THE MOLECULAR AND CELLULAR DIFFERENCES BETWEEN TENDONS AND LIGAMENTS Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy By Yalda Ashraf Kharaz March 2015 ABSTRACT Tendons and ligaments play key roles in the musculoskeletal system in both man and animals. Both tissues can often be injured as result of contact based accidents or ageing and disease, causing discomfort, pain and increased susceptibility to degenerative joint disease. To date, tendon and ligament biology is relatively under-studied in healthy, non-diseased tissues. This information is essential to understand the pathology of these tissues and vital for future development of tendon and ligament tissue-engineered structures. This thesis aims to investigate the molecular and cellular differences between tendons and ligaments around the canine stifle joint. The biochemical composition, structural, and morphological characteristics were identified between the different regions of the intra- articular cranial cruciate ligament (CCL) and extra-articular medial collateral ligament (MCL), and the positional long digital extensor tendon (LDET) and energy storing superficial digital flexor tendons (SDFT). Differences in proteome composition were also assessed between CCL and LDET. Cells isolated from canine CCL and LDET were cultured in a 3D in vitro fibrin culture model and measured for differences in structural, biochemical and proteome composition. Statistical significant differences in extracellular matrix (ECM) composition in terms of glycosaminoglycan (GAG) and elastin content were primarily detected in CCL in comparison to the other three tissues. The CCL was also found to have morphological differences including less compact collagen architecture, differences in cell nuclei phenotype, and increased (GAG) and elastin content. Proteomic comparison between CCL and LDET resulted in significantly abundant fibrocartilage proteins such as collagen type II, aggrecan, versican and chondroadherin in CCL, while the LDET was more abundant in asporin and thrombospondin-4. 3D tendon and ligament constructs were able to recapitulate tendon and ligamentous tissue characteristics particularly with regards to ECM proteins present, however both construct were less abundant in ECM protein and contained a greater proportion of cellular proteins, corresponding with low collagen and high level of DNA content measured in both constructs. 3D tendon and ligament constructs derived from tendon and ligament cells had similar ECM, proteomic and structural composition, indicating that cell source may not be an important factor for tendon or ligament tissue engineering. ii TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii TABLE OF CONTENTS ....................................................................................................... iii INDEX TO FIGURES ............................................................................................................ v INDEX TO TABLES .......................................................................................................... viii LIST OF ABBREVIATIONS .................................................................................................. ix ACKNOWLEDGEMENT ...................................................................................................... xi CHAPTER 1: GENERAL INTRODUCTION .............................................................................. 1 1.1 CONNECTIVE TISSUE .................................................................................................... 2 1.2 TENDON ....................................................................................................................... 6 1.3 LIGAMENT ................................................................................................................... 7 1.4 STRUCTURE OF TENDON AND LIGAMENT ................................................................... 8 1.5 TENDON AND LIGAMENT COMPOSITION ................................................................. 10 1.6 LIGAMENTS AND TENDONS OF THE KNEE JOINT ...................................................... 24 1.7 TENDON AND LIGAMENT INJURY AND DISEASE ....................................................... 28 1.8 HEALING OF TENDINOUS AND LIGAMENTOUS TISSUE ............................................. 31 1.9 CURRENT METHOD OF TENDON AND LIGAMENT REPAIR AND TISSUE ENGINEERING …………………………………………………………………………………………………………………………….34 1.10 SUMMARY OF TENDON AND LIGAMENT COMPARISON STUDIES ............................ 36 1.11 HYPOTHESIS AND AIMS ............................................................................................. 37 CHAPTER 2: GENERAL MATERIAL AND METHODS ............................................................ 38 2.1 TISSUE COLLECTION ................................................................................................... 39 2.2 TISSUE PREPARATION ................................................................................................ 42 2.3 DEVELOPMENT OF TISSUE ENGINEERED LIGAMENT AND TENDON CONSTRUCTS .. 43 2.4 BIOCHEMICAL ANALYSIS ............................................................................................ 45 2.5 HISTOLOGY STAINING AND SCORING........................................................................ 50 2.6 TISSUE IMMUNOSTAINING ....................................................................................... 56 2.7 TRANSMISSION ELECTRON MICROSCOPY ................................................................. 59 2.8 PROTEOMIC ANALYSIS ............................................................................................... 60 2.9 GENERAL STATISTICAL ANALYSIS .............................................................................. 64 CHAPTER 3: A BIOCHEMICAL COMPARISON OF THE EXTRACELLULAR MATRIX COMPOSITION OF TENDONS AND LIGAMENTS AROUND THE CANINE STIFLE JOINT ......... 65 3.1 INTRODUCTION ......................................................................................................... 66 3.2 HYPOTHESIS & AIM ................................................................................................... 68 3.3 EXPERIMENTAL PROCEDURE ..................................................................................... 69 3.4 RESULTS ..................................................................................................................... 72 3.5 DISCUSSION ............................................................................................................... 78 3.6 CONCLUSION ............................................................................................................. 82 iii CHAPTER 4: THE MORPHOLOGICAL AND STRUCTURAL DIFFERENCES AND EXTRACELLULAR MACROMOLECULES DISTRIBUTION BETWEEN TENDONS AND LIGAMENTS AROUND THE CANINE STIFLE JOINT ...................................................................................................... 83 4.1 INTRODUCTION ......................................................................................................... 84 4.2 HYPOTHESIS AND AIMS ............................................................................................. 85 4.3 EXPERIMENTAL PROCEDURES ................................................................................... 87 4.4 RESULTS ..................................................................................................................... 89 4.5 DISCUSSION ............................................................................................................. 109 4.6 CONCLUSION ........................................................................................................... 120 CHAPTER 5: A COMPARISON OF THE EXTRACELLULAR MATRIX COMPOSITION OF NATIVE TENDON/LIGAMENT AND 3D TENDON/LIGAMENT CONSTRUCTS .................................. 121 5.1 INTRODUCTION ....................................................................................................... 122 5.2 HYPOTHESIS & AIMS ................................................................................................ 123 5.3 EXPERIMENTAL PROCEDURES ................................................................................. 124 5.4 RESULTS ................................................................................................................... 127 5.5 DISCUSSION ............................................................................................................. 138 5.6 CONCLUSION ........................................................................................................... 142 CHAPTER 6: PROTEOMIC COMPARISON OF TENDON, LIGAMENT AND 3D TENDON/LIGAMENT CONSTRUCTS ............................................................................... 143 6.1 INTRODUCTION ....................................................................................................... 144 6.2 HYPHOTHESIS & AIMS ............................................................................................. 147 6.3 EXPERIMENTAL PROCEDURE ................................................................................... 148 6.4 RESULTS ................................................................................................................... 151 6.5 DISCUSSION ............................................................................................................. 173 CHAPTER