University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 3-31-2008 The ffecE ts of Non-Surgical Interventions on Osteoarthritis-Like Changes in the Mouse Knee Wendy K. Anemaet University of South Florida Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the American Studies Commons Scholar Commons Citation Anemaet, Wendy K., "The Effects of Non-Surgical Interventions on Osteoarthritis-Like Changes in the Mouse Knee" (2008). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/121 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. The Effects of Non-Surgical Interventions on Osteoarthritis-Like Changes in the Mouse Knee by Wendy K. Anemaet A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy School of Aging Studies College of Arts and Sciences University of South Florida Co-Major Professor: Anna Plaas, Ph.D. Co-Major Professor: William Haley, Ph.D. Katalin Mikecz, Ph.D. Keiba Shaw, Ed.D. Brent Small, Ph.D. Date of Approval: March 31, 2008 Keywords: cartilage, degradation, exercise, hyaluronan, transforming growth factor-beta, treadmill © Copyright 2008, Wendy K. Anemaet Dedication I dedicate this to my daughter, Aviendha, who has been right there every step of the way. You have experienced and endured more than most 10 year olds in this process. My hope is that it inspires (not disheartens) you to continually ask questions and seek answers. You are a truly special person and a great gift from God. Thanks for your patience, flexibility, encouragement, fun- loving personality, smiles, and hugs. I couldn’t ask for a better daughter. I hope that this research leads to more research and many more answers, so that one day your generation will not experience the debilitation that can come with osteoarthritis. And, above all else, I dedicate this to God who provides strength beyond measure and encouragement at just the right time. I have clung to Joshua 1:9 over the past years. Every day it is more apparent to me that we truly are fearfully and wonderfully made. What a great blessing it is to “discover” parts of Your marvelous creation. The more I learn, the more I am humbled by Your greatness. Wendy Acknowledgments This research would not have been possible without the support of numerous people at both the University of South Florida School of Aging Studies and Rush University Medical Center Departments of Biochemistry and Rheumatology. Most notably, Dr. Cathy McEvoy (Director, School of Aging Studies, University of South Florida) and Dr. William Haley (School of Aging Studies, University of South Florida); Dr. Ted Oegema (Chairman, Department of Biochemistry, Rush University Medical Center) and Dr. Joel Block (Director, Section of Rheumatology, Rush University Medical Center) for provision of laboratory space and access to shared equipment; Dr. Anna Plaas and Dr. John Sandy (Rush University Medical Center) for sharing their expertise and insights in osteoarthritis research, thought provoking discussions, poster and thesis editing, and their help in procuring financial support from the Arthritis Foundation (National Office and Florida Chapter) and Seikagaku Corporation, and the provision of the ADAMTS5-/- KO mouse strain through Pfizer Incorporated; Dr. Katalin Mikecz and Dr. Tibor Glant (Rush University Medical Center) for advice on mouse breeding, intra-articular injections, mouse handling, and use of the Kodak Whole Body Imager; Dr. Carol Muehleman (Rush University Medical Center) for use of dissecting microscope and camera; Dr. Eugene Thonar and Mary Ellen Lenz (Rush University Medical Center) for assistance with HA ELISA technique; Barbara Osborn, Mike Diaz, and Dr. Jun Li for biochemical and histological assays; Dr. Tannin Schmidt and Dr. John Sandy for help with cartilage grading; the Staff of the Comparative Research Centers at the University of South Florida and Rush University Medical Center, especially Dr. Marge Piel and Anthony Davis (Rush University Medical Center) for help in animal care and husbandry. Table of Contents List of Tables iv List of Figures v List of Appendices viii Abstract ix Chapter One: Background and Literature Review 1 Overview of Osteoarthritis 1 Definition 1 Incidence and Prevalence 2 Etiology of Osteoarthritis 3 Genetics 3 Injury 3 Obesity 4 Aging 5 Pathogenesis of Osteoarthritis 6 The Human Knee Joint 8 The Synovium 11 Bone 14 Articular Cartilage 18 Assessment of Cartilage Degeneration 22 Histological Grading of Cartilage in Human OA and in Animal Models 23 Clinical Treatment for Osteoarthritis 24 Psychosocial Interventions 25 Pharmacological Interventions 28 Oral Medications 28 Injectable Agents 29 Physical Therapy Interventions 33 Orthotics and Bracing 33 Modalities 34 Exercise 35 Aerobic Exercise 36 Range of Motion/Flexibility Exercise 38 Resistance Exercise 38 Water-Based Exercise 41 Functional Exercise 41 Mode of Action of Exercise 42 i Cell Based Therapies 45 Surgical Interventions 45 Animal Model Research 47 Overview of Animal Models 47 Murine Models 48 Spontaneous OA 48 Surgically-Induced OA 50 Manipulation of Cartilage Specific Genes 52 Chemical 54 Animal Models and Therapeutic Hyaluronan Injections 56 Animal Models and Treadmill Exercise 56 Chapter Two: Specific Aims and Research Hypotheses 60 Research Hypotheses 61 Specific Aims 61 Chapter Three: Development of Mouse Model of Knee OA 63 Research Design 63 Materials 67 Methods 68 Mouse Breeding and Husbandry 68 Intra-articular TGF-β1 Injections 69 Blood Collection and Plasma Preparation 71 TGF-β1 Enzyme Linked Immunosorbance Assay (ELISA) 71 Mechanical Overuse Through High Intensity Treadmill Running 74 Tissue Harvesting 77 Radiography 77 India Ink Cartilage Surface Evaluation 77 Hematoxylin/Eosin Histopathology 82 Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Analyses 83 Data Evaluation: Statistical Parameters 84 Results and Analyses 85 Effect of TGF-β1 Injection and Mechanical Overuse on Bone Structure 85 Anabolic Stimulation of Joint Tissues by Intra-Articular Injection of TGF-β1 87 Synovial Lining Fibrosis and Cartilage Degeneration at Two Weeks Post TGF-β1 Injection 89 India Ink Scoring of Cartilage Surfaces 91 FACE Analysis of Cartilage Chondroitin Sulfate Content 99 ii Effect of TGF-β1 Injections and Mechanical Overuse on ADAMTS-5 KO Mice 101 Treadmill Performance 101 Discussion and Conclusions 104 Chapter Four: Intra-Articular HA Injection Intervention 110 Research Design 110 Materials 112 Methods 112 Animal Husbandry 112 Intra-Articular HA Injections 113 Tissue Harvesting 113 Determination of Clearance Time of HA from the Knee Joint Space 113 Blood Collection and Plasma Preparation 116 Determination of Plasma HA Concentration by HA ELISA 116 Data Evaluation: Statistical Parameters 119 Results and Analyses 119 Clearance Time of HA from the Knee Joint Space 119 HA Concentration in Plasma Following Intra-Articular Injections of HA 124 Effect of Intra-Articular HA on Joint Pathology 127 Discussion and Conclusions 131 Chapter Five: Aerobic Exercise Intervention 134 Research Design 134 Methods 136 Aerobic Exercise via Alternate Day, Low Intensity Treadmill Running 136 Data Evaluation: Statistical Parameters 137 Results and Analyses 139 Effect of Exercise Intervention on Joint Pathology 139 Treadmill Performance 142 Discussion and Conclusions 144 Chapter Six: Summary and Conclusions 147 Significance and Implications 147 Limitations 149 Future Directions 150 References 153 Bibliography 186 Appendices 191 About the Author End Page iii List of Tables Table 1 Studies investigating the effects of aerobic exercise in mice 59 Table 2 Mouse utilization for the induction of OA-like changes 66 Table 3 Mean cartilage scores for right femurs by quadrant 92 Table 4 Mean cartilage scores for right tibias by quadrant 93 iv List of Figures Figure 1. Representation of complex relationship between environmental and endogenous risk factors for joint damage, osteoarthritis, and joint pain and their consequences 7 Figure 2. Schematic of the knee joint 9 Figure 3. Location of the synovium and synovial fluid in the knee joint 13 Figure 4 Structure of bone 15 Figure 5 Bone cell associations 16 Figure 6 Zones of articular cartilage 21 Figure 7 Sequential pyramidal approach to OA management 26 Figure 8 Research design for the development of a non-surgical mouse model of OA 64 Figure 9 Site of needle entry for intra-articular injection of the knee 70 Figure 10 Endogenous (mouse) TGF- β1 concentrations for mice injected with human TGF-β1 and mice injected with BSA 73 Figure 11 Mechanical overuse through high intensity treadmill running 75 Figure 12 Example grading of femoral and tibial cartilage surfaces after India ink staining 80 Figure 13 Cartilage surfaces were divided into four quadrants 81 Figure 14 Anterior/posterior and medial/lateral radiographs of right knees at day 18 86 Figure 15 Histopathological evaluation of right knee medial compartments and synovial lining for C57Bl/10 mice day 5 following H/E staining 88 v Figure 16 Histopathological evaluation of right knee medial compartments and synovial lining for C57Bl/10 mice day 18 following H/E staining 90 Figure 17 Mean cartilage scores for right femurs by quadrant 94 Figure 18 Mean cartilage scores for right tibias