechanics Historically, tendons were simply described as transmitting forces from muscle to bone, allowing for motion of the bone More recently, research has focused on the effect of tendon elasticity on motion a) Elasticity Tendons function as springs, which... damage of the tendon [81,95] 13 b) Stiffness The stiffness of an object describes its rigidity, or the extent to which it resists deformation in response to an applied force Stiffness can be measured by calculating the slope of a force-displacement curve and represents the ratio of force applied to the tendon to its elongation in response to the force Tendon stiffness is dependent upon the location and . point. Finally, I am thankful for my husband, Jeremy, who has stood by me with encouragement, patience, and support throughout all of my ups and downs. iv ABSTRACT Rachel Candace Dirks. COMBINED EFFECTS OF EXERCISE AND COLLAGENASE ON THE RODENT ACHILLES TENDON Rachel Candace Dirks Submitted to the faculty of the University Graduate School in partial fulfillment. made of loose fibrillar tissue and prevents the tendon from moving against surrounding tissues. Finally, the tendon bursae exist to prevent bony prominences from compressing the tendon. 1.2.2