USF Libraries
USF Digital Collections

The effects of non-surgical interventions on osteoarthritis-like changes in the mouse knee

MISSING IMAGE

Material Information

Title:
The effects of non-surgical interventions on osteoarthritis-like changes in the mouse knee
Physical Description:
Book
Language:
English
Creator:
Anemaet, Wendy K
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
Cartilage
Degradation
Exercise
Hyaluronan
Transforming growth factor-beta
Dissertations, Academic -- Aging Studies -- Doctoral -- USF   ( lcsh )
Genre:
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: Osteoarthritis (OA) is a debilitating condition affecting over 21 million persons in the United States. This number is expected to rise in the coming decades. Treatment approaches for OA focus on symptom modifying measures (i.e., pain relief) as disease modifying interventions do not currently exist. However, some of the interventions used to alleviate the symptoms of OA are also thought to have disease-modifying benefits. Two such non-surgical interventions for OA are intra-articular hyaluronan (HA) injections and physical exercise. In order to effectively study their effects in human OA, animal models that are amenable for studying intervention outcomes are needed. The research focused on developing and characterizing a progressive non-surgical model of knee OA in adult mice.This model was used to firstly, examine the capacity of intra-articular HA injections to prevent knee joint degeneration, and secondly to examine the capacity of moderate exercise to prevent onset and progression of joint degeneration. Intra-articular injections of TGF-beta1 into murine knees produce synovial hyperplasia, osteophyte formation, and fibrotic changes on cartilage surfaces and joint capsules. However, additional exposure of the joints to high intensity treadmill running (biomechanical overuse) results in more widespread and focal OA-like cartilage erosions of both the tibial and femoral surfaces, similar to that described for the pathological appearance of late human knee OA. Taken together, these data support that synovitis and soft-tissue activation in early OA joints may precede and/or accelerate the process cartilage degeneration characteristic of progressive and late stage osteoarthritis.Intra-articular injections of high molecular weight HA one day following TGF-beta1 injections resulted in decreased synovial hyperplasia, minimized osteophyte formation, and significantly decreased severity of cartilage lesions. A four week, alternate day, low intensity aerobic treadmill running program prior to TGF-beta1 injections and overuse also resulted in decreased severity of cartilage lesions.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2008.
Bibliography:
Includes bibliographical references.
System Details:
Mode of access: World Wide Web.
System Details:
System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Wendy K. Anemaet.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 200 pages.
General Note:
Includes vita.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001990972
oclc - 311521055
usfldc doi - E14-SFE0002357
usfldc handle - e14.2357
System ID:
SFS0026675:00001


This item is only available as the following downloads:


Full Text
xml version 1.0 encoding UTF-8 standalone no
record xmlns http:www.loc.govMARC21slim xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.loc.govstandardsmarcxmlschemaMARC21slim.xsd
leader nam Ka
controlfield tag 001 001990972
003 fts
005 20090302105431.0
006 m||||e|||d||||||||
007 cr mnu|||uuuuu
008 090302s2008 flu s 000 0 eng d
datafield ind1 8 ind2 024
subfield code a E14-SFE0002357
035
(OCoLC)311521055
040
FHM
c FHM
049
FHMM
090
HQ1061 (Online)
1 100
Anemaet, Wendy K.
4 245
The effects of non-surgical interventions on osteoarthritis-like changes in the mouse knee
h [electronic resource] /
by Wendy K. Anemaet.
260
[Tampa, Fla] :
b University of South Florida,
2008.
500
Title from PDF of title page.
Document formatted into pages; contains 200 pages.
Includes vita.
502
Dissertation (Ph.D.)--University of South Florida, 2008.
504
Includes bibliographical references.
516
Text (Electronic dissertation) in PDF format.
520
ABSTRACT: Osteoarthritis (OA) is a debilitating condition affecting over 21 million persons in the United States. This number is expected to rise in the coming decades. Treatment approaches for OA focus on symptom modifying measures (i.e., pain relief) as disease modifying interventions do not currently exist. However, some of the interventions used to alleviate the symptoms of OA are also thought to have disease-modifying benefits. Two such non-surgical interventions for OA are intra-articular hyaluronan (HA) injections and physical exercise. In order to effectively study their effects in human OA, animal models that are amenable for studying intervention outcomes are needed. The research focused on developing and characterizing a progressive non-surgical model of knee OA in adult mice.This model was used to firstly, examine the capacity of intra-articular HA injections to prevent knee joint degeneration, and secondly to examine the capacity of moderate exercise to prevent onset and progression of joint degeneration. Intra-articular injections of TGF-beta1 into murine knees produce synovial hyperplasia, osteophyte formation, and fibrotic changes on cartilage surfaces and joint capsules. However, additional exposure of the joints to high intensity treadmill running (biomechanical overuse) results in more widespread and focal OA-like cartilage erosions of both the tibial and femoral surfaces, similar to that described for the pathological appearance of late human knee OA. Taken together, these data support that synovitis and soft-tissue activation in early OA joints may precede and/or accelerate the process cartilage degeneration characteristic of progressive and late stage osteoarthritis.Intra-articular injections of high molecular weight HA one day following TGF-beta1 injections resulted in decreased synovial hyperplasia, minimized osteophyte formation, and significantly decreased severity of cartilage lesions. A four week, alternate day, low intensity aerobic treadmill running program prior to TGF-beta1 injections and overuse also resulted in decreased severity of cartilage lesions.
538
Mode of access: World Wide Web.
System requirements: World Wide Web browser and PDF reader.
590
Co-advisor: Anna Plaas, Ph.D.
Co-advisor: William Haley, Ph.D.
653
Cartilage
Degradation
Exercise
Hyaluronan
Transforming growth factor-beta
0 690
Dissertations, Academic
z USF
x Aging Studies
Doctoral.
773
t USF Electronic Theses and Dissertations.
856
u http://digital.lib.usf.edu/?e14.2357



PAGE 1

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

PAGE 2

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 answe rs. You are a truly special person and a great gift from God. Thanks for your patience, flexibility, encouragement, funloving personality, smiles, and hugs. I couldn’t ask for a better daughter. I hope that this research leads to more res earch 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 mo re 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

PAGE 3

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

PAGE 4

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.

PAGE 5

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

PAGE 6

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 TGF1 Injections 69 Blood Collection and Plasma Preparation 71 TGF! 1 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! 1 Injection and Mechanical Overuse on Bone Structure 85 Anabolic Stimulation of Joint Tissues by Intra-Articular Injection of TGF! 1 87 Synovial Lining Fibrosis and Cartilage Degeneration at Two Weeks Post TGF! 1 Injection 89 India Ink Scoring of Cartilage Surfaces 91 FACE Analysis of Cartilage Chondroitin Sulfate Content 99 ii

PAGE 7

Effect of TGF! 1 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

PAGE 8

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

PAGE 9

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! 1 concentrations for mice injected with human TGF! 1 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

PAGE 10

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 by quadrant 95 Figure 19 Mean cartilage scores for left femurs by quadrant 96 Figure 20 Mean cartilage scores for left tibias by quadrant 97 Figure 21 Mean cartilage scores by group for right and left knee cartilage surfaces 98 Figure 22 Chondroitin sulfate content of right knee cartilage as determined by FACE analyses 100 Figure 23 Histopathological evaluation of right knee medial compartments and synovial lining for ADAMTS 5 KO mice following H/E staining 102 Figure 24 Percentage of time spent on front half of treadm ill days 3 through 13 for mice in mechanical overuse and mechanical overuse + TGF! 1 groups 103 Figure 25 Research design for intra-articular HA injection intervention 111 Figure 26 Selection of regions of interest on fluorescent image corresponding to radiographic images for determining net Intensity of fluorescence within the knee joint 115 Figure 27 Schematic representation of HA ELISA 118 Figure 28 Fluorescent imaging with FITC labeled HA and Alexa 580 H ABP 120 Figure 29 HA fluorescence in knee joints of mice 122 Figure 30 HA clearance from knee joints of mice 123 Figure 31 Plasma HA concentrations 126 v i

PAGE 11

Figure 32 Mean cartilage scores for right femurs by quadrant after HA intervention 128 Figure 33 Mean cartilage scores for right tibias by quadrant after HA intervention 129 Figure 34 Percentage of time spent on front half of treadm ill days 3 through 13 for mice in the mechanical overuse, mechanical overuse + TGF! 1, and mechanical overuse + TGF! 1 + HA groups 130 Figure 35 Research design for aerobic exercise intervention 135 Figure 36 Mean cartilage scores for right femurs by quadrant after exercise intervention 140 Figure 37 Mean cartilage scores for right tibias by quadrant after exercise intervention 141 Figure 38 Percentage of time spent on front half of treadm ill days 3 through 13 by group for mice in the mechanical overuse, mechanical overuse + TGF! 1, and mechanical overuse + TGF! 1 + exercise groups 143 vii

PAGE 12

List of Appendices Appendix 1 Abbreviations used 191 Appendix 2 Reported outcomes of exercise in persons with OA 193 Appendix 3 Mouse models used to study OA 196 Appendix 4 Poster presented at the International Conference on Preclinical Models of Osteoarthritis, May 2006 197 Appendix 5 Poster Presented at the Annual Meeting of the Orthopedic Research Society, February 2007 198 Appendix 6 Poster presented at the American College of Rheumatology Annual Scientific Meeting, November 2007 199 Appendix 7 Poster presented at the Annual Meeting of the Orthopedic Research Society, March 2008 200 viii

PAGE 13

The Effects of Non-Surgical Interventions on Osteoarthritis-Like Changes in the Mouse Knee Wendy K. Anemaet ABSTRACT Osteoarthritis (OA) is a debilitating condition affecting over 21 million persons in the United States. This number is expected to rise in the coming decades. Treatment approaches for OA focu s on symptom modifying measures (i.e., pain relief) as disease modifying interventions do not currently exist. However, some of the interventions used to alleviate the symptoms of OA are also thought to have disease-modifying benefits. Two such non-surgical interventions for OA are intra-articular hyaluronan (HA) injections and physical exercise. In order to effectively study their effects in human OA, animal models that are amenable for studying intervention outcomes are needed. The research focused on developing and characterizing a progressive non-surgical model of knee OA in adult mice. This model was used to firstly, examine the capacity of intra-articular HA injections to prevent knee joint degeneration, and secondly to examine the capacity of moderate exercise to prevent onset and progression of joint degeneration. ix

PAGE 14

Intra-articular injections of TGF1 into murine knees produce synovial hyperplasia, osteophyte formation, and fi brotic changes on cartilage surfaces and joint capsules. However, additional exposure of the joints to high intensity treadmill running (biomechanical overuse) results in more widespread and focal OA-like cartilage erosions of both the tibial and femoral surfaces, similar to that described for the pathological appearance of late human knee OA. Taken together, these data support that synovitis and soft-tissue activation in early OA joints may precede and/or accelerate the process cartilage degeneration characteristic of progressive and late stage osteoarthritis. Intra-articular injections of high molecular weight HA one day following TGF1 injections resulted in decreased synovial hyperplasia, minimized osteophyte formation, and significantly decreased severity of cartilage lesions. A four week, alternate day, low intensity aerobic treadmill running program prior to TGFinjections and overuse also resulted in decreased severity of cartilage lesions. x

PAGE 15

Chapter One Background and Literature Review Overview of Osteoarthritis Definition Osteoarthritis (OA), also known as degenerative joint disease (DJD), is a musculoskeletal disease that is diagnosed both structurally and clinically. Structurally, synovitis (Benito et al., 2005), progressive articular cartilage loss (Cahue et al., 2004; Cerejo et al., 2002), osteophyte formation (Boegard et al.,1998), and subchondral sclerosis (Yamada et al., 2002), generally in weight bearing joints such as knee and hip, give rise to biomechanically unstable joints that result in loss of function (Sharma et al., 2001). The resulting abnormal biomechanical forces on the joint tissues play a role in the erosion of cartilage surfaces as well as subchondral sclerosis. Clinically, patients with OA experience pain, stiffness, loss of motion, weakness, and joint instability, all leading to functional limitation and disability (Altman et al., 1986). However to date, criteria to precisely stage the severity of the disease for treatment options remain ill defined (Boegard et al., 1998; Creamer et al., 1999; Felson et al., 2000a; McAlindon et al., 1992; O’Reilly et al., 1998; Peterson et al., 1996; Weidow et al., 2006). For example, persons may report a high level of pain with only mild radiographic changes, or conversely, may have dramatic radiographic changes with minimal reports of pain. 1

PAGE 16

Development and application of assays of OA-specific biomarkers for synovial fluid, serum and urine have been the focus of active clinical and basic science research efforts, but have not yet resulted in clear marker panels that could be readily used in diagnosis and prognosis of OA in the clinic or in translational research studies (Bauer et al., 2006; Giles et al., 2007; Girling et al., 2006; Kloppenburg et al., 2007; Krause et al., 2006; Lohmander and Eyre, 2005; Nemirovskiy et al., 2007; Sumer et al., 2006; Thonar et al, 1993). Incidence and Prevalence Clinical diagnosis of OA is made in one in three persons over the age of 50 years, and women are more frequently affected than men (Lawrence et al., 1998). OA is the leading cause of chronic disability in the elderly (Felson et al, 2000), owing in part to improved longevity. One in two persons over the age of 70 years and over 85% of persons aged 80 years or more have a clinical diagnosis of OA (National Centers for Health Statistics, 2004). The most commonly affected joint is the knee, but OA also affects the hip, spine, hands, and feet (Glass, 2006). Incidence and prevalence vary slightly by the affected area. Women have higher incidence and prevalence of hand and knee OA and men have higher prevalence of hip OA (Jordan, 1996). 2

PAGE 17

Etiology of Osteoarthritis Genetics Genetic defects of connective tissue structural and regulatory proteins, such as bone morphogenetic protein 2 (BMP2), cartilage intermediate layer protein (CILP), frizzled related protein (FRZB),and TNF alpha-induced protein (TNFAIP6) have been clearly implicated in the development of OA and are often associated with increased risk of OA (Lane et al., 2006; Valdes et al., 2004). For example, mutations in humans that increase susceptibility to OA involve collagens II (Vikkula et al, 1994), IX (Olsen, 1997), and XI (Jacenko and Olsen, 1995) genes and are often manifested as multiple joint involvement (Abel et al., 2006; Bateman, 2005; Clements et al., 2006; Hakim and Sahota, 2006; Lopponen, et al., 2004; Roughley et al., 2006; Williams and Jimenez, 2003; Zhang and Doherty, 2005). However, while genetic linkages to OA exist, the disease is rarely caused by a single genetic defect (Zhang et al., 1998), but instead can be considered a multi-genetic, multi-factorial class of diseases. (See Figure 1). Injury About 10% of all OA cases are post-traumatic in origin (Marsh, 2004), including articular fractures (Furman et al ., 2006), work or sports related overuse of joints (Hansen and Reed, 2006; Hunt, 2006; McMillan and Nichols, 2005), and injuries to the anterior cruciate ligament and/or menisci (Bartz and Laudicina, 2005; Koh and Dietz, 2005; Roos, 2005). 3

PAGE 18

The precise etiology of post-traumatic OA remains to be determined as individuals with no joint trauma and little repetitive activity also develop OA (Hannan et al., 1993; Schrier, 2004). Moreover, exercising or involvement in sports in the absence of muscular imbalanc es or physical damage to joint tissues does not result in increased OA (Cymet and Sinkov, 2006; Schrier, 2004). Thus, it is more likely that the extensive remodeling of joint tissues (synovium, cartilage, bone, ligament, meniscus) which occurs in response to injury generates the metabolic disturbance in the whole joint that eventually leads to the pathological manifestation of OA (Felson, 2004). Obesity Obesity has been found to be strongly linked to knee OA (Cooper et al., 2000; Schouten et al., 1992). Persons who were overweight at age 37 (when knee OA is uncommon) had an increased risk of developing knee OA at age 70 or older (Felson et al., 1988). In women between 20-89 years with a body mass index (BMI) of > 25 kg/m2, a new diagnosis for symptomatic knee OA was fourto ninefold higher than for age-matched controls with BMI < 25 kg/m2 (Felson, 1990). In addition, obesity is linked to enhanced progression of OA as defined by radiographic changes (Schouten et al., 1992; Spector et al., 1994) and may result in increased joint pain and disability (Jinks et al., 2006; Mallen et al., 2007; Verbrugge et al., 1991). It has been suggested that accumulation of adipose tissue results in altered levels of hormones, cytokines, and growth factors, increased bone 4

PAGE 19

density, and changes in immune res ponse (such as prolonged low grade inflammatory response), and all of the above may have a role in OA development (Nevitt and Felson, 1996; Olson et al., 2007). In addition, weight gain is common in persons with decreased functional status and/or disability, all hallmarks of OA and aging, potentially enhancing disease progression further in such individuals (Lievense et al., 2002). Aging Both the incidence (number of new cases in a specific period of time) and prevalence (the number of cases in a population at a given time) of OA increase in the elderly (Kopec et al., 2007). A common initiating factor in OA is the inability of the joint to repair mechanical (trauma) or metabolic (obesity) injuries (Olson and Marsch, 2004) during which cellular and biochemical pathways, including immune responses, are activated for tissue repair (Polyzois et al., 2006). Immunosenescence, therefore, may be an important factor in promoting pathogenesis of OA in the elderly. Age-related changes in the innate and acquired immune pathways manifest themselves in chronic inflammation and inefficiency in immune surveillance (Senchina and Kohut, 2007), including agerelated changes in function of neutrophils and macrophages (Fulop et al., 1997; Gomez et al., 2005: Lord et al., 2001; Plackett et al., 2004; Stout and Suttles, 2005; Wenisch et al., 2000). As a result, older adults are more vulnerable to infection (Crichton and Puppione, 2006), and wound healing is slow or even impaired. Moreover, circulating levels of pro-inflammatory mediators including 5

PAGE 20

interleukin (IL)-1, IL-6, tumor necrosis factor-alpha (TNF" ), prostaglandin, and C-reactive protein (CRP) can occur in the elderly (Franceschi et al., 2000). Pathogenesis of Osteoarthritis The pathogenesis of OA involves interactions of joint tissues, joint biomechanics, and biochemical pathways. The predominant pathological features of OA are synovitis and fibrosis, accelerated bone remodeling with ostoephyte development, and articular cartilage degeneration. As outlined above, underlying characteristics, such as age, genetic background, and comorbidities (obesity) predispose an individual to the development of OA. In conjunction with aberrant joint biomechanics in the form of injury, overload, or joint instability, this predisposition may lead to altered biochemical pathways including those involved in cytokine and growth factor signaling, or matrix biosynthesis and turnover. These altered pathways lead to the development and progression of tissue damage in OA which present clinically as radiographic changes in bone and cartilage and joint pain. The presentation of pain is also affected by psychosocial and socioeconomic factors (Thumboo et al., 2002) as well as the presence of comorbidities (Tuominen et al., 2007). The presence of pain, in turn, may act to alter biochemical pathways leading to further development and progression of OA. In addition, pain leads to disability and distress which lead to increased pain perception (Figure 1). 6

PAGE 21

Figure 1: Representation of complex relationship between environmental and endogenous risk factors for joint damage, osteoarthritis, and joint pain and their consequences (From: Dieppe and Lohmander, 2005) 7

PAGE 22

The Human Knee Joint The mammalian knee joint is comprised of two long bones the tibia and the femur and a sesamoid bone, the patella (Figure 2). The patella serves as a pulley allowing the quadriceps muscle to act more efficiently, sliding in the femoral sulcus Three major muscle groups control movement at the knee. The quadriceps is comprised of four muscles (the vastus lateralis the vastus intermedius the rectus femoris and the vastus medialis ). This muscle group is responsible for knee extension and is contiguous with the extensor mechanism (comprised of the patella tendon, patella, and patella ligament). Muscle imbalances, especially weakness of the vastus medialis, results in abnormal tracking of the patella. Abnormal pulling of the quadriceps muscle (from muscle imbalance or structural abnormalities) in other joint tissues results in the patella moving out of the femoral sulcus. The distal end of the femur, proximal end of the tibia, and posterior aspect of the patella are lined with articular cartilage. When the patella tracks abnormally, damage to the articular cartilage on the femur and patella may occur (Cahue et al., 2004; Sharma et al., 2001; Sharma et al., 2003). The hamstrings consist of the semimembranosus the semitendinosus and the biceps femoris and all three are responsible for knee flexion. In addition, the hamstrings protect the integrity of the anterior cruciate ligament by counteracting shear through controlling anterior movement of the tibia on the femur. The third muscle acting at the knee is the gastrocnemius which is primarily responsible for ankle plantarflexion, but also assists with knee flexion. 8

PAGE 23

Figure 2: Schematic of the knee joint (patella reflected) (Diagram by Arnheim and Prentice accessed at http://factotem.org/library) 9

PAGE 24

Ligaments about the joint provide stability. The patella ligament connects the patella and quadriceps muscle to the tibia at the tibial tuberosity and provides anterior-posterior stability to the joint. The anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) cross at the center of the joint (Figure 2) connecting the tibia to the femur. They are intra-articular ligaments because they are within the joint capsule and surrounded by synovial fluid. They protect the knee against twisting and control forward sliding of the tibia on the femur (shear). The medial collateral (MCL) and lateral collateral (LCL) ligaments are located on the medial and lateral sides of the knee, respectively (Figure 2). They connect the femur to the tibia on the medial side and fibula on the lateral side. The MCL protects the knee against valgus (inwar d) forces and the LCL protects against varus (outward) forces. Menisci are two crescent-shaped pieces of fibrocartilage between the femoral and tibial surfaces (Figure 2). They cushion the joint from impact and distribute compressive and shear forces across the articular cartilage surfaces. Biomechanically the knee is at risk for injury as a result of the high torque moments created by the long bones and the frequency of use of the lower extremities. Dynamic loading of the joint (walking, running, and jumping) can increase joint forces up to 20 times relative to just standing. In addition, rapid changes of motion that occur with many acti vities as well as balance corrections require quick changes in velocity and impose additional stresses on supporting structures of the knee. 10

PAGE 25

The Synovium The synovial membrane is the delicate, vascularized tissue that covers the non-articular surfaces of the synovial joint cavity, including intra-articular ligaments (the anterior and posterior cruciate and patellar ligaments), the patellar tendon, and the intra-capsular bone surfaces (Figure 3). It is composed of an epithelial-like cell lining facing the joint cavity (intima), and a deeper, vascularized fibrous layer (subintima) that can also contain groups of differentiated fat cells. Synovial functions include innate immune surveillance, lubrication, and intraarticular joint tissue nutrition, representing a metabolic protection of joint function (Berumen-Nafarrate et al., 2002). The synoviocytes in the lining have been defined ultra-structurally as Type A (macrophage-like) cells and Type B (fibroblast-like) cells (Pavolich and Lubowitz, 2008). Type A cells function as resident tissue macrophages to remove cell debris and products of macromolec ular turnover processes which include synovial fluid hyaluronan (HA; Bondeson et al., 2006) lubricin, collagen, and aggrecan fragments. Type B cells are the major producers of HA and lubricin for the synovial fluid that decrease the joint friction coefficient reducing forces on the articular cartilage (Schmidt et al., 2007; Smith et al., 2003). Alteration in cell and matrix constituents of the synovium, which is also known as synovitis, has been reported to correlate with progression of OA. Thus the pathological synovium may to a large extent be responsible for production of pro-inflammatory cytokines (interleukins, TNF and oncostatin M) and growth factors (platelet derived growth factor [PDGF], transforming growth factor-beta 11

PAGE 26

[TGF! 1]). (Bondeson et al., 2006; Sutton et al., 2007) These in turn may regulate the expression of other pro-inflammatory pathways leading to the production of tissue destructive proteases including collagenases and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteases (Fukui et al., 2005; Youn et al., 2002). 12

PAGE 27

Figure 3: Location of the synovium and synovial fluid within the knee joint From http://www.healthcare.utah.edu/healthinfo/images anterior posterior 13

PAGE 28

Bone Bone matrix is primarily composed of calcium hydroxylapatite deposited within a collagen matrix. The mineral provides compressive strength and the collagen provides elasticity. The outer layer of cortical bone accounts for 80% of the total bone mass and a cell-rich porous network of rodand plate-like trabecular bone accounts for the remaining 20% of the total bone mass (Figure 4). Trabecular bone, however, has about ten times the surface area of cortical bone and its porous network harbors blood vessels and marrow. The periosteum is a thin layer of connective tissue on the outer surface of bones. It has nociceptive nerve endings, provides the blood supply to the cortical bone, and contains progenitor cells that are activated during fracture healing (Fiedler et al., 2002). Bone contains two major cell types (Figure 5): osteoblasts are boneforming cells that make osteoid, a protein mixture of type I collagen, proteoglycans, and glycoproteins which mineralizes to become bone; and, osteoclasts are large multi-nucleated cell on the bone surfaces responsible for bone resorption (You et al., 2008). 14

PAGE 29

Figure 4: Structure of bone 15

PAGE 30

Figure 5: Bone cell associations 16

PAGE 31

Bones function to protect internal organs, build the skeleton to support the body, and produce movement in conjunction with muscles. In addition bones act as storage sites for minerals, especially calcium and phosphorus, and contain the hemapoeitic (blood) cell development within the marrow. Bone can also buffer the blood against pH changes by absorbing or releasing alkaline salts and can chelate heavy metals thereby removing them from the blood and minimizing their toxicity on other organs of the body (Mutlu et al., 2007). Several investigators have proposed that alteration in bone metabolism may play a role in the initiation and progression of OA (Botter et al., 2007; Muraoka et al., 2007; Radin, 1999; Sniekers et al., 2008). For example, recent studies looking at collagen synthesis and degradation reported an imbalance in those processes that may cause some of the OA-associated subchondral bone changes including subchondral sclerosis (Bailey et al., 2004; Day et al., 2004). Osteophyte formation at joint margins in OA joints (Boegard et al., 1998) are another indication of bone adaptation associated with OA. Osteophytes develop in joints constrained by a joint capsule lined with synovium (van der Kraan and van den Berg, 2007). Mesenchymal progenitor cells within the periosteum (Figure 4) and the synovial lining (Figure 3) are involved in the development of osteophytes (Shirasawa et al., 2006). Their proliferation and migration followed by chondrogenic differentiation and cartilaginous matrix accumulation at the epiphyseal margins of bone and cartilage have been well described (van der Kraan and van den Berg, 2007). (See Figure 5). Subsequent 17

PAGE 32

chondrocyte hypertrophy and endochondral ossification forms the mature osteophyte. TGF! 1 and insulin-like growth factor (IGF)-1 are both involved in the formation of osteophytes (van der Kraan and van den Berg, 2007; Okazaki et al., 1999), as are growth factors such as BMP-2 and BMP-4, and these are also typically products of macrophages (Blom et al., 2004; van Lent et al., 2004). Articular Cartilage Articular cartilage is a specialized connective tissue composed of an abundant extracellular matrix (ECM) and metabolically active cells, the chondrocytes The chondrocytes in adult articular cartilage are responsible for producing and organizing the extracellular matrix in response to the normal physical-chemical demands generated by usage of the tissue during joint movement (Iannone and Lapadula, 2003). The extracellular matrix of cartilage is composed primarily of collagens II, IX, and XI and the proteoglycan aggrecan. In turn, by forming supramolecular complexes (collagen fibrils and proteoglycan/hyaluronan aggregrates) they provide the tissue with its viscoelastic properties to resist compression and dissipate sheer forces imposed on cartilage surfaces during articular joint usage. Four distinct, histological zones have been identified in human cartilages (Figure 6). The superficial tangential zone is the outer layer facing the synovial cavity. It has the highest collagen content (85% by dry weight) and comprises 1020% of the cartilage thickness. Chondrocytes in this superficial zone are 18

PAGE 33

elongated and collagen fibrils are oriented parallel to the joint surface which may help to resist shear forces (Carter and Wong, 2003). The transitional zone makes up about 60% of the cartilage thickness and contains less collagen (68% by dry weight) but abundant proteoglycan compared to the superficial tangential zone. Collagen fibers are larger in diameter and are more randomly oriented. Residual chondrocytes have a round morphology. The deep zone comprises 30% of the cartilage thickness and has collagen fibers oriented perpendicular to the subchondral bone. Chondrocytes are round and arranged in columns. A calcified cartilage zone is at the interface with the bone, with few chondrocytes. This region also contains the calcified “tidemark” region that functions as a barrier to vascular penetration from the subchondral bone region (Langworthy et al., 2004). Substantial efforts over the past several decades were directed towards understanding the molecular and cellular mechanisms by which the structural components of articular cartilage are irreversibly destroyed and result in progression of OA. Briefly, collagen fibrils are degraded by matrix metalloproteinase collagenases (MMPs) (Davoli et al., 2001; Pratta et al., 2003; Smith, 2006; Tardif, et al., 2004), whereas aggrecan molecules are substrates for ADAMTS proteases (Gao et al., 2004; Nagase and Kashiwagi, 2003; Sandy, 2006; Sandy and Verscharen, 2001; Song et al., 2007). Despite the extensive work on the protease identification, little is known about the temporal and spatial regulation of their activities in normal tissue turnover and OA (Cawston and Wilson, 2006; Sandy, 2006). 19

PAGE 34

ADAMTS metalloproteases cleave aggrecan, a major component of cartilage extracellular matrix in human OA. It remains open which aggrecanase is responsible for aggrecan destruction during articular cartilage degeneration in human OA (Song et al., 2007; Tortorella and Malfait, 2008). However, the generation and use of ADAMTS-5 knockout (KO) mice demonstrate that activity of this enzyme is required for progression of OA-like cartilage lesions in mouse knees surgically destabilized by MCL transection (Glasson et al., 2005) or challenged by antigen-induced arthritis (Stanton et al., 2005). Increased accumulation of ADAMTS-5 in human OA cartilage compared to normal agematched tissues has also been reported and supports a role for this enzyme in the pathogenesis of human OA (Plaas et al., 2007). 20

PAGE 35

Figure 6: Zones of articular cartilage From http://www.chelationtherapyonline.com/articles/p179.htm S = superficial zone; M= middle zone; D = deep zone From: http://www.nomranmarcusmd.com/research.html 21

PAGE 36

Assessment of Cartilage Degeneration Several in vivo techniques have been developed to diagnose and monitor the progression of cartilage degeneration. Radiographs are commonly taken, but they are unable to delineate cartilage directly because of lack of sensitivity to detect soft tissue contrast. They are commonly used to measure joint space width, an indirect assessment of cartilage thickness (Scott et al., 2007). Arthroscopy allows a direct view of cartilage and is considered the most reliable method for cartilage assessment (Lee et al., 2007). However, it is an invasive procedure potentially leading to post-surgical complications of infection and unintended joint injury. In addition, arthroscopy is expensive making it unsuitable as a global evaluative technique (Forssblad et al., 2004). Non-invasive imaging methods include computed tomography (CT), magnetic resonance imaging (MRI), and ultrasonography (US). CT, with and without contrast, produces images in the transverse plane (perpendicular to the direction of weight bearing), but cartilage over the weight bearing area of the knee cannot be directly viewed (Mackay et al., 2006). Current MRI techniques allow measurement of cartilage thickness (Burstein and Gray, 2006). They provide detailed images of tissue structure regardless of the plane by measuring levels of metabolites in cartilage and achieve superior soft tissue contrast compared to CT. MRI is able to detect cartilage abnormalities and meniscal injuries with the same resolution and clarity as arthroscopy (Hyusse and Verstraete, 2007). MRI can also be enhanced by injection of ionic gadolinium into the joint (Roos and Dahlberg, 2005). Healthy cartilage which is rich in negatively charged aggrecan repels the contrast while 22

PAGE 37

degenerating cartilage with substantial aggrecan loss shows signal enhancement because it does not repel the contrast (Roos and Dahlberg, 2005). The usefulness of MRI in detecting and classifying cartilage degradation may also be improved by the development of on-line databases, such as morphological atlases of knee cartilage, and using shape indices as a means of classification (Tameem et al., 2007). Ultrasound correlates significantly with histological grading and continues to be studied to determine if it provides a non-invasive, cost-effective technique for accurately assessing cartilage quality (Lee et al., 2007). Histological Grading of Cartilage in Human OA and in Animal Models Cartilage tissue taken in animal studies or post-surgically in human studies can be graded based on its macroscopic and microscopic appearance. Noted pathological features include surface roughening, fissure development, delamination of the surface, cavity formation, tissue fragmentation, and fibrocartilage growth (Pritzker et al., 2006). A macroscopic classification system grades OA as Grades I-IV based on qualitative descriptions of the texture of the cartilage surface, the size of cartila ge lesions, and changes in the subchondral bone (Collins et al., 1949). A 14-point microscopic grading system using histological stains, such as Safranin O with light green counterstain, is based on cellular changes, presence of Safranin O cartilage matrix staining, and changes such as erosion and vessel penetration. The Osteoarthritis Research Society International (OARSI) system is a 24-point score using six grades (based on 23

PAGE 38

depth of progression into the cartilage) and/or four stages (based on percentage of involvement; Pritzker et al., 2006). India ink wash of the joint surfaces allows further observation of cartilage surfaces and provides a means of quantifying the degradation based on the amount of India ink present (Chang et al., 1997; Kobayashi et al., 2000; Lewis et al., 2005; Stoker et al., 2006). Applying India ink highlights surface abnormalities of the articular cartilage. The India ink carbon particles (diameter 40-100 nm; Madsen et al., 1992) do not enter normal articular cartilage due to its small average pore size of ~6 nm (Maroudas, 1979 ) but does become entrapped in surface irregularities and adhere to the fibrillated cartilage (Meachim et al., 1972) thus appearing as darkly stained areas. Clinical Treatment for Osteoarthritis The current treatment modalities for OA are largely intervention-based, as they address symptoms only. Disease “cures” have not been successfully developed. Therapeutic approaches range fr om psychosocial, pharmacological, and physical therapy interventions to surgical replacements of degenerated joints. Effective long-term management of the disease proposes involvement of a sequential approach, the details of which are based on disease severity at the time of diagnosis. Such treatment strategies ideally begin with information and education at the earliest stages of disease diagnosis. This is followed by self help interventions (including analgesics) then progresses to simple [non-steroidal antiinflammatory (NSAID) medications, corticosteroids, and physical and 24

PAGE 39

occupational therapy] and advanced (injections) non-surgical interventions. Finally, application of surgical procedures may be warranted (Figure 7). These treatment interventions and studies that have evaluated their efficacy are reviewed briefly in the following sections Psychosocial Interventions Psychosocial interventions such as patient education techniques and lifestyle modifications are commonly used in other chronic diseases such as diabetes (Delamater et al., 2001), obesity (Cresco et al., 2007), and cardiovascular (Patel and Adams, 2008) and neurodegenerative (Martial and Donahue, 2006) disorders. Similar approaches have been developed for OA and are utilized in many parts of the world in the treatment of OA. An education self help program was compared with “care as usual” in 273 persons with hip and/or knee OA to determine the effects on pain, other complaints (such as stiffness and loss of movement), and functional limitations. The patient group using the self help program reported decreased pain and improved function when compared to the “care as usual” controls as early as three months after starting. They reported improvements throughout a 21 month follow-up (Heats et al., 2005). Another study investigated the effects of coping skills training on 25

PAGE 40

Figure 7: Sequential pyramidal approach to OA management (From: Dieppe and Lohmander, 2005) 26

PAGE 41

descending modulation of nocioception via the nociceptive flexion reflex (NFR) in persons with knee OA. The NFR results in withdrawing from a noxious stimulus mediated along the A-delta and C fibers which extend to the dorsal horn spinal neurons where the signal is modulated. The NFR threshold is correlated with subjective pain threshold. The intervention significantly increased NFR thresholds and decreased pain ratings (Emery et al., 2006). Educational interventions such as therapeutic education and functional readaptation (TEFR) have also been tested for positive effects on health related quality of life. For example, a study of persons with knee OA on a wait list for total knee replacement comparing TEFR plus pharmacological treatment to pharmacological treatment alone found significant improvements in the Western Ontario and MacMaster’s University Osteoarthritis Index (WOMAC) function score, pain, and physical function as measur ed by the Short Form Health Survey General Questionnaire (SF-36; Nunez et al., 2006). In another study the effects of the Arthritis Self Management Program (Lorig et al., 1985) and the Chronic Disease Self Management Program (Lorig et al., 1999) on quality of life, health behaviors, self efficacy, and health care utilization was assessed. Both intervention programs demonstrated positive outcomes in all measures. However, the arthritis-specific program had better results at earlier time points than did the general chronic disease program (Lorig et al., 2005). Specific lifestyle modifications such as weight loss, have been shown to be efficacious in some persons with OA. One study involving a group of women 27

PAGE 42

with a BMI < 25 showed that neither weight loss nor weight gain affected the risk of knee OA later in life. In a second group of women with BMI > 25, a weight loss of 12 pounds significantly lowered (>50%) the rate of knee OA. Further, in the group with BMI > 25 weight gain was associated with an increased rate of knee OA later in life (Felson, 1990), Similar findings were reported in a study of women who experienced knee pain and were overweight. An average weight loss of 15 pounds using a six month diet and walking intervention resulted in improved knee pain, decreased lower extremity disability, better vO2 maximum, and greater six minute walk distance compared to baseline (Martin et al., 1996). A study of older adults (men and women aged 60 and older) with obesity and symptomatic knee OA reported that diet plus exercise and exercise alone led to decreased knee pain and less disability after the six month interventions (Messier et al., 2004). Furthermore, individuals in the diet plus exercise group lost 15 pounds more than those in the exercise alone group and had better improvements in knee-related disability. Pharmacological Interventions Oral Medications Acetaminophen has been used for decades to treat symptoms of OA. Studies on less than 60 subjects for 6 weeks or less published in the 1980s and 1990s reported acetaminophen was better than placebo for pain relief in persons with OA (Amadio and Cummings, 1983; Zoppi et al., 1995). Due to its excellent safety record, large clinical trials which found a benefit of acetaminophen over 28

PAGE 43

placebo (Miceli-Richard et al., 2004; Pincus et al., 2004), and meta-analyses showing a small effect size (Neame et al., 2004; Zhang et al., 2004), acetaminophen is still recommended as a first line treatment for pain associated with OA. NSAIDS (ibuprofen, naproxen, nabumetone) are generally effective for pain relief in persons with OA. However, they may induce gastrointestinal or other complications (such as electrolyte imbalance, dizziness, and increased blood pressure) associated with long term use (Richy et al, 2004). NSAIDS have been reported to be marginally better than acetaminophen for pain relief (Eccles et al., 1998; Zhang et al., 2004), but little or no difference has been reported between formulations (Gotzsshe 2003; Scott et al., 2003). Given the higher rate of adverse effects associated with NSAIDS, they are added only if acetaminophen does not sufficiently relieve symptoms. To address the issue of gastrointestinal side effects, cyclo-oxygenase 2 (COX-2) selective inhibitors were introduced as a new generation of NSAIDS. While studies demonstrated effectiveness in pain relief, the moderately selective COX inhibitors did not have better gastrointestinal safety (Ju’ni et al., 2002) and the highly selective COX inhibitors led to increased cardiovascular adverse events (Ju’ni et al., 2004). Injectable Agents Pharmacologic agents can also be delivered directly into the affected joint directly via an intra-articular injection. For example, corticosteroid injections for 29

PAGE 44

short term pain relief during “inflammatory flares” of OA have been used to assist with physical therapy (Arroll and Goodyear-Smith, 2004). A meta-analysis of randomized controlled trials showed 1-4 weeks of pain relief after intra-articular injections of corticosteroids into OA knee joints (Arroll and Goodyear-Smith, 2004). However, a poor understanding of the detrimental effects of corticosteroid injections on the metabolic and structural integrity of joint tissues impedes on their usefulness as safe and reliable therapeutics (Divine et al., 2006). A large number of studies have shown that intra-articular injections of HA decrease pain and improve functiona l outcomes in knee OA (Altman and Moskowitz, 1998; Huskisson and Donnelly, 1999; Kotz and Kolarz, 1999; Leardini, et al., 1987; Puhl et al., 1993; Wobig et al., 1998). HA is a high molecular weight polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. It is produced by all cells in the body and secreted into the extracellular space with molecular weight s from 1-7 million Daltons. In articular joints, Type B cells in the synovial lining synthesize and secrete HA into the synovial fluid (Laurent et al., 1996) at a steady state concentration of 2-4 mg/ml (Balazs and Denlinger, 1993). Its high viscoelastic properties provide lubrication during slow joint movements and shock absorption during rapid joint movements (Brandt et al., 2000). Studies in the 1980s reported that HA concentration and size were reduced in synovial fluid collected from persons with OA of the knee (Dahl et al., 1985). Similar findings were reported in human temporomandibular joint disease as well (Takahashi et al., 2004). This led to postulation that intra-articular injection of HA may act to restore the 30

PAGE 45

viscoelasticity and the lubricating functions in an HA-depleted joint (Balazs and Denlinger, 1993). HA can also exert regulatory effects on cells through interaction with a range of HA receptors (CD44, toll-like receptors, L YVE-1, stabilin) (Nehls and Hayen, 2000). Such receptor interactions may lead to downstream inhibition of prostaglandin E2 production (Yasui et al., 1992) and also provide protection against free radical cytotoxicity (Presti and Scott, 1994), altered inflammatory cell adherence, proliferation, migration, and phagocytosis (Ghosh, 1994). The potential disease-modifying effects of intra-articular injections of HA may also work through the cell modifying pathways. Pain relief after three to five weekly, intra-articular injections of HA is reported to last from six months to a year (Altman and Moskowitz, 1998; Arrich et al., 2005; Bellamy et al., 2006; Huskisson and Donnelly, 1999; Kotz and Kolarz, 1999; Leardini, et al., 1987; Lo et al, 2003; Modawal et al., 2005; Puhl et al., 1993; Wang et al., 2004; Wobig et al., 1998) This is longer than its half-life (18-24 hours) within the joint (Kotz and Kolarz, 1999). In vivo studies of human synovial fluid demonstrated decreased numbers of activated macrophages and lymphocytes (Corrado et al., 1995) and decreased synovial effusion (Dougados et al., 1993) with HA injections. HA may therefore have a local or systemic anti-inflammatory effect. Addition of HA to human articular cartilage explants ex vivo showed inhibition of IL-1 stimulated production of MMPs like MMP-1, MMP-3 and MMP-13 (Julovi et al., 2004). Addition of HA to fibroblast-like synoviocytes resulted in decreased expression of 31

PAGE 46

ADAMTS-5 mRNA and this was accompanied by down-regulation of TNF" IL8,and inductible nitric oxide synthase (iNOS) (Wang et al., 2006). It should be noted that HA is also used clinically to prevent post-surgical adhesion development (Urman et al., 1991) in abdominal (Tsai et al., 2005) and ophthalmic (Arshinoff et al., 2002) surgery. In addition, increased concentration of HA in fetal wounds is one of the reasons for scarless wound healing (Mast et al.,1992). The wound healing actions of HA have been attributed to a proinflammatory effect necessary for the granulation, re-epithelialization, and remodeling stages of wound healing. Its cellular effect enhances proinflammatory cell infiltration (Wisniewski et al., 1996), and increases local production of TNF" IL-1 and IL-8 (Kobayashi and Terao, 1997). In addition HA can also augment the granulation phase of healing through enhancing cell migration (Ellis and Schor, 1996) and angiogenesis at the site of tissue damage (Deed et al.,1997). In summary, the symptom-modifying effe ct of intra-articular HA in persons with OA may alter synovial cell proliferation, differentiation, and apoptosis and secondarily inhibit expression of pro-inflammatory signaling molecules and proteases in the joint that eventually lead to matrix destruction, as opposed to repair, in an OA joint (Goldberg and Buckwalter, 2005). 32

PAGE 47

Physical Therapy Interventions Physical therapy for the treatment of OA aims to correct joint biomechanics and provide symptom relief via orthotics and bracing, modalities, and exercise. The overall goal is to maximize functional capabilities while minimizing pain and trauma to soft tissues, bone, and cartilage surfaces (American Physical Therapy Association, 2003). These interventions are rarely used in isolation, but instead are used in conjunction with psychosocial and pharmacologic interventions. Orthotics and Bracing Orthotics and braces can change joint biomechanics and redistribute joint forces. The most frequently used orthotic in OA treatment is a lateral wedge which places the ankle and foot in less supination resulting in decreased stress on the medial compartment of the knee. Outcomes from studies using orthotic interventions vary depending on whether or not there was standardization of shoes in which the orthotics were inserted. Using lateral wedge insoles in a twoyear study with persons with knee OA led to neither symptomatic (change in WOMAC scores, need for injections or lavage, or self-assessed activity) nor structural (joint space width) improvements, but a decreased number of days of NSAID usage was reported (Pharm et al., 2004). A four week intervention of lateral wedge insoles and standardized shoes gave significant improvements in WOMAC pain, stiffness, and function scores (Fang et al., 2006). A third study using an eight week intervention with elastic subtalar strapping reported 33

PAGE 48

radiographically detected decreases in femoritibial angle. Significant improvement in pain during bed rest with full knee extension, upon getting up from a seated position, and after getting up, as well as increased maximum distance walked, and aggregate severity score were also reported (Toda and Segal, 2002). Knee bracing has also been utilized in an attempt to correct joint biomechanics at the knee and thereby reduc e unusual forces at this joint. Indeed, knee valgus bracing improved disease-specif ic quality of life and function (Scott et al., 2003), and taping the patella in a more medial orientation also resulted in reduced knee pain (Cushnaghan et al.1994). Modalities Physical therapy modalities for the treatment of OA are used to decrease pain and inflammation (Brousseau et al., 2004). They include thermal modalities and electrical stimulation. Thermal modalities are primarily in the form of hot packs, cold packs, and ice massage. While a single application of ice packs placed on the anterior and posterior knee did not significantly affect edema, ten sessions of cold pack application resulted in a significant effect (Hecht, 1983). Hot pack application did not have this effect. Twenty minutes of ice massage five times a week for two weeks was also found to decrease pain and improve knee extension strength, range of motion, and time to walk 50 feet (Yurtkuran, 1999). 34

PAGE 49

Electrical stimulation elicits muscle contraction via the placement of electrodes on the skin (American Physical Therapy Association, 2003). This can assist in muscle strengthening as well as improving blood flow to the treated area. It has been suggested that the presenc e of the electrical current can attract and repel the positively and negatively charged ions in the underlying tissues and these may regulate a variety of cellular responses including inflammation and pain pathways (Kloth, 2005). Pulsed electrical stimulation has been shown to improve patient and physician global evaluation and patient reported pain and reduce NSAID use by > 50% (Farr et al., 2006; Garland et al., 2007). Neuromuscular electrical stimulation has also been shown to decrease pain in persons with knee OA (Gaines, et al., 2004). Exercise Therapeutic exercise is any activity designed to improve joint and/or muscle performance (American Physical Therapy Association, 2003). It includes aerobic/cardiovascular exercise, range of motion and flexibility, resistance training, and functional exercise. Exercise has long been used as a treatment intervention for persons with OA as it decreases joint pain and stiffness and increases functional abilities (Ettinger et al., 1997). Appendix 3 summarizes OArelated exercise studies and their reported outcomes. 35

PAGE 50

Aerobic Exercise Aerobic exercises include walking programs or stationary bicycling, are performed for at least 20 minutes at a time, and involve active movement that increases the heart rate. While aerobic exercise utilizes muscle action, muscle strengthening is not the primary objective. Aerobic exercise is generally performed to improve fitness level by increasing the efficiency of the heart and lungs with increasing activity. However, aerobic exercise also results in movement of the joints, loading of the bones, and muscle use, all of which may impact knee OA. A large number of studies using aerobic exercise for persons with knee OA are reported. They all show significant improvements in a variety of functional, psychosocial, and pain m easures. These measures include timed chair rise, six minute walk test, (Mangione et al., 1999), medication use, walking distance, function as measured by the Arthritis Impact Measurement Scale (AIMS) physical activity subscale (Kovar et al., 1992), 50 feet walking time, depression, anxiety, physical activity (Minor et al., 1989), walking speed, disability scores (Penninx et al., 2002; Ettinger et al., 1997), depressive symptoms as measured by the Center for Epidemiological Studies—Depression Scale (Penninx et al., 2002), self-efficacy for stair climbing (Rejeski et al., 1998), aerobic capacity (Mangione et al., 1999; Minor et al., 1989), and pain (Ettinger et al., 1997; Kovar et al., 1992; Mangione et al., 1999; Penninx et al., 2002). 36

PAGE 51

Aerobic exercise did not increase daily reported pain, indicating this repetitive lower extremity exercise does not exacerbate pain symptoms (Mangione et al., 1999). Moreover, one study suggested that continuation of aerobic exercise is necessary in order to maintain benefits as cessation lead to outcome reports that were not were not significantly different from the nonexercising control group (Sullivan et al., 1998). Indeed, several professional panels and consensus groups advocate aerobic exercise for persons with OA. The American Geriatric Society (AGS) Panel on Exercise and Osteoarthritis recommends aerobic exercise three to four times a week for 20-30 minutes a day at a low to moderate intensity which is further defined as 50-75% of the maximum heart rate or a rating of perceived exertion (RPE) of 10-13 (AGS Panel on Exercise and Osteoarthritis, 2001). The American College of Rheumatology (ACR) workgroup recommends 30 minutes of moderate intensity aerobic exercise (50-70% of maximum heart rate) at least three times a week (Altman et al., 2000). The MOVE consensus and the European League Against Rheumatism (EULAR) also recommend the inclusion of aerobic exercise for persons with OA, but do not make any recommendations on intensity, frequency, or volume (Jordan et al., 2003; Roddy et al., 2005). According to Bennell and Hinman, strengthening exercises appear superior to aerobic exercise for persons with knee OA in improving physical symptoms, such as pain, whereas aerobic exercise may be more effective for functional outcomes over the longer term (Bennell and Hinman, 2005). However, a systematic review of 13 randomized controlled trials found no difference 37

PAGE 52

between aerobic and strengthening exercise programs in their ability to reduce pain and disability in the same population (Roddy et al., 2006). Since aerobic exercise has components of the other types of exercise (joint motion as in range of motion/flexibility exercise, strengthening to some degree as in resistance training, and activities of daily living as in functional exercise) investigating the benefits of aerobic exercise may provide a broad picture of the effect exercise can have on OA. Range of Motion/Flexibility Exercises Range of motion and flexibility exercises involve movement of extremities without resistance as well as stretching exercises aimed at increasing muscle length and flexibility to increase the range of motion within a joint. There are no studies specifically evaluating the effect of range of motion/flexibility exercises on persons with OA. Instead, these exercises are typically combined with other types of exercise (aerobic, resistance, and functional) for individuals with OA (Minor et al., 1989) Resistance Exercises Resistance exercises for OA involve moving a body part against an external force. This force ranges from bodyweight to low intensity (less than 20% of the maximum load a person can move through the full arc of movement) to high intensity (up to 85% of the maximum load a person can move through the 38

PAGE 53

full arc of movement). Examples of such exercises are tightening the quadriceps muscle with the knee extended, standing up from a chair, using a knee extension or leg curl machine, and extending the knee in a seated position with weights around the ankle are all examples of resistance exercise. The amount of resistance is quantified by determining the maximum force an individual can generate or maximum load an individual can move during the exercise. Resistance exercises strengthen the muscles around an affected joint and thereby may improve muscle balance and joint biomechanics lessening stress on the joint tissues. In addition, resistance exercises result in joint movement and bone loading which may impact knee OA. Resistance exercises have shown positive benefits for persons with OA in the following outcome categories: increased strength (Ettinger et al., 1997; Eyigor et al., 2004; Gur et al., 2002; Penninx et al., 2002; Schilke et al., 1996; Topp et al., 2002; Topp et al., 2005), decreased pain (Ettinger et al., 1997; Eyigor et al., 2004; Gur et al., 2002; Penninx et al., 2002; Schilke et al., 1996; Topp et al., 2002), decreased stiffness, and arthritis activity as measured by the Osteoarthritis Severity Index (OASI) and the AIMS (Schilke et al., 1996), improved function (Ettinger et al., 1997; Eyigor et al., 2004; Gur et al., 2002; Penninx et al., 2002; Schilke et al., 1996; Topp et al., 2002; Topp et al., 2005 ), less disability, decreased depressive symptoms (Ettinger et al., 1997; Penninx et al., 2002), decreased disease severity, and improved WOMAC, SF-36, and AIMS-2 scores (Eyigor et al., 2004). 39

PAGE 54

Improving muscle strength, especially of the quadriceps, may be very important in preventing disease progression. Slemenda et al., found that women with stronger quadriceps had a reduced risk of developing radiographic evidence of knee OA as compared to those with weaker quadriceps (Slemenda et al., 1998). This finding was confirmed by Hootman et al, who looked at 3081 community dwelling adults without OA and found that women with moderate to high isokinetic quadriceps strength had a 55% reduced risk of developing hip OA and a 64% reduced risk of developing knee OA (Hootman et al., 2004). A strong quadriceps muscle may provide joint protection and decrease abnormal wear on the cartilage surfaces. Increased hip abduction strength is also associated with decreased knee OA progression (Chang et al., 2004). Hip abductors originate on the pelvis above the hip joint and attach on the lateral leg just below the knee. As a result, they have a stabilizing effect on the pelvis and influence toe out angle during gait minimizing abnormal stress on the knee. Conversely, increased hip adduction strength is associated with increased disease progression and severity in the knee (Yamada et al., 2001) indicating that perhaps this muscle group should not be subject to strengthening exercises in persons with OA. Although lower extremity muscle strengthening is beneficial in most persons with OA, the benefits of simply improving strength may be compromised by the enhanced quadriceps strength which in itself has been associated with increased disease progression in persons with malaligned or lax knees (Sharma 40

PAGE 55

et al., 2003). Thus a careful screening of patients prior to strengthening exercise and appropriate modification of exercises is recommended to prevent joint trauma as a result of malalignment or laxity. While some studies have been conducted to determine the most efficacious type of resistance training (isometric, isotonic, isokinetic), this area is still largely unstudied (Eyigor et al., 2004; Gur et al., 2002; Topp et al., 2002). In addition, evidence does not exist regarding the most appropriate load at which to perform resistance exercise in order to get the best gains for persons with OA. Water-Based Exercise Studies comparing water-based exercise to land-based exercise find functional gains achieved with both interventions. However, persons in the waterbased program had better improvement in pain (Cochrane et al., 2005), walking distance and physical component on SF-12 (Foley et al., 2003) and persons in the land-based program had better improvements in strength, walking speed, and self-efficacy (Foley et al., 2003). Functional Exercise Functional exercises include activities, such as getting up from a chair and climbing stairs. If resistance is added to the activities, they are considered resistance exercises as in the case where an individual wears a weighted belt or vest while performing sit to stand or stair climbing. If the duration of the activity is sustained, it is considered aerobic exercise, such as climbing a flight of stairs five times. 41

PAGE 56

Their use has been found to significantly improve times for performing activities of daily living, isokinetic quadriceps muscle strength, WOMAC scores, stair climbing, walking time, (Diracoglu e al., 2005; Lin et al., 2005) and proprioceptive sensation (Diracoglu e al., 2005). Mode of Action of Exercise Despite increasing consensus and evidence for exercise as an effective intervention in OA progression, the underlying physiologic mechanisms have not been determined. This is most likely due to the complex and pleiotropic effects of such a therapy on the physiology of the organism. A commonly held view is that exercise improves joint biomechanics by restoring muscular balance and strengthening supporting structures around the joint, resulting in diminished stress and structural trauma to the cartilage. In vivo biomechanical studies showed that exercise might influence the integrity of muscle tissue. Thus, muscular balance and strengthening has been shown to diminish stress and structural trauma to the soft tissues (Hootman et al., 2004; Slemenda et al., 1998). In vitro studies using tissue explants and isolated cells have revealed mechano-sensitive pathways, such as mi togen activated protein kinase and gene expression pathways for both degenerative and reparative actions of chondrocytes (Fitzgerald et al., 2006; Lee et al., 2005; Sah et al., 1989). In vitro studies of mechanical strain on chondrocytes show that low magnitude 42

PAGE 57

mechanical strain on cartilage suppresses IL-1 and TNF alpha (both proinflammatory cytokines) and up regulates proteoglycan and collagen synthesis. However, high magnitude mechanical strain on cartilage is pro-inflammatory, initiating cartilage destruction and inhibiting matrix synthesis. (Deschner et al., 2003) This suggests a dose-response relationship between exercise and collagen synthesis in humans as well. However, what constitutes low magnitude and high magnitude mechanical strain with exercise is not clear, nor is the difference in response in humans with s hear forces alone versus compressive forces alone versus a combination of forces. The extent to which these are mechanistically related to events in the whole joint and in combination with soluble mediators (cytokines and growth factors) remains to be fully defined. Moreover, it is not entirely understood how the complex function of joint movement translates into specific types of biophysical signals such as compression, shear, fluid flow and streaming potentials (Fitzgerald et al., 2006; Gray et al., 1989; Kerin et al., 2002; Kim et al., 1995; Lee et al., 2005; Sah et al., 1989). The development of a simplified in vivo animal model of mechanical overuse and detailed cell and biochemical examination of the ensuing cartilage and bone response will aid greatly to this area of research. The availability of high resolution imaging on tissue structures, such as delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC), an in vivo technique that relies on the principle of the negatively charged contrast agent distributing inversely to the negatively charged 43

PAGE 58

glycosaminoglycans (GAGs) in the cartilage shows promising approaches to evaluate the effect of exercise on structural and biochemical changes in joint tissues such as cartilage (Link et al., 2006; Roos and Dahlberg, 2005; Tiderius et al., 2004). Roos et al. utilized dGEMRIC to investigate the effect of moderate exercise on knee cartilage quality and found that performing aerobic and weight bearing strengthening exercises three times a week for 4 months resulted in a significant improvement in cartilage quality as demonstrated by increased GAG content. This correlated with change in the Knee Osteoarthritis Outcomes Survey (KOOS) indicating an improvement in clinical status as well (Roos et al., 2005). Physical exercise may also affect inflammation and the immune response. A study of 274 persons with knee OA participating in an exercise and nutrition program looked at serum levels of inflammatory markers including IL-6, CRP, TNF" and the soluble receptors (sR) IL-6sR, IL-2sR, TNF-sR1 and TNFsR2. Higher serum levels of TNF-sR1 and TNF-sR2 (soluble receptors for a proinflammatory cytokine) were associated with lower physical function (Penninx et al., 2004). Further, these persons had more pain and stiffness and more reported physical disability as well as slower walking speeds and worse radiographic scores. Isometric exercises may induce a beneficial inflammatory response in the joint tissue. For example, studies have shown that isometric exercises result in increased molecular weight of hyaluronan, increased viscosity of joint fluid, and decreased chondroitin sulfate concentra tion (Miyaguchi et al., 2003). Further, a 44

PAGE 59

rise in serum HA, a marker of acute inflammation (Elliott et al., 2005), was noted with physical activity in persons with OA (Criscione et al., 2005). Cell Based Therapies Cartilage lesions have little capacity to spontaneously heal (Buckwalter and Mankin, 1997). As a result, cell-based approaches have been developed to assist in repairing cartilage defects. With these approaches, cells are manipulated in vitro then implanted in the cartilage defect (Richter, 2007). Mesenchymal stem cells have been used fo r this purpose because they can be expanded by in vitro culture and still maintain their multilineage potential (DeBari, 2006). Cartilage cells from autologous cartilage elsewhere in the body have also been removed, cultured, then implanted into the damaged area (Brittberg et al., 1994). This type of therapy may be enhanced by use of resorbable biomaterials for scaffolds into which chondrocytes are seeded and cultured, prior to implantation (Marlovits, et al., 2006). These methods have undergone extensive pre-clinical investigations using animal models of cartilage disease and defect, but are now being used in humans. Surgical Interventions Surgical interventions are generally implemented only after conservative measures have been unsuccessful (See Figure 2, Dieppe and Lohmander, 2005). They include both joint preserving (arthroscopy and osteotomy) and joint replacement techniques. 45

PAGE 60

A commonly utilized surgical procedure for mild to moderate OA is arthroscopy. This technique can range from simple lavage and debridement to cartilage defect repair attempts and cartilage transplantation. Effectiveness of arthroscopic intervention is controversial (Chang et al., 1993; Gross et al., 1991; McEldowney and Weeker, 1995; McLaren et al., 1991; Moseley et al., 2002). If OA is contained to one side of the joint, a correction osteotomy is an option in order to transfer load-bearing from the a ffected to the non-affected region of the knee. A Cochrane review of 13 studies involving 693 persons concluded that osteotomy improves knee function and pain, but there is no evidence to support that osteotomy is more effective than conservative treatment (Brouwer et al., 2007). Joint replacement techniques remove the pathological tissues and replace them with prostheses. While there are no randomized controlled trials comparing joint replacement to sham treatment or standard care, there are individual studies on sham interventions demonstrating a m oderate effect on pain (Hrobjartsson and Gotzsche, 2001) and studies of joint replacement demonstrating a large effect on pain (Ashworth et al., 2002; Fortin et al., 2002). 46

PAGE 61

Animal Model Research Overview of Animal Models Mechanistic studies of human disease progression and therapeutic efficacies have utilized animal models since the 1950s. Human OA is generally not diagnosed until it has progressed with pronounced pathological alterations in the joint leading to pain and radiographic changes. Further, obtaining diseased tissue prior to end-stage joint replacement surgery without potentially damaging the joint is not possible, and non-invasive methods of assessing changes in joint structure (i.e., MRI, radiographs) do not provide enough information on cellular and metabolic dynamics in the diseased joint. For these reasons, animal models of OA have been developed to provide a known cause of induction and time points of disease progression (Bendele, 2001). Since injury is a common predisposing factor, most models involve surgically-induced insults in order to cause mechanical stress to the joint. Models have been developed in sheep (Cake et al., 2004), dogs (Mastberger et al., 2006), rabbits (Inouye et al., 1973), guinea pigs (Bendele et al., 1991), rats (Hayami et al., 2006; Laurent et al., 2006; Moore et al., 2005), and mice (Bendele, 2001). Direct comparison with age-matched, healthy tissue taken from animals of the same or similar genetic and environmental backgrounds can be readily obtained for radiographic, histologic, and biochemical evaluation methods. More recently murine (mouse) models have gained popularity with benefits of lower cost, homogeneity of genetic background, ability to inbreed, and 47

PAGE 62

the capacity to obtain an increasing number of genetically modified strains. Creating surgically-induced injuries in mice has been difficult due to the small size of the joints and therefore requires microsurgical techniques (Kamekura et al., 2005). Murine Models Spontaneous OA Age-onset development of OA occurs in STR, DBA/1, and C57 Black mouse strains. These “spontaneous” models may reveal mechanisms that underlie some forms of human OA (Yamamoto et al., 2005). The highest incidence of this type of OA occurs in the STR/1N strain (Jay, 1951) and the related STR/ort strain (Sokoloff and Jay, 1956). Cartilage lesions are detected histologically as early as 8 weeks of age and appear at the insertion of the medial collateral ligament and the medial tibial plateau (Mason et al., 2001). Males have a higher incidence than females, and cartilage loss is most prevalent in the medial compartments of the knee joint. Interestingly, spontaneous obesity develops at three mont hs of age in these mice, even when kept on a standard diet. Peak bone mass is reached at 12 weeks compared to 16 to 24 weeks in non-affected strains. Other investigators using this mouse strain have reported high incidence of patella subluxation (Walton, 1979), decreased anterior cruciate ligament strength, soft tissue calcification (Evans et al., 1994) and increased biochemical turnover of collagen in cartilage (measured by gel zymography) all of which may 48

PAGE 63

contribute to the spontaneous OA-like pat hologies in these mice (AndersonMackenzie et al., 1999). Histological evaluation of knee joints from STR/ort strain using TUNEL assays showed an increasing number of apoptotic cells in cartilage covering medial tibial and femoral bone with advanced histological lesions (Mistry et al., 2004). This finding may be consistent with preceding biomechanical or metabolic stress on the cartilage (Mason et al., 2001). In addition, cartilage of STR/ort mice has been reported to be deficient in extracellular superoxide dismutase, a reactive oxygen species (ROS) scavenger (Regan et al., 2005). This may predispose to oxidative damage to cells and accelerated apoptosis. C57 Black mice are also susceptible to developing OA lesions in knee joints as a result of a genetic mutation with recessive Mendelian inheritance (Silberberg and Silberberg, 1960). Incidence and severity of OA based on histopathological cartilage damage assessed by hematoxy llin and eosin stain and decreased toludine blue staining (Yamamoto et al., 2005) increases with age in both males and females from 20% at 2 months to 80% at 16 months Cartilage erosion appeared in the subchondral layers of cartilage Decreased collagen content and fibril alignment as well as decreased proteoglycan contents accompanied the cartilage destruction (Yamamoto et al., 2005). The development of OA at relatively predictable time points would render these “spontaneous” models appropriate for the evaluation of therapeutic interventions for disease progression. Ho wever, they have not been widely used 49

PAGE 64

mainly due to the need to house and maintain these animals for up to a year which increases the cost of experimentation. Surgically-Induced OA Post-trauma models of OA models are typically induced surgically to create joint instability and have been applied in large animals, such as sheep (Cake et al., 2004), dogs (Mastberger et al., 2006), rabbits (Inouye et al., 1973), guinea pigs (Bendele et al., 1991), and rats (Hayami et al., 2006; Laurent et al., 2006; Moore et al., 2005). The mouse presents a challenge for surgically-induced OA because of its small size (Figure 9a) However, microsurgical techniques for meniscal and ligament destabilizations have been successfully utilized to induce OA-like joint changes in the knee (Kamekura et al., 2005). MCL transection and/or complete or partial meniscectomy are the most commonly used techniques, but other ligaments including the PCL, LCL, or ACL have also been transected (Kamekura et al., 2005). For example, MCL transection and partial medial menisectomy lead to development of mild cartilage lesions (fibrillations and loss of superficial flattened cell layer) on the medial plateau in ~50% of treated mice by 2 weeks post surgery, and by 3-4 weeks all mice with this injury had developed OA-like changes (Clements et al., 2003). The various surgical procedures are now typically classified on the severity and direction of instability: a) severe, after transection of all ligaments, including the patellar ligament, and removal of both menisci; b) moderate, after 50

PAGE 65

ACL transection and medial menisectomy; c) mild, after ACL transaction; and d) medial, after MCL transection and medial menisectomy (Kamekura et al., 2005). While many of these changes may be consistent with joint changes seen in humans after ligament and/or meniscal injury, the medial model most closely resembles changes seen in the majority of persons with OA. However, the severe, moderate, and mild models may be helpful in investigating the development and progression of certain subtypes of OA. Surgical models have been widely used by the pharmaceutical industry for pre-clinical studies on disease modifying OA drug development because of the rapid, reproducible changes that result. However, several limitations need to be considered. Firstly, OA is not always the result of significant trauma to the joint. While some patients with OA can pinpoint specific injuries in which ligaments or menisci were injured, many cannot. Hence OA may often be the result of cumulative microtrauma which is not well represented by surgically-induced models. Secondly, in surgically-induced models, cartilage damage occurs in a relatively short time (1-2 weeks post surgery), whereas progression of human OA occurs over the course of years. In addition, when humans sustain such joint injuries it is rare for them to continue loading the joint. Most undergo surgical repair in an attempt to restore joint stability. In instances where stability cannot be successfully restored, activity is severely limited and therefore, joint loading is minimized. Thirdly, surgical models have only limited use for evaluation of therapies because the amount of trauma induced in the absence of surgical repair does not allow “normal” joint loading and leads to continued joint damage 51

PAGE 66

even in the presence of interventions. Fourthly, surgeries can easily result in direct damage to other joint structures, such as synovium and cartilage, at the time of surgery that may initiate additional wound healing pathways (including scarring) that are not normally part of the human OA pathology. Fifthly, the biomechanics of joint loading in the mouse is quite different from that in the human and may not provide accurate information regarding the effects of joint instability. Manipulation of Cartilage Specific Genes Genetic models provide insight into possible causative factors and generate strains that consistently develop OA (Clements et al., 2003; de Hooge et al., 2005; Giancotti and Rusoslahti, 1999; Saamanen et al., 2000; Xu et al., 2003; Zemmyo et al., 2003). Mutations known to make humans susceptible to OA involve collagens II (Vikkula et al., 1994), IX (Olsen, 1997), and XI (Jacenko and Olsen, 1995). As a result, many of the genetic models focus around these candidate genes. Spontaneous single nucleotide deletion in the ColXIa1 gene ( 1 chain of type XI collagen) in the cho/+ strain results in thickened collagen fibrils in the cartilage (Xu et al., 2003) and leads to destruction of cartilage and biochemical alterations, such as increased chondrocyte metabolism and increased proteoglycan degradation, similar to human OA (Tetlow et al., 2001). Del 1 (+/-) strain has a deletion mutation in the transgene coding for type II collagen (Saamanen et al., 2000). These mice produce shortened pro-alpha 1 52

PAGE 67

chains of collagen II and develop superficial fibrillation of cartilage at 12 weeks of age. This progresses to cartilage erosion, meniscal degeneration, bony sclerosis, and exposure of subchondral bone (Saamanen et al., 2007). Deletion of an alpha 1 integrin, a transmembrane protein that binds components of the extracellular matrix and may be involved in cell activation, differentiation, proliferation, and survival (Giancotti and Rusoslahti, 1999) results in synovial hyperplasia, increased cellularity and apoptotic cells in the cartilage, and finally severe cartilage degeneration (Zemmyo et al., 2003). The primary benefit of the genetic models is the known molecular etiology and the controlled severity and incidence of OA pathogenesis. However, limitations exist. These mice can be very difficult to breed and may need specialized housing, both increasing the cost of experiment. Further, it is not known if the gene is directly involved in OA or whether developmental changes due to the deletion predisposes an individual animal to OA. For example, many of these models develop skeletal deformations such as chondroplasias. These models are not readily amenable to treatment interventions unless conditional knock-outs are generated that display the gene abnormality only during adulthood or upon induction of the disease. In addition disease progression may not always mimic that which occurs in human disease because of a more selective activation of pathogenetic pathways by genetic alterations and limited representation of the many facets of OA development and progression (Figure 1) may result. 53

PAGE 68

Chemical OA-like changes can also be induced in joints by injection of noxious agents such as, quinolone antibiotics (Christ et al., 1988), iodoacetate (Guingamp et al., 1997; Janusz et al., 2001), collagenase (Blom et al., 2004) and pro-inflammatory agents such as TGF! 1(Van Beuningen et al., 2000). Quinolone, an antibiotic that targets bacterial topoisomerase, causes articular cartilage degeneration in juvenile animals. These changes occur rapidly (within 24 hours) and include focal swelling, chondrocyte death, and blister-like lesions in the mid zone cartilage (Bendele, 2001). With time the cartilage surrounding these lesions also deteriorates, with fibrillation and proteoglycan loss similar to that seen in other models of OA (Bendele, 2001). While this model has been used in guinea pigs (Bendele et al., 1990) and dogs (Gough et al., 1992), it is rarely used because it affects only growth cartilage, but not articular cartilage, and its mechanism is not well understood (Simonin et al., 1999). Bacterial collagenase induces osteophyte development at the periarticular margins followed by fibrosis and an influx of macrophages into the synovial lining. This model is representative of some of the early changes seen within OA, but does not reflect pathology seen in late stages of OA other than osteophyte formation (Blom et al., 2004). Iodoacetate causes cartilage thinning and fibrillation (Janusz et al., 2001) as well as osteophyte development (Guingamp et al., 1997), but also produces hyperalgesia and allodynia within one week of injection (Fernihough et al., 2004). Because of the pain presentation, this method is primarily used to study pain. 54

PAGE 69

The pain symptoms produced can be controlled by oral administration of Naproxen, rofecoxib, and acetaminophen which are also used for pain relief in persons with OA (Bove et al., 2003). Intra-articular injections of TGF! 1, a multifunctional modifier of skeletal tissue growth (Heino et al., 2002; Yan et al., 2001), was first reported by Van Beuningan et al. to induce OA-like changes in mouse knees. After multiple (up to three) injections into the knee joint on alternate days, they reported osteophyte formation (Van Beuningen et al., 2000) and influx of macrophage-like cells in the synovial lining, which are important producers of cytokines and growth factors regulating mesenchymal cell activities (Rappolee and Werb, 1992). Recent studies using this model investigated the role of macrophages in osteophyte formation (van Lent et al., 2004) and the role of interleukin-1 in OA development (Blamey Davidson et al., 2005). The primary benefit of this model is that OA-like changes can be initiated within the joint in a very short period of time (less than a week in some instances). Joint anatomy is maintained making it highly suitable to evaluating therapeutic intervention effects. As is the case for the surgical models, the injection models require specialized, technical expertise to perform the targeted injections. The injection (needle) may damage tissues within the joint cavity leading to additional injury responses and substances may not be quantitat ively delivered to the joint space. 55

PAGE 70

Animal Models and Therapeutic Hyaluronan Injections The effects of intra-articular injections of HA on OA have not been well studied in mice. However, some work has been done in this area on other animal models. Rabbits receiving five weekly HA injections one month after anterior cruciate ligament transection to induce OA demonstrated less severe cartilage damage than the non-HA injected controls (Amyl et al., 2003; Takahashi et al., 1999). In addition, a down-regulation of proteolysis enzymes such as MMP-3 and cytokines, such as IL-1 in the synovium was reported (Takahashi et al., 1999). This process appears to be partially mediated by CD44, a cell surface receptor for HA (Waddell et al., 2007). In another report, rabbits with anterior cruciate ligament transection that had five or ten weekly HA injections showed less disease progression. This could be improved by increasing the therapy to ten weekly injections (Amyl et al., 2003). After bilateral partial medial meniscectomies in rabbits, the application of five weekly HA injections resulted in significantly lower nitric oxide production and decreased cell apoptosis in the central region of the meniscus (Kobayashi et al., 2002; Takahashi et al., 2000) as well as increased collagen remodeling in the peripheral region (Sonada et al., 2000) Animal Models and Treadmill Exercise There are limited studies using aerobic exercise with mice (Table 1). Although treadmill running has not been utilized in mice with OA it has been used 56

PAGE 71

in non-OA mouse models. In a study of the effect of short term moderate exercise on tumor metastases C57Bl/6 mice ran for one hour a day for 6 days at a speed of 36 meters per minute (Murphy et al., 2004). G93A transgenic mice with a mutation causing the development of amyotrophic lateral sclerosis ran 3045 minutes per day three to five times a week for eight weeks at speeds of 15-37 cm per second (Mahoney et al., 2004). The same mouse strain was used to demonstrate that regular exercise was associated with an increased lifespan after running the mice 30 minutes per day, five days a week at a speed of 21 cm per second for 10 weeks (Kirkinezos et al., 2003). Lightfoot et al. studied aerobic capacity variations in ten inbred strains by running mice at 36-72 cm per second until the mice were unable to continue (Lightfoot et al., 2001). C57Bl mice ran an average of eight minutes on testing which is significantly lower than four other strains and places them on the lower end of the spectrum of aerobic capacities in the strains tested (Lightfoot et al., 2001). Massett and Berk also investigated exercise training response in six inbred and hybrid mouse strains by running them at speeds of 25-32 cm per minute one hour per day, five days per week for four weeks (Massett and Berk, 2005). Subsequently exercise performance was assessed by running at speeds of 14-62 cm/sec until exhaustion. Endurance exercise performance of C57Bl mice was similar to Balb/c but significantly lower than other strains with only small improvements in endurance exerci se performance (22-33% increase in distance) occurring after four weeks of training (Massett and Berk, 2005). 57

PAGE 72

Finally, critical running speed (the speed that can be sustained for 40-60 minutes) was studied in three mouse strains by running at 30-85 cm per second until exhaustion (Billat et al., 2005). For C57Bl mice, critical speed was determined to be 20 cm per sec. Maximum aerobic speed has been determined for mice and although it has been shown to vary slightly between mouse strains (Billat et al., 2005; Massett and Berk, 2005) it can be used to determine maximum oxygen uptake (vO2 max), the maximum capacity to transport and utilize oxygen during incremental exercise (Baker and Gleeson, 1999). A percentage of vO2 max is the best method utilized in prescribing exercise with 40-60% vO2 max coinciding with low intensity, 60-80% coinciding with moderate intensity, and greater than 80% coinciding with high intensity (Swain et al., 2004). Using the formula vO2 = 3.99 + 1.94v where v is the speed, low intensity aerobic exercise for the mouse at 50% vO2 is achieved at approximately 14 cm/sec (Baker and Gleeson, 1999). The effect of treadmill exercise on OA in mouse models has not been studied. Only voluntary wheel running in normal and collagen IIa1 knockout mice was reported (Lapvetalainen et al., 2002). Running did not affect OA development in normal mice but reduced incidence of spontaneous OA in the knockouts (Lapvetalainen et al., 2002). 58

PAGE 73

Table 1: Studies investigating the effects of aerobic exercise in mice Study Strain Mode of Exercise Effect Investigated Outcome Lapvetalainen et al., 2002 Col 2a1 KO Voluntary wheel running OA development Reduced OA development Murphy et al., 2004 C57Bl/6 Treadmill running Lung tumor metastases Decreased metastases Mahoney et al., 2004 G93A Treadmill running ALS onset and survival with high intensity endurance training No affect on onset of symptoms; faster decrease in motor performance and death in male mice only Kirkinezos et al., 2003 G93A Treadmill running ALS lifespan with moderate intensity endurance training Increased lifespan; males greater than females Lightfoot et al., 2001 10 strains of inbred mice Treadmill running Interstrain variation in aerobic capacity Strong genetic contribution to aerobic capacity in mice—Balb/c mice had 3 times greater aerobic capacity than C57/Bl6 mice Massett and Berk, 2005 C57BlackTreadmill running Exercise training response 22-33% increase in distance after 4 weeks of training Billat et al., 2005 C57BlackTreadmill running Critical running speed Critical running speed is 20 cm/sec; v02 max is approximately 8.0 ml O2/gh 59

PAGE 74

Chapter Two Specific Aims and Research Hypotheses OA is a debilitating condition affecting over 21 million persons in the United States. This number is expected to rise in the coming decades. Current treatment approaches for OA focus on symptom modifying measures (i.e., pain relief) as disease modifying interventions do not currently exist. However, some of the interventions used to relieve and improve the symptoms of OA may actually have disease-modifying benefits. Two such non-surgical interventions for OA are intra-articular HA injections and exercise. In order to effectively study the mechanistic aspects of such treatment options, an animal model of OA that is amenable for studying such intervention outcomes is needed. Mouse models of OA have become increasingly popular tools for this type of research due to rapidity, ease of disease assessment, costeffectiveness, and availability of targeted gene knock-in and knock-out strains. The overall aims of this dissertation project were to: a) develop a rapidly progressing mouse model of knee OA that does not need surgical intervention or noxious chemical agents; and, b) test the response of the model to diseasemodifying interventions such as HA injections or exercise. The data obtained from this study will provide novel information to refine and implement such interventions in studies using larger animals and human patients. They can also 60

PAGE 75

be incorporated into educational materials for patient education in psychosocial and physical therapy interventions. Research Hypotheses 1. Comprehensive OA-like changes in muri ne knee joints (synovitis, soft tissue fibrosis, osteophyte formation and cartilage and meniscus degeneration) can be induced by anabolic stimulation of joint tissues by intra-articular TGF! 1 followed by mechanical overuse of the activated joint. 2. Cartilage degeneration in the above model can be inhibited by therapeutic intervention during the anabolic stimulation phase, using intra-articular HA injections. 3. Cartilage degeneration in the above model can be decreased by four weeks of alternate day, low intensity aerobic exercise preceding the anabolic stimulation phase. Specific Aims The research was conducted under three specific aims: Aim 1 Develop and characterize a progressive non-surgical model of knee OA in adult mice. C57Bl/10 male mice were subjected to two alternate day intraarticular injections of TGF! 1 into one knee joint, followed by high intensity treadmill running at 32 cm/sec for 13 days. Joint pathology was evaluated by radiographs, India ink cartilage grading, and histopathology. The validity of the 61

PAGE 76

OA model was established by demonstrating resistance of ADAMTS 5-/mice to cartilage degeneration. Aim 2 Examine the capacity of intra-articular HA injection to inhibit progression of knee joint degeneration in the mouse model established under Specific Aim 1. High molecular weight HA (SupartzTM, Clinical Grade) was injected intraarticularly during the anabolic /inflammatory phase of the model. Metabolic turnover routes of injected HA in the joint were examined by in vivo imaging of fluorescently labeled HA in the knee joint and by quantitative ELISA of HA in plasma. Joint pathology was evaluated by radiographs, India ink cartilage grading, and histopathology. Aim 3. Examine the capacity of low intensity aerobic exercise to prevent onset and progression of joint degeneration in the mouse model established under Specific Aim 1. C57Bl/10 male mice underwent four weeks of alternate day, low intensity treadmill running at 14 cm/sec prior to inducing OA by TGF! 1 injection and mechanical overuse through high intensity treadmill running. Joint pathology was evaluated by radiographs and India ink cartilage grading. Each specific aim and corresponding research hypothesis will be addressed in separate chapters following. The research design, materials, methods, results and analyses, and discussion and conclusions for the development of the model will be presented in chapter 3, the HA intervention in chapter 4, and the aerobic exercise intervention in chapter 5. 62

PAGE 77

Chapter Three Development of Mouse Model of Knee OA All in vivo experimental protocols described below have been approved by the IACUC of Rush University, Medical Center, under Protocol 07-009. Research Design (Figure 8 and Table 2) Experimental groups : 1.1 Cage Control Day 5 (n=14): no TGF! 1 injections, cage activity, sacrificed day 5 2.1 Cage + TGF! 1 Day 5 (n=19): TGF! 1injections, cage activity, sacrificed day 5 3.1 Cage + Sham Day 5 (n=19): BSA injections in lieu of TGF! 1 injections, cage activity, sacrificed day 5 4.1 Cage Control Day 18 (n=18): no TGF! 1 injections, cage activity, sacrificed day 18 5.1 Cage + TGF! 1 Day 18 (n=23): TGF! 1 injections, cage activity, sacrificed day 18 63

PAGE 78

Figure 8: Research design for the development of a non-surgical mouse model of OA Day 5 Day 18 Overuse through high intensity treadmill running • Speed = 32 cm/sec • Duration = 20 minutes • Frequency = daily x 13 days Mechanical Overuse TGF1Injection Right Leg (2x 48 h interval) Day 1 Day 3 Day 5 TG F1Injection Right Leg (2x 48 h interval) Day 1 Day 3 Sacrifice Sacrifice SacrificeCage Day 18 A B 64

PAGE 79

6.1 Cage + Sham Day 18 (n=19): BSA injections in lieu of TGF! 1 injections, cage activity, sacrificed day 18 7.1 Mechanical Overuse Day 18 (n=20): no TGF! 1 injections, mechanical overuse via high intensity treadmill running, sacrificed day 18 8.1 Mechanical Overuse + TGF! 1 Day 18 (n=19): TGF! 1 injections, mechanical overuse via high intensity treadmill running, sacrificed day 18 9.1 Mechanical Overuse + Sham Day 18 (n=18): BSA injections in lieu of TGF! 1 injections, mechanical overuse via high intensity treadmill running, sacrificed day 18 In addition to C57Bl/10 mice, 129SvEvBrd Agg 2-/mice (ADAMTS-5 Knockouts) and their controls (129S6/SvEvTac) were used to determine if ADAMTS-5 plays a role in the cartilage changes. Only males 12-16 weeks of age were used. 65

PAGE 80

Table 2: Mouse utilization for the induction of OA-like changes StrainC57Bl/10 n=169 Agg 2-/n=36 129S6/SvEv n=42 EvaluationH/EIndia ink and FACE H/EIndia ink and FACE H/EIndia ink and FACE Cage Control Day 5 8 6 2 0 2 0 Cage + TGF! 1 Day 5 13 6 2 0 2 0 Cage + Sham Day 5 13 6 0 0 0 0 Cage Control Day 18 12 6 2 6 3 6 Cage + TGF! 1 Day 18 17 6 2 6 3 6 Cage + Sham Day 18 13 6 0 0 0 0 Mechanical Overuse Day 18 14 6 2 6 4 6 Mechanical Overuse + TGF! 1 Day 18 13 6 2 6 4 6 Mechanical Overuse + Sham Day 18 12 6 0 0 0 0 66

PAGE 81

Materials TGF! 1 (PeproTech, Inc., Rocky Hill NJ); Bovine Serum Albumin (Sigma Aldrich, St. Louis MO); India ink (Speedball Arts Product Co., Statesville NC); ketamine (Phoenix Pharmaceutical Inc., St. Joseph MO); xylazine (Phoenix Pharmaceutical Inc., St. Joseph MO); injection needles (Tyco Healthcare Group, Mansfield MA); 5 mm and 3 mm micro dissection spring scissors (Roboz Surgical Instrument Co. Inc., Gaithersburg MD); Human TGF! 1 ELISA kit (Bender Med Systems, Burlingame CA); eppendorf tubes (DOT Scientific, Inc., Burton MI); heparin (Becton Dickinson Vacutainer Systems, Rutherford NJ); hematoxylin (Richard Allan Scientific, Kalamazoo MI); eosin (Richard Allan Scientific, Kalamazoo MI); HA ELISA materials were obtained and prepared as described (Li et al., 1989; Rayan et al., 1998; Thonar et al., 1992); FACE chemicals were obtained and prepared as described (Plaas et al., 2001). General purpose, two-lane mouse treadmill with single belt construction and dividing wall outfitted with an electrical stimulus system composed of two shock grids with stimulus intensity (163 V at a range of 0-1.5 mA, for a max of 0.5 seconds per shock) and monitored by LED lamps to indicate which stimulus grid is active (Stoelting, Inc., Wood Dale IL). The manufacturer’s design of the electrical stimulus grid reflects special attention to avoid injuries to animals. 67

PAGE 82

Methods Mouse Breeding and Husbandry The following strains of mice were used for experimentation: C57Bl/10 male mice procured from Jackson Laboratories or National Cancer Institute at 711 weeks of age and housed until they were 12 weeks old; 129S6/SvEvTac breeding pairs from Taconic; 129SvEvBrd Agg 2-/(ADAMTS 5-/exon 2 back crossed into the C57B./6 strain) breeding pairs obtained from Charles River Laboratories through Dr. Micky Tortorella and Dr. Anne-Marie Malfait (Pfizer, Inc.). Twelve week old 129S6/SvEvTac and 129SvEvBrd Agg 2-/(ADAMTS 5-/-) mice used for experiments were obtained through an in-house breeding program. All mice were housed in polycarbonate shoebox rodent cage (five mice per cage) with corncob pelleted bedding and were given free access to food (Harlan Tekland Global Protein Rodent Diet) via wire bar lid and acidified water via water bottle and/or automated watering system. Animals were monitored for health on a daily basis and received cage changes every two weeks. For procedures requiring restraint, mice were held by the scruff of the neck with thumb and forefinger and the tail was secured between the fourth and fifth fingers. Intraarticular injections and blood collections were performed under general anesthesia consisted of a ketamine/xylazine cocktail (100mg/kg ketamine + 10 mg/kg xylazine) in sterile PBS delivered intraperit oneally via a 28G x inch needle on 0.5 ml syringe. Upon termination of the protocol mice were humanely euthanatized via carbon dioxide followed by cervical dislocation to confirm death. 68

PAGE 83

Intra-Articular TGF! 1 Injections Twelve-week old adult male mice were anesthetized as described above, right legs shaved with enough border to prevent fur from contaminating the injection site and the knee joint area rinsed with dilute iodine solution. TGF! 1 (PeproTech, Inc., Rocky Hill, NJ) (200 ng in 6 l of sterile PBS/0.1% purified BSA) or 6 l BSA solution al one was injected with a 28G x inch needle through the patellar ligament into intra-articular space of the knee joint (Figure 9). Animals were allowed to recover and then transferred back to their home cage. Injections were repeated after a 48 hour interval to achieve repeatedly high levels of this factor in the joint. 69

PAGE 84

Figure 9: Site of needle entry for intra-articular injection of the knee between the femur and tibia just below the patella through the patella ligament and into the joint capsule. a) Mouse shown actual size; and b) right knee joint enlarged to show a more detailed injection site a) b) 70

PAGE 85

Blood Collection and Plasma Preparation Blood (5-7 drops for in vivo time points and terminal bleeds at time of sacrifice) was collected from the submandibular vein at baseline using a sterile 18G needle, two days post first TGF! 1injection (day 3), two day post second TGF! 1 injection (day 5), one week post second TGF! 1injection (day 12), and 14 days post second TGF! injection (day 18). Prior to all blood draws anesthesia was administered as described previously. Blood was collected into heparinized, sterile Eppendorf tubes, cells removed by centrifugation at 13,000 rpm for ten minutes at room temperat ure, plasma supernatants removed and stored at -80 C until further analyses. TGF! 1 Enzyme Linked Immunosorbance Assay (ELISA) Twelve-week old adult male mice were divided into control and treatment groups. The treatment group (n = 6) received TGF! 1 injections as described above. The control group (n =6) received intra-articular injections of BSA. Blood was drawn daily and prepared as described previously. Mouse TGF! 1 ELISA and human TGF! 1 ELISA was performed according to manufacturer’s directions (Bender Med Systems, Burlingame CA). ELISA detection of circulation human TGF! 1 in the mouse was negative and consistent with the injected protein being utilized within the joint and/or cleared by the lymphatic system. Interestingly, plasma levels of 71

PAGE 86

endogenous (mouse) TGF! 1 were elevated following each intra-articular TGF! 1 injection (Figure 10). In comparison, intra-articular injection of BSA resulted in a temporary decrease in endogenous plasma TGF! 1. 72

PAGE 87

Figure 10: Endogenous (mouse) TGF! 1 concentrations for mice injected with human TGF! 1 and mice injected with BSA as measured by mouse TGF! 1 ELISA and expressed as a percentage of predose. Each time point represents two assays. 0 20 40 60 80 100 120 140 160Pre-dose1 Day post 1st injection 2 Days post 1st injection 1 Day post 2nd injectionPercentage of Pre-doseTime +TGFbeta +BSA 73

PAGE 88

Mechanical Overuse Through High Intensity Treadmill Running Treadmill running was initiated one day after the second TGF! 1 injection (Figure 11). The mice were familiarized with running on the treadmill for two days at speeds of 24-32 cm per second for 20 minutes per day. Strenuous treadmill running was started on day three and continued for 20 minutes per day for 13 days. Running speed was calculated as follows: using the formula vO2max = 3.99 + 1.94v where v is the speed (km/hour) and vO2max for C57Bl mice is 8.0 ml O2/ghour, high intensity aerobic exercise for the mouse at approximately 80% vO2 is achieved at a speed of 32 cm/second (Baker and Gleeson, 1999; Billat et al, 2005). A daily record was kept for each animal that included: duration of the run (minutes), distance run (calculated from the run time and speed of the treadmill), and number and time of interactions with the shock grid. In addition, treadmill performance was measured by the amount of time each mouse stayed on the front half of the running treadmill (Figure 11). Mice readily adapted to the daily treadmill running. However, if an animal refused to run continuously during the initial familiarization period, or up to 2 consecutive days during therapeutic exercise, it was excluded from further studies. No signs of pain or distress even at the early inflammatory stage after TGF! 1 injection were noted, excluding the need for analgesics. 74

PAGE 89

Figure 11: Mechanical overuse through high intensity treadmill running 75

PAGE 90

The C57Bl/10 mice ran 381.7 meters per day on average and the 129SvEvBrd Agg2-/mice ran 379.2 meters per day on average. A difference was noted between C57Bl/10 TGF! 1 injected and non-injected mice in the time spent on the front half of the treadmill. Initially both groups spent an average of 19.5 minutes on the front half of the treadmill. The non-TGF! 1 injected mice continued to spend an average of at least 18 minutes on the front half of the treadmill throughout the 13 day period. However, by day 5 of running (day 9 of the protocol) the TGF! 1 injected mice spent an average of 18 minutes or less, and by day 17 they spent an average of 14.6 minutes. This difference between TGF! 1 injected and non-injected mice was not apparent in the 129SvEvBrd Agg2-/(ADAMTS 5 KO) strain. However, the knockout mice did not run as well as the C57Bl/10 mice. The 129SvEvBrd Agg2-/(ADAMTS 5 KO) mice came in contact with the shock panels more frequently and remained in contact with the shock panels for longer periods of time. The most notable difference was in the time spent on the front half of the treadmill. The C57Bl/10 mice ran on the front half of the treadmill (away from the shock panel) an average of 19.5 minutes while the 129SvEvBrd Agg2-/(ADAMTS 5 KO) mice ran on the front half of the treadmill an average of 0.7 minutes. In addition, the 129SvEvBrd Agg2-/(ADAMTS 5 KO) mice developed abrasions on their feet as a result frequent contact with the shock panel. These abrasions were treated with silvadene cream. 76

PAGE 91

Tissue Harvesting Hind limbs were harvested from euthanatized animals for histopathological evaluation via transection of the mid shaft of the femur. Fur, skin, and excess muscle tissue was removed, limbs placed in saline soaked gauze, and stored at 20 C evaluation as below. Radiography Animals were sacrificed on day 5 and day 18 and placed on a transparent platform with lower extremities in full knee extension in an anterior/posterior view and in a 90 flexed position in a medial/lateral view. Radiographs of the lower body were taken using the KODAK Molecular Image Station-In Vivo FX with Kodak Molecular Imaging Software with settings of 60 second x-ray exposures, no filters, f-stop 5.8, and a field of view of 40. Imaging allowed visualization of osteophyte formation and bony alignment. India Ink Cartilage Surface Evaluation Animals were sacrificed on day 5 and day 18, hind limbs harvested as described above, and tissues stored in saline soaked gauze at-20 degrees C. Prior to India ink staining joints were dissected under a Nikon SMZ1000 microscope as follows: 1) the patella ligament was transected and diagonal cuts along medial and lateral joint lines were made to allow the patella to be pulled superiorly away from the joint; 2) medial and lateral collateral ligaments were then transected allowing joint distraction and transection of the cruciate 77

PAGE 92

ligaments; 3) remaining joint capsule was cut to allow full separation of the tibia and femur; 4) residual synovial tissue and menisci were removed from tibial plateaus and femoral condyles to allow full exposure of the cartilage surfaces. Cartilage surfaces were then rinsed with PBS and photographed with a Nikon SMZ 1000 camera on a dissecting microscope and SPOT Basic software version 3.5.9 for Windows. India ink was applied to femoral condyles and tibial plateaus with a small brush and allowed to set for two minutes. Unbound ink was removed with saline washes (up to 30 seconds) and gently dabbing with a cotton-tipped applicator. Marked cartilage surfaces were re-photographed. Images were viewed in Adobe Photoshop for scoring (Figure 12). Femoral and tibial surfaces were divided into four quadrants each and cartilage damage assessed in each quadrant by the following grading system (Kobayashi et al., 2000). 0 = Grade 1 (intact surface): surface normal in appearance and does not retain ink 1 = Grade 2 (minimal fibrillation): surface appeared normal before staining, but the India ink showed fibrillation 2 = Grade 3 (overt erosion): surface fibrillation was apparent before staining and areas retained ink as intense black patches 3 = Grade 4 (ulceration): loss of cartilage exposes underlying bone Thus for each tibial or femoral surface a scoring range of 0-12 can be determined with higher scores denoting increased cartilage damage. 78

PAGE 93

Cartilage scores for all four quadrants were analyzed individually then summed to obtain the overall cartilage score for each cartilage surface (right femur, left femur, right tibia, and left tibia). In addition, quadrants 1 and 2 were summed for the anterior surface score, quadrants 3 and 4 for the posterior score. Finally quadrants 1 and 3 were summed for the lateral cartilage score and quadrants 2 and 4 for the medial cartilage score (Figure 13). 79

PAGE 94

Figure 12: Example grading of femoral and tibial cartilage surfaces after India ink staining 80

PAGE 95

Figure 13: Cartilage surfaces were divided into four quadrants. Anterior cartilage = quadrant 1 + quadrant 2; posterior cartilage = quadrant 3 + quadrant 4; lateral cartilage = quadrant 1 + quadrant 3; medial cartilage = quadrant 2 + quadrant 4 81

PAGE 96

Hematoxyllin/Eosin Histopathology Hind limbs were dissected at the mid-femur, skin and muscle were removed prior to fixation in 10% neutral buffered formalin for 48 hours, and specimens were decalcified for 2 weeks in 5% EDTA (in phosphate buffered saline, pH 7.0), prior to processing and paraffin embedding as described (Plaas et al., 2007). Sections (6 m thin) were cut, stained with hematoxyllin and eosin (H/E) using the following sequence of steps: two deparaffinization steps in xylene (2 minutes each), rehydration through graded alcohols (100% for 2 minutes, 95% for 2 minutes, DI water for 2 minutes), hematoxyllin for 5 minute washes (DI water for 30 seconds, Scott solution for 30 seconds, DI water for 30 seconds, 1% acid alcohol wash 15 seconds, 95% EtOH for 2 minutes), eosin for 5 minute washes (95% EtOH for 2 minutes, 95% EtOH for 2 minutes, 100% EtOH for 2 minutes, 100% EtOH for 2 minutes), dehydrated through xylene and cover slipped in mounting medium. Stained sections were viewed with an Olympus BH-2 microscope, images captured via a CCD camera, and analyzed by Adobe Photoshop software. This method provides a view of the tissue morphology highlighting cellularity and tissue types. Data were analyzed observationally and used for interand intragroup comparisons. 82

PAGE 97

Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Analyses FACE is a sensitive, specific and rapid method for the detection of carbohydrates including glycosaminoglycans (GAGs) such as chondroitin sulfate and hyaluronan, both of which are abundant in cartilage tissue (Calabro et al., 2001; Plaas et al., 2001) The amount of each saccharide in the starting mixture is reflected by the fluorescence intensity of the resulting band on the gel (Plaas et al., 2001). GAGs are synthesized as polymers of repeating disaccharides covalently bound to core proteins to form proteoglycans. When GAG chains are cleaved, a free reducing group that can be fluoro-tagged is created. These fluorotagged products are separated on polyacrylamide gels and fluorescent images recorded by CCD camera (Calabro et al., 2001). Tibial and femoral cartilages were removed at the epiphyseal junction and GAGs solubilized by digestion for four hours at 60 C in 200 ul of 0.1 M sodium acetate, pH 7.0 containing proteinase K. The digest was cleared by centrifugation, the supernatant heated at 100 C for ten minutes to inactivate the proteinase. Salt and peptides were removed from the GAGs by centrifugation on MicroCon3 filters, and then digested with 5 mUnits of chondroitinase ABC in 200 ul of ammonium acetate pH 7.4 at 37 C for 18 hours. Buffer salts were removed by speedvac evaporation, the dried disa ccharide products derivatized with 2aminoacridone/cyanoborohydride separated by electrophoresis on 20% polyacrylamide gels and fluorescent bands imaged and quantitated as described (Plaas et al., 2001). 83

PAGE 98

Data Evaluation: Statistical Parameters Statistical analyses were performed using Statistical Package for Social Sciences (SPSS) version 15.0 for Windows. Data was analyzed for descriptive statistics (means, medians, standard deviations) and fixed effect one way analysis of variance (ANOVA) with Tukey post hoc testing and level of significance at p value less than 0.05. Group comparisons were made for overall cartilage scores, total scores for each cartilage surface (right femur, left femur, right tibia, and left tibia), anterior, posterior, medial and lateral areas of each cartilage surface, and each quadrant (anteromedial, anterolateral, posteromedial, and posterolateral) on each cartilage surface. Power analysis based on the 4 point grading system and with the assumption based on preliminary studies of a between group mean difference of at least one point revealed a sample size of 3 in control groups and 6 in treatment groups per time point sufficient for power > 80%. In addition Repeated Measures ANOVA with Tukey post hoc testing and level of significance at p value less than 0.05 was used to analyze data on treadmill performance data. The time each mouse spent on the front half of the treadmill each day was expressed as a percentage of the total time and data were analyzed for descriptive statistics and differences within and between groups and within and between days. 84

PAGE 99

Results and Analyses Effect of TGF! 1 Injection and Mechanical Overuse on Bone Structure Radiographic assessment of right knee joints in anterior/posterior and medial/lateral views failed to detect any gross changes in bone remodeling with TGF! 1 alone or in combination with mechanical overuse through high intensity treadmill running (Figure 14). Furthermore, the mechanical overuse did not result in generation of bone fractures that could contribute to the pathological joint degeneration. 85

PAGE 100

Figure 14: Anterior/posterior and medial/lateral radiographs of right knees at day 18 86

PAGE 101

Anabolic Stimulation of Joint Tissues by Intra-articular Injection of TGF! 1 Transection of the anterior cruciate ligament in animal models is the most commonly used injury that results in biochemical, biomechanical and morphological changes in the articular cartilage, resembling OA pathogenesis in the human knee. The joint pathologies in these models is comprised of an anabolic (hypertrophic) phase followed by a catabolic (degenerative) phase (Adams and Brandt, 1991, Adams, 1989; Adams et al., 1983). There is a progressive increase in the amount of articular cartilage and meniscal tissue hypertrophy in the unstable knee, indicative of an active synthetic response by resident cells. This together with formation of osteophytes and capsular fibrosis precedes the cartilage degeneration phas e of the disease (Adams and Brandt, 1991, Adams, 1989; Adams et al., 1983). Van Beunigen et al. reported similar pathologies in soft tissues of mouse knee joints following multiple(4-6), consecutive intra-articular injections of TGF! 1 into murine knee joints (Van Beuningen et al., 2000). Similarly, we observed that even after two consecutive injections of TGF! 1, extensive synovial hyperplasia as well as meniscal and articular cartilage hypertrophy developed (Figure 15). In particular, the synovial lining in nave mice was about 1-3 cells deep and cell densities were increased dramatically by TGF! 1. In addition, cellular infiltration and granulation tissue formation in the subintimal layers of injected knees was apparent. 87

PAGE 102

Figure 15: Histopathological evaluation of right knee medial compartments and synovial lining for C57Bl/10 mice day 5 following H/E staining. (A) 4x magnification of saggital sections (medial compartment); (B) 20 x (left hand panels) and 40x (right hand panels) of synovial lining showing extensive hyperplasia induced by TGF! 1. SF = synovial fluid; A = adipocyte (fat cell) Cage Controls, and (b) Cage + TGF! 1 day 5 (A) (B) Cage Control Cage + TGF! 1 SFSF SF SF A Hyperplasia Hyperplasia Hyperplasia 88

PAGE 103

Synovial Lining Fibrosis and Cartilage Degeneration at Two Weeks Post TGF! 1 Injection Mice maintained for two weeks after the second TGF! 1 injection either at cage activity or with mechanical overuse through high intensity treadmill running, developed advanced OA pathologies, cartilage lesions, and osteophyte development (Figure 16). Synovial hyperplasia was still present, but less pronounced compared to acute TGF! 1 treatment. In addition, fibrosis developed in the menisci [Figure 16 a (iv) and b (iv)] and femoral joint margins [Figure 16 b (ii)]. These findings were not apparent in any of the control groups 89

PAGE 104

Figure 16: Histopathological evaluation of right knee medial compartments and synovial lining for C57Bl/10 mice day 18 following H/E staining. Cage +TGF! 1 Day 18:(a) 4x magnification, whole joint saggital section; (b) 20x magnification cartilage degeneration on femoral condyle; (c) 20x magnification cartilage degeneration on tibial plateau; (d) 20x magnification of medial meniscus Mechanical overuse +TGF! 1 Day 18:(e) 4x magnification, whole joint saggital section; (f) 20x magnification synovial fibrosis; (g) 20x magnification cartilage degeneration on tibial plateau; (h) 20x magnification of medial meniscus a b c d e f g h Cage + TGF! 1 Mechanical Overuse + TGF! 1 Lesion 90

PAGE 105

India Ink Scoring of Cartilage Surfaces In keeping with the histological evaluation, India ink staining after intraarticular injections of TGF! 1 followed by 14 days of cage activity or treadmill running showed the development of cartilage degeneration (lesions and fibrillation/fibrotic changes) on both the right femoral cartilage surfaces (Table 3) and the right tibial cartilage surfaces (Table 4). For all 3 control groups (noninjected, BSA injected, cage, or tr eadmill running only) cartilage surfaces scored <1. Minimal fibrillation was seen in some, but not all animals. By comparison, for the group of mice that received intra-articular TGF1 and were maintained with cage activity, scores on anterior surfaces only were significantly higher than for the control group—posterior surfaces did not differ significantly (Figure 17). Mice receiving TGF! 1 injections and mechanical overuse averaged scores of > 2.00 in all four quadrants on femurs and > 1.83 in all four quadrants on tibias. These results confirm previously published data (Van Beunigen et al., 2000) that cartilage fibrillation and erosion developed after prolonged administration of intra-articular TGF! 1 Notably, exposure to treadmill running after intra-articular TGF1 resulted in significantly more cartilage damage on all anterior surfaces of femurs and tibias than cage activity after injection and gave increased India ink scores in these regions (Figures 17-18). Damage to cartilage surfaces in contralateral (left) legs after TGF1 injection was also noted at day 5 and day 18 with both cage activity and mechanical overuse (Figures 19-21) and may indicate the involvement of systemic or neuronal (pain) pathways during joint disease development. 91

PAGE 106

Table 3: Mean cartilage scores for right femurs by quadrant (standard deviations in parentheses) denotes statistically significant difference from non-TGFinjected groups at p< 0.05; ** denotes statistically significant difference from non-TGFinjected groups at p< 0.01; *** denotes statistically significant difference from non-TGFinjected groups at p< 0.005 AnteroMedial Quadrant AnteroLateral Quadrant PosteroMedial Quadrant PosteroLateral Quadrant Cage Control Day 5 0.00 (0.00) 0.67 (0.58) 0.00 (0.00) 0.67 (0.58) Cage + Sham Day 5 0.00 (0.00) 0.50 (0.71) 1.00 (0.00) 1.00 (0.00) Cage + TGF! 1 Day 5 1.80* (0.45) 1.40* (0.55) 1.60 (0.55) 1.80 (0.45) Cage Control Day 18 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.67 (0.58) Cage + Sham Day 18 0.00 (0.00) 0.00 (0.00) 1.00 (0.00) 1.50 (0.71) Cage + TGF! 1 Day 18 2.00* (0.89) 2.50*** (0.55) 1.33 (0.52) 2.00 (0.62) Mechanical Overuse Day 18 0.00 (0.00) 0.00 (0.00) 0.33 (0.58) 1.00 (1.00) Mechanical Overuse + Sham Day 18 0.00 (0.00) 0.00 (0.00) 0.67 (0.58) 0.67 (1.16) Mechanical Overuse + TGF! 1 Day 18 2.00* (0.89) 2.50*** (0.55) 2.33* (0.82) 2.67** (0.52) 92

PAGE 107

Table 4: Mean cartilage scores for right tibias by quadrant (standard deviations in parentheses) AnteroMedial Quadrant AnteroLateral Quadrant PosteroMedial Quadrant PosteroLateral Quadrant Cage Control Day 5 0.67 (0.58) 0.33 (0.58) 0.00 (0.00) 0.00 (0.00) Cage + Sham Day 5 0.00 (0.00) 0.00 (0.00) 0.50 (0.71) 0.00 (0.00) Cage + TGF! 1 Day 5 1.00 (0.71) 0.40 (0.55) 0.80 (0.45) 1.00 (0.71) Cage Control Day 18 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) Cage + Sham Day 18 0.50 (0.71) 0.50 (0.71) 0.50 (0.71) 0.00 (0.00) Cage + TGF! 1 Day 18 2.00* (0.00) 1.83** (0.41) 1.33 (0.82) 1.33 (0.52) Mechanical Overuse Day 18 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.33 (0.58) Mechanical Overuse + Sham Day 18 0.00 (0.00) 0.00 (0.00) 0.33 (0.58) 0.67 (0.58) Mechanical Overuse + TGF! 1 Day 18 2.33*** (0.52) 2.33*** (0.52) 1.83** (0.75) 2.50*** (0.55) denotes statistically significant difference from non-TGFinjected groups at p< 0.05; ** denotes statistically significant difference from non-TGFinjected groups at p< 0.01; *** denotes statistically significant difference from non-TGFinjected groups at p< 0.005 93

PAGE 108

Figure 17: Mean cartilage scores for right femurs by quadrant. 1 = control; 2 = +TGF! 1 injection; 3 = + sham injection: 0 depicts no cartilage damage denotes statistically significant difference from non-TGFinjected groups at p< 0.05; ** denotes statistically significant difference from non-TGFinjected groups at p< 0.01; *** denotes statistically significant difference from non-TGFinjected groups at p< 0.005 Cage Day 5 Cage Day 18 Mechanical Overuse Day 18 Antero-Medial Antero-Lateral Postero-Medial Postero-Lateral * * ** * ** * 0 94

PAGE 109

Figure 18: Mean cartilage scores for right tibias by quadrant. 1 = control; 2 = +TGF! 1 injection; 3 = + sham injection; 0 depicts no cartilage damage denotes statistically significant difference from non-TGFinjected groups at p< 0.05; ** denotes statistically significant difference from non-TGFinjected groups at p< 0.01; *** denotes statistically significant difference from non-TGFinjected groups at p< 0.005 Mechanical Overuse Day 18 Cage Day 18 Cage Day 5 Antero-Medial Antero-Lateral Postero-Medial Postero-Lateral * *** ** * ** 0 95

PAGE 110

Figure 19: Mean cartilage scores for left femurs by quadrant. 1 = control; 2 = +TGF! 1 injection; 3 = + sham injection; 0 depicts no cartilage damage denotes statistically significant difference from non-TGFinjected groups at p< 0.05; ** denotes statistically significant difference from non-TGFinjected groups at p< 0.01 Antero-Medial Mechanical Overuse Day 18 Cage Day 5 Cage Day 18 Antero-Lateral Postero-Lateral Postero-Medial * 96

PAGE 111

Figure 20: Mean cartilage scores for left tibias by quadrant. 1 = control; 2 = +TGF! 1 injection; 3 = + sham injection; 0 depicts no cartilage damage denotes statistically significant difference from non-TGFinjected groups at p< 0.05 Mechanical Overuse Day 18 Cage Day 18 Cage Day 5 Antero-Medial Antero-Lateral Postero-Medial Postero-Lateral * * 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 97

PAGE 112

Figure 21: Mean cartilage scores by group for right and left knee cartilage surfaces. 1 = control; 2 = +TGF! 1 injection; 3 = + sham injection: 0 depicts no cartilage damage denotes statistically significant difference from non-TGFinjected groups at p< 0.05; ** denotes statistically significant difference from non-TGFinjected groups at p< 0.005; *** denotes statistically significant difference from non-TGFinjected groups at p< 0.0001 Cage Day 5 Mechanical Overuse Day 18 Cage Day 18 Right Knee Surfaces Left Knee Surfaces * ** ** ** * 98

PAGE 113

FACE Analysis of Cartilage Chondroitin Sulfate Content To confirm the usefulness of India ink scoring as a reliable means of assessing loss of murine knee joint cartilage, the total chondroitin sulfate (CS) contents of dissected femoral and tibial cartilages was determined using FACE (Figure 22). Total chondroitin sulfate content was 6.7 + 1.2 g per joint in nave, untreated animals. This was decreased to 5.6 + 1.5 g per joint in TGF! 1 treated joints with cage activity and to 3.4 + 2.2 g after TGF! 1 and mechanical overuse. Mechanical overuse alone for 14 days showed a slight increase in CS content per joint to 8.2 + 1.8 g compared with untreated cage control joints. 99

PAGE 114

Figure 22: Chondroitin Sulfate content of right knee cartilage as determined by FACE analyses 100

PAGE 115

Effect of TGF! 1 Injections and Mechanical Overuse on ADAMTS-5 KO Mice Cartilage degeneration in surgically induced models of OA is dependent on the activity of ADAMTS 5 protease in joint tissue. To determine if such a mechanism also operates in the progression of cartilage damage observed in this model, ADAMTS 5 KO mice underwent TGF! 1 injection and mechanical overuse through high intensity treadmill running. Knockout mice showed no damage to either tibial plateau or femoral condyle cartilage when injected with TGF! 1 without or with subsequent mechanical overuse. Thus, India ink scores remained low, similar to those in nave wildtype mice. Histological assessment confirmed lack of cartilage degeneration as well as synovial fibrosis (Figure 23). However, synovial hyperplasia was present as seen with the C57Bl/10 mice at day 5 (Figure 23). Treadmill Performance While both the number of shocks encountered during the daily run and the amount of time spent on the front half of the treadmill were recorded for the mice, the number of shocks encountered was not a reliable measure of performance as the shock panel became soiled at times resulting in inaccurate recording of the number of shocks. Mice in the TGF! 1 injected group spent significantly less time on the front half of the treadmill beginning at day 5 and progressively deteriorating through day 13 as compared to those mice not treated with TGF! 1 (Figure 24). 101

PAGE 116

Figure 23: Histopathological evaluation of right knee medial compartments and synovial lining for ADAMTS 5 KO mice following H/E staining. (A) 4x magnification of saggital sections (medial compartment); (B) 20 x (left hand panels) and 40x (right hand panels) of synovial lining. SF = synovial fluid; Cage + TGF! 1 Day 5 Mechanical Overuse + TGF! 1 Day 18 Cage Controls (A) (B) SF SF SF SFHyperplasia Hyperplasia Hyperplasia 102

PAGE 117

Figure 24: Percentage of time spent on front half of treadmill days 3 through 13 for mice in mechanical overuse and mechanical overuse + TGF! 1 groups 103

PAGE 118

Discussion and Conclusions Recommendations from the Osteoarthritis Research Society for designing of clinical trials to investigate disease-modifying activity state the primary outcome should be a measure of joint structure or morphology—studies making use of surrogate markers of cartilage destruction as an outcome are considered helpful, but not sufficient alone (Altman et al., 1996). The two intra-articular injections of TGF! 1 resulted in meniscal and synovial hyperplasia and fibrotic changes on cartilage surfaces and joint capsule. These alterations in tissue cellularity and structure persisted up to 14 days and led to variable formation of chondro/osteophytes at the bone/cartilage margins, and to extensive fibrotic remodeling of the synovial lining, the meniscal attachment regions, and the cartilage surfaces (Figures 15 and 16). Most notably, tissue remodeling in the vicinity of meniscal and cartilage fibrosis resulted in the OA-like pathologies in the TGF! 1 treated joints. This is in keeping with and extending the previously published data by others (Bakker et al., 2001; Blaney Davidson et al., 2007; van Beuningen et al., 2000). Additional exposure of TGF! 1 treated joints to controlled high intensity treadmill running representing a biomechanical overuse resulted in more severe cartilage pathology. Particularly, cartilage surfaces showed structural irregularities that were more extensive and covered a greater percentage of the surfaces (Figures 17-18). Individual lesions in certain regions, particularly the medial tibial plateau were deeper and exposed subchondral bone. In addition, the mechanical overuse induced damage to ca rtilages on the anterior surfaces of 104

PAGE 119

the TGF! 1 activated joint is consistent with the involvement of a biomechanical factor in the development of the cartilage degeneration. Altogether, the pathologies seen in this non-surgical model bear a closer resemblance to the human disease pathogenesis and pathology than the surgically or chemically induced mouse models of OA (Blom et al., 2004; Christ et al., 1988; Clements et al., 2003; Guingamp et al., 1997; Janusz et al., 2001; Kamekura et al., 2005). Interestingly, increased cartilage damage was also noted on the cartilage surfaces of the contralateral knee in mice injected with TGF! 1 in both cage and mechanical overuse groups. The degeneration was less severe than in the injected joint, as assessed by India ink, but may point to involvement of systemic factors in the initiation and progression of the joint tissue degeneration. Indeed, involvement of contralateral and/or other joints has been frequently reported in the human disease as well (Mazzuca et al., 2003; Spector et al., 1994). In keeping with the notion that exercise alone does not play a part in the pathogenesis of OA (Ettinger et al., 1997; Hootman et al., 2004; Roos et al., 2005; Slemenda et al., 1998), mechanical overuse alone in the absence of TGF! 1 activated joint tissue remodeling did not result in detectable cartilage damage In the context of joint tissue homeostasis, TGF! 1 has been proposed to act through its anabolic effects on matrix. In vitro it is reported to stimulate chondrocytes to increase production of proteoglycans and type II collagen (Darling and Athanasiou, 2005; Pujol et al., 1991). In vivo stimulation of proteoglycan synthesis has also been reported (van Beuningen et al., 1994). In 105

PAGE 120

addition TGF! 1 counteracts IL-1 up regulation of MMP-13 and MMP-14, which are important in cartilage degradation (Blaney Davidson et al., 2007a). TGF! 1 also plays an important role in wound healing, angiogenesis, immunofunction, and cancers (Deed et al., 1997; Ellis and Schor, 1996; Kobayashi and Terao, 1997; Wisniewski et al., 1996). Its overproduction leads to many pathological conditions which include a range of fibrotic diseases such as pulmonary fibrosis (Koli et al., 2008), glomerulosclerosis (Wang et al., 2007), renal interstitial fibrosis (Ravinal et al., 2005), cirrhosis (Seki et al., 2007), Crohn’s disease (Burke et al., 2007), cardiomyopathy (Khan and Sheppard, 2006), scleroderma (Pannu et al., 2006), and chronic graft-vs-host disease (Lunn et al., 2005). Moreover, its involvement in the formation and progression of cancers is twofold. Firstly, it suppresses the progression of early transformations and tumorogenesis, but subsequent production of the factor by cancer cells themselves promotes inflammatory matrix remodeling, cell migration, and metastasis (Buijs et al., 2007). Similarly, the role of TGF! 1 in our model of OA might be similar to such dual actions on tissue remodeling. Firs tly, high concentrations of TGF! 1 in the joint clearly resulted in cell proliferation, migration, matrix production, and remodeling converging in a more generalized fibrotic response. This in turn permits the generation, activation, and survival of an altered cell type, such as a myofibroblast, which in turn perpetuates a pro-inflammatory environment in the joint (Bauer et al., 2006). 106

PAGE 121

Mechanical overuse is likely to produce mild injuries (or micro-trauma) to cartilage surfaces. Whereas these superficial damages can be rapidly restored in a healthy joint, the pro-inflammatory environment in a TGF! 1 activated joint will perpetuate and progress into more advanced lesions. In addition to cellmediated perpetuation of damage in such joints, osteophyte development was noted both on histology and radiography and these structures are well known to impair normal joint mechanics (Felson et al., 2005). In some surgically induced animal models, histopathology showed increased cellularity and swelling of the collagen fibrillar structures of the collateral, cruciate, and patellar ligaments. However these histologies were not verified by quantitative parameters and therefore confirmative data to implicate the involvement of ligament and tendon remodeling in the pathogenesis of this model need to be confirmed biomechanically (Wang and Flatow, 2005). The cartilage damage seen by India ink scoring was also confirmed by FACE analysis. Mice receiving TGF! 1 injections had decreased total chondroitin sulfate content in the knee joints compared to the controls indicating changes in the cartilage structure. It appears that increased activity in the absence of insult may actually be beneficial to cartilage health as the chondroitin sulfate content of joints of mice who were subjected to high intensity treadmill running without prior TGF! 1 injection was actually higher than that of the control group. ADAMTS-5 also plays a role in the development of OA in this model. When subjected to the model, mice lacking the active form of this protease did 107

PAGE 122

not develop significant cartilage damage on India ink staining and histology. Synovial hyperplasia was present at day 5 as was seen with the wild type C57Bl/10 mice. However, at day 18 the cartilage surface integrity remained intact. Determination of the mode of action of this enzyme during the TGF! 1 induced changes, such as synovial hyperplasia, will be important to further the understanding of possible mechanisms of joint protection against OA in this model. Mice in the TGF! 1 injected group spent significantly less time on the front half of the treadmill beginning at day 5 and progressively deteriorating through day 13. This may indicate that their ability or motivation to run was impaired, since the likelihood of encountering the shock panel is reduced by running on the front half of the treadmill. Mice in this group endured more frequent shocks rather than increasing their running speed to avoid sliding onto the shock grid. In comparison, mice subjected to high intensity treadmill running in the absence of TGF! 1 injections spent greater than 90% of the time on the front half of the treadmill. Data from the development of this non-surgical OA model are in support of synovitis and soft-tissue activation in pre-OA joints possibly preceding and/or accelerating the process of cartilage degeneration characteristic of progressive and late stage osteoarthritis. ADAMTS-5 appears to play some role in the development of these cartilage changes, but its role is not well understood. The rapidity (13 days) and reproducibility of this non-surgical model of murine OA, together with the rapid assessment of monitoring cartilage damage reported 108

PAGE 123

here, provides a platform for further work in evaluating both oral and intraarticularly administered therapeutics to modify joint pathologies leading to advanced stage OA. In addition, the model presents a novel way of evaluating non-pharmacologic interventions while maintaining the integrity of the supporting joint structures. 109

PAGE 124

Chapter Four Intra-Articular HA Injection Intervention Research Design (Figure 25) C57Bl/10 male mice aged 12 weeks old (n=36) were divided into the following groups: 1.2 Cage + TGF! 1 +HA Day 5 (n=12): TGF! 1 injections, HA injection, cage activity, sacrificed day 5 2.2 Cage +TGF! 1+HA Day 18(n=12): TGF! 1 injections, HA injection, cage activity, sacrificed day 18 3.2 Mechanical Overuse+ TGF! 1+HA (n=12): TGF! 1injections, HA injection, mechanical overuse via high intensity treadmill running, sacrificed day 18 Mice from the previous experiments on the induction of OA-like changes were used as the controls. 110

PAGE 125

Figure 25: Research design for intra-articular HA injection intervention Day 5 Day 18 Overuse through high intensity treadmill running • Speed = 32 cm/sec • Duration = 20 minutes • Frequency = daily x 13 days Mechanical Overuse TGF1Injection Right Leg (2x 48 h interval) Day 1 Day 3 Day 5 TGF1Injection Right Leg (2x 48 h interval) Day 1 Day 3 Sacrifice Sacrifice SacrificeCage Day 18 A B HA Injection Right Leg Day 4 Day 4 HA Injection Right Leg 111

PAGE 126

Materials The following materials were used in addition to those listed in Chapter 3: microplate reader (Bio-Tek Instruments, Inc., Winooshi VT); Multichannel pipettedigital (ICN ImmunoBiologicals, Costa Mesa CA); Mini-Orbital microplate shaker (Bellco biotechnology, Vineland NJ); BioTek Automated Microplate Washer Model EL 403 (BioTek Instruments, Inc., Winooshi VT); Nunc-Immuno Plate I F (VWR Scientific, Philadelphia PA); Microwell Plates (VWR Scientific, Philadelphia PA); Microplate sealing tape VWR Scientific, Philadelphia PA); microplate plastic lid (VWR Scientific, Philadelphia PA); reagent reservoir for multichannel pipette (ICN ImmunoBiologicals, Costa Mesa CA); monoclonal anti-keratan sulfate antibody 5D4 (ICN ImmunoBiologicals, Costa Mesa CA); horseradish peroxidase-conjugated anti-mouse IgG (Pierce Chemical Company, Rockford IL); chondroitin ABC Lyase (ICN ImmunoBiologicals, Costa Mesa CA); Tween 20 (Sigma Chemical Company, St. Louis MO); Bovine serum albumin (Sigma Chemical Company, St. Louis MO); O-phenylenediamine (Sigma Chemical Company, St. Louis MO); SupartzTM high molecular weight HA (Seikagaku, Inc.). Methods Animal Husbandry In vivo experiments were conducted with C57Bl/10 male mice procured from Jackson Laboratories at 7-11 weeks of age and housed at the Rush or the University of South Florida animal facility until they were 12 weeks old. All 112

PAGE 127

procedures for mouse handling, intra-articular TGF! 1 injections and mechanical overuse through high intensity treadmill running were as described in Chapter 3. Intra-Articular HA Injections To determine the effects of intra-articular injections of HA, mice received two doses of HA (10 l of 5mg/ml Supartz in 6 l saline) into the intra-articular space of the right knees on the same day with approximately 30 minutes between injections. These were performed using a 28G x inch needle on 0.5 ml syringe. Prior to HA injections, anesthesia was administered as described previously. Tissue Harvesting Hind limbs were harvested from euthanatized animals for histopathological evaluation via transection of the mid shaft of the femur as described in Chapter 3 and fixed for histology or stored at -20 C until evaluation with India ink. Determination of Clearance Time of HA from the Knee Joint Space Mice were injected with 6 l (5mg/ml) fluorescein isothiocyanate (FITC)labeled Supartz (endotoxin free and sterile, provided by Seikagaku Corporation, Tokyo, Japan) into the joint space of both knees in nave (n = 5) and TGF! 1 treated C57Bl/10 mice (n = 5) In addition a metabolically inactive (humanely euthanatized via carbon dioxide) mouse was also included to determine the requirement of live-tissue for HA clearance from the joint and the extent to which 113

PAGE 128

FITC fluorescent signal intensity was decreased by the repeated UV exposures during multiple imaging time points. -injection. Whole body and knee joint imaging was performed on the KODAK Molecular Image Station-In Vivo FX and Kodak Molecular Imaging Software. Fluorescent images were obtained under UV transillumination using a 465 nm excitation filter, a 535 nm emission filter, f-stop (focal length divided by the diameter of the lens) at 5.8, and a field of view of 60 mm. Images were obtained at baseline (immediately following injection), and 45 minutes, 1 hour, 3 hours, 6 hours, 12 hours, and 22 hours post injection. Radiographs were also taken at each imaging time point as described previously and overlays with fluorescent images used to determine the spatial distribution of fluorescence relative to the intra-articular space. Quantification of the fluorescence was determined on overlaid images within five mm diameter circular regions of interest (ROI) surrounding the intraarticular space (Figure 26). Using the Kodak Molecular Imaging Software, the net intensities (pixels) of the ROIs were imported into an Excel spreadsheet. Averages and standard deviations were calculated for each group at each time point. 114

PAGE 129

Figure 26: Selection of regions of interest on fluorescent image corresponding to radiographic images for determining net intensity of fluorescence within the knee joint in FITC-labeled HA injected mice and overlay of radiograph and fluorescent images 115

PAGE 130

Blood Collection and Plasma Preparation Animals were anesthetized as described previously and blood (5-7 drops for in vivo time points and terminal bleeds at time of sacrifice) was collected from the submandibular vein after puncture with a sterile 18G needle. Collections were done at baseline, two days after the first TGF! 1injection (day 3), two day after the second TGF! 1injection (day 5), one week after the second TGF! 1injection (day 12), and 14 days after the second TGF! injection (day 18). Blood was collected into sterile 500 l eppendorf tubes containing 5 l of heparin, cells were removed by centrifugation at 13,000 rpm for ten minutes at room temperature, plasma supernatants removed, and stored at -80 C until further analyses. In addition, blood was collected as described above at the time of sacrifice from 56 male C57Bl/10 nave and TGF! 1 injected mice to determine HA concentration in the plasma at the following time points (n=4): baseline (prior to HA injection), 1 hour, 3 hours, 6 hours, 24 hours, 7 days, and 14 days after injection of HA. Determination of Plasma HA Concentration by HA ELISA Plasma samples were analyzed for HA content using ELISA, essentially as described (Li et al., 1989; Rayan et al., 1998; Thonar et al., 1992) Briefly, keratan sulfate-free aggrecan from rat chondrosarcoma (RCS D1) was digested with chondroitinase ABC, then coated onto immunoplates in a sodium carbonate/sodium bicarbonate/sodium azide buffer at a concentration of 6 g/ml. HA (present in the standard and the samples) will bind specifically to the 116

PAGE 131

immobilized RCS-D1 G1 domain. A second aggrecan preparation from bovine articular cartilage D1 (BAC D1), which contains keratin sulfate is then introduced into the reaction mixture to bind to the immobilized HA. Finally, the bound keratin sulfate containing aggrecan is quantitated using an anti-keratin sulfate antibody (5-D-4) and horseradish peroxidase labeled anti-mouse IgG (an enzyme-linked secondary antibody). After adding O-phenylenediamine substrate, a color reaction occurred turning the samples orange (Figure 27). The intensity of the color reaction was proportional to the amount of HA antigen present in the sample. The concentration of HA present in each sample was then calculated by comparison of the absorbance values in each case to a standard curve generated from a known concentration of HA treated in the same way and incorporated in the same plate. Data from the ELISA was used for comparisons of change over time within and between groups. 117

PAGE 132

Figure 27: Schematic representation of HA ELISA. (1) RCS-D1 is immobilized on immunoplate; (2) HA from standard and samples binds specifically to the RDS-D1 coated plate; (3) BAC D1 detects HA; (4) anti-keratin sulfate antibody (5-D-4) binds to BAC D1; horseradish peroxidase labeled anti-mouse IgG binds to 5-D4; (5) o-phenylenediamine substrate is added and substrate-enzyme color development occurs RCS-D1 RCS-D1 RCS-D1 RCS-D1 RCS-D1 BACD1-KS BACD1-KS BACD1-KS 5-D-4 5-D-4 HRP OPD Substrate Product “Orange”HA HAHAHA 118

PAGE 133

Data Evaluation: Statistical Parameters Statistical analyses were performed using Statistical Package for Social Sciences (SPSS) version 15.0 for Windows as descri bed in Chapter 3. Results and Analyses Clearance Time of HA from the Knee Joint Space In situ whole body imaging of mice that received injections of FITC-labeled HA was firstly used to determine the accuracy and quantitative delivery of HA into the joint space (Figure 28). The total amount of fluorescence detected immediately after injection into nave joints was 19,000,000 + 4,000,000 pixel density units compared with 7,500,000 + 1,250,000 pixel density units for TGF! 1 injected joints and the proportional difference remained essentially constant over the entire range of image points. The lower intensity of fluorescence obtained with TGF! 1 treated knees using the same amount of starting material injected might have resulted from enhanced quenching of fluorescence bound up by de novo accumulated tissues and matrix after TGF! 1 injection, such as hyperplastic patellar fat pad (see Figure 16). An altered partitioning of macromolecules injected into TGF! 1 treated joints was confirmed by injecting Alexa 680 labeled hyaluronan binding protein (H ABP) into naive and treated joints, which showed a concentration of fl uorescence in region of the infrapatellar fat pad of mice injected with TGF! 1 (Figure 28). 119

PAGE 134

Figure 28: Fluorescent imaging with FITC labeled HA and Alexa 580 H ABP shows concentration of fluorescence in region of the infrapatellar fat pad of mice injected with TGF! 1 and more diffuse fluorescence throughout the knees of mice not injected with TGF! 1 naive TGF! 1-treated Alexa 580 HABP FITCHA 120

PAGE 135

Using the in vivo imaging technique, it was determined that the half life (t1/2) of the injected FITC-HA is approximately 6 hours in nave joints and is increased to 12 hours in the TGF! 1 injected joint (Figure 29). Furthermore, the clearance kinetics displayed a fast phase (0-6 hours) followed by a slower phase (6-22 hours; Figure 30). The fast turnover pool might represent lymphatic clearance of a freely diffusible pool of injected HA (Sabaratnam et al., 2003). The second, slower turnover pool is most likely HA molecules that have become incorporated into the ECM, remain bound to HA-specific receptors on the cell surface, or accumulate intracellularly in endocytotic vesicles (Tammi et al., 2001). 121

PAGE 136

Figure 29: HA fluorescence in knee joints of mice injected with TGF! 1, mice not injected with TGF! 1, and mice that are metabolically inactive (MI) Fluorescence Unit (Pixels per ROI) 122

PAGE 137

Figure 30: HA clearance from knee joints of mice injected with TGF! 1 and mice not injected with TGF! 1. Half life indicated as t1/2 t1 / 2~ 6 hours t1/2 ~ 12 hours 123

PAGE 138

HA Concentration in Plasma Following Intra-Articular Injections of HA ELISA is a widely used, sensitive and highly specific biochemical technique for the detection of macromolecules (proteins, polysaccharides) in a given sample. The particular ELISA method used here was developed for the detection of polymeric HA in biological fluids and tissue digests (Li et al., 1989; Rayan et al., 1998; Thonar et al., 1992). The range of detection of this assay is between 10 and 1000 ng of polymeric HA per ml of sample. A recent study employing this method to determine HA levels in serum of healthy individuals and persons diagnosed with OA revealed an increase of serum HA in persons with OA. Moreover, significant correlations existed between elevated HA levels and disease duration, citing it as a possible bi omarker of disease activity (Turan et al., 2007). Baseline (pre-HA injection) concentrations of HA showed a variation between the two groups of mice used. The TGF! 1 injected mice had lower preHA injection (baseline) plasma concentrations of HA than non-TGF! 1 injected mice (Figure 31). All animals were maintained at the same activity level and bled at the same time of day after receiving the same amount of anesthesia. The only difference between these mice at baseline (before HA injection) was previous activation of the joints TGF! 1 injection in one group. Both groups demonstrated low levels of plasma HA between three and twelve hours after HA injection. These time points correspond to the times during which maximal rate of clearance of HA from the joint occurs (see Figure 30). This might indicate diurnal variations or activation of hepatic mechanisms by 124

PAGE 139

the HA cleared from the plasma (Laurent et al., 1996). These interesting fluctuations in plasma HA warrant further examination to determine the underlying biochemical mechanisms. However, such experiments are outside the scope of this project due to the number of animals required for such studies. Moreover, the objective of this experiment in the context of this thesis has been addressed, which was to determine if intra-articularly injected HA is cleared via a systemic route. 125

PAGE 140

Figure 31: Plasma HA concentrations for TGF! 1 injected and non-TGF! 1 injected mice from pre-injection (baseline) to 14 days after HA injection as measured by HA ELISA Concentration (ng/ml) 126

PAGE 141

Effect of Intra-Articular HA on Joint Pathology India ink staining of both the right femoral and tibial cartilage surfaces revealed that intra-articular injections of HA resulted in less cartilage damage (lesions and fibrillation/fibrotic changes) after both cage activity and mechanical overuse via high intensity treadmill running (Figures 32-33). Groups treated with HA had significantly less cartilage damage (< 1.33 on femoral surfaces and < 0.67 on tibial surfaces) than those not treated with HA. Scores were virtually identical to those obtained for nave animals (Figures 17-18). To examine whether the protective effects of HA were due to systemic changes, in another group of mice HA was injected into the contra lateral joint. However, administration of HA via this route did not show any effect on prevention of cartilage lesions, cell proliferation in the synovial tissues, or osteophyte formation in the TGF! 1 injected knee. Moreover, doubling the dosage of HA from 10 l to 20 l into the affected knee did not reveal any additional joint protective effects when compared to the single dose. 127

PAGE 142

Figure 32: Mean cartilage scores for right femurs by quadrant after HA intervention. 1= TGF! 1 + HA; 2 = TGF! 1 with no HA; 0 depicts no cartilage damage denotes statistically significant difference from controls and non-HA injected groups at p< 0.05; ** denotes statistically significant difference from controls and non-HA injected groups at p< 0.005 Antero-Medial Antero-Lateral Postero-Medial Postero-Lateral C age Day 5 C age Day 18 M echanical Overuse Day 18 0 * * * * * 0 0 128

PAGE 143

Figure 33: Mean cartilage scores for right tibias by quadrant after HA intervention. 1= Cage + TGF! 1 + HA; 2 = Cage + TGF! 1 denotes statistically significant difference from controls and non-HA injected groups at p< 0.05; ** denotes statistically significant difference from controls and non-HA injected groups at p< 0.01; *** denotes statistically significant difference from controls and non-HA injected groups at p< 0.005 Antero-Medial Antero-Lateral Postero-Medial Postero-Lateral M echanical Overuse Day 18 C age Day 18 C age Day 5 * * ** ** * ** 129

PAGE 144

Figure 34: Percentage of time spent on front half of treadmill days 3 through 13 for mice in the mechanical overuse, mechanical overuse + TGF! 1, and mechanical overuse + TGF! 1 + HA groups 130

PAGE 145

Discussion and Conclusions Intra-articular injection of HA at a single, early time point in this OA model reduced the formation of cartilage lesions induced by TGF! 1 alone or in combination with mechanical overuse (Figures 32-33). The mean cartilage scores in treated animals were decreased from approximately 2.4 to 0.6 in each quadrant. This supports similar findings in studies done on surgical models of OA in rabbits (Amiel et al., 2003; Takahashi et al., 1999). Histological examination of the mouse joints (data not shown) revealed that a significant effect of the chondroprotection in this model might be the result of inhibition of fibrotic tissue formation within the synovium and at the articular cartilage surface, as well as reduced ost eophyte development. In support of the conclusion that the injected HA had a direct effect on the pathological cell proliferation and tissue remodeling induced by TGF! 1 was the finding that HA injected into the contra lateral control joint had no effect on the development of fibrosis and cartilage lesions in the affected joint. Moreover, injection of saline alone into either the diseased or contra lateral control knee also provided no therapeutic effect. Further work is needed to identify the cell types and their receptors that mediate the joint protection in this model. The inhibition of fibrosis seen here is consistent with anti-adhesion properties of HA reported in abdominal and ophthalmic surgeries. In addition, HA is reported to enhance angiogenesis and cell migration, thereby augmenting the granulation phase of wound healing. Similar mechanisms may come into play in 131

PAGE 146

the chondroprotective role of HA observed here. A largely direct therapeutic efficacy of HA on the tissues within the joint, as opposed to an indirect systemically mediated effect, was supported by the data that rapid clearance of HA from the joint within 24 hours (Figure 30) was not accompanied by concurrent increase in the levels of circulating HA (Figure 31). This would be keeping with clearance pathways involving the lymphatic route to the liver (Johnson et al., 2007). Interestingly, serum levels of HA were decreased during the maximal clearance activity from the joint which happened in the first six hours post injection (Figure 29). As systemic HA is also removed by the liver (Fraser and Gibson, 2005), this finding might indicate a direct coupling of lymphatic and systemic HA clearance pathways by the liver. Mice that had been injected with TGF! 1 spent significantly less time on the front half of the treadmill beginning at day 5 (<90%) and progressively deteriorating through day 13 (<75%). This would seem to indicate that their ability or desire to run was affected since the likelihood of encountering the shock panel is reduced by running on the front half of the treadmill. The mice in this group endured more frequent shocks rather than increasing their running speed to avoid the noxious stimuli. The HA intervention mitigated this functional consequence as HA treated mice spent greater than 95% of the time on the front half of the treadmill (Figure 34) and suggests an analgesic effect of HA in this model. Clearly, intra-articular injections of HA may not function solely to restore viscoelastic and protective functions of HA in the joint as previously thought 132

PAGE 147

(Balazs and Denlinger, 1993). The beneficial effects seen here last longer than the half life of the injected HA, which is 18-24 hours (Kotz and Kolarz, 1999), and support findings in human studies that pain relief from intra-articular injections of HA occurs, and lasts 26-52 weeks (Altman and Moskowitz, 1998; Huskisson and Donnelly, 1999; Kotz and Kolarz, 1999; Leardini, et al., 1987; Puhl et al., 1993; Wobig et al., 1998). Thus, in addition to the application of the mouse model for studying the mechanistic effects of intra-articular HA therapy, it will be useful for pre-clinical testing of existing and new formulations of HA preparations for their efficacy as well as disease modifying products during the progression of tissue destruction. The potential benefits of combination of such injections with physical therapy can also be investigated. 133

PAGE 148

Chapter Five Aerobic Exercise Intervention Research Design (Figure 35) C57Bl/10 male mice aged 12 weeks old (n=54) were divided into the following groups: 1.3 Cage +TGF! 1+ exercise Day 5 (n=12): 4 weeks aerobic exercise, TGF! 1 injections, cage activity, sacrificed day 5 2.3 Cage + exercise Day 5 (n=6): 4 weeks aerobic exercise, cage activity, sacrificed day 5 3.3 Cage +TGF! 1 + exercise Day 18 (n=12): 4 weeks aerobic exercise, TGF! 1 injections, cage activity, sacrificed day 18 4.3 Cage + exercise Day 18 (n=6): 4 weeks aerobic exercise, cage activity, sacrificed day 18 5.3 Mechanical Overuse + TGF! 1 +exercise Day 18 (n=12): 4 weeks aerobic exercise, TGF! 1 injections, mechanical overuse via high intensity treadmill running, sacrificed day 18 6.3 Mechanical Overuse + exercise Day 18(n=6): 4 weeks aerobic exercise, overuse via high intensity treadmill running, sacrificed day 18 134

PAGE 149

Figure 35: Research design for aerobic exercise intervention Day 5 Day 18 Overuse through high intensity treadmill running • Speed = 32 cm/sec • Duration = 20 minutes • Frequency = daily x 13 days TGF1Injection Right Leg (2x 48 h interval) Day 1 Day 3 Day 5 TGF1Injection Right Leg (2x 48 h interval) Day 1 Day 3 Sacrifice Sacrifice SacrificeCage Day 18 A B 4 Week Alternate Day Exercise Low intensity aerobic exercise via treadmill running • Speed = 14 cm/sec • Duration = 20 minutes • Frequency = every other day x 28 days Mechanical Overuse 4 Week Alternate Day Exercise Aerobic exercise through low intensity treadmill running • Speed = 14 cm/sec • Duration = 20 minutes • Frequency = every other day x 28 days 135

PAGE 150

Methods Details are described in Chapters 3 and 4. C57Bl/10 male mice procured from Jackson Laboratories at 7-11 weeks of age were housed at the Rush animal facility until they were 12 weeks old. All mouse husbandry and handling experimentations were carried out as described.. Aerobic Exercise via Alternate Day, Low Intensity Treadmill Running The experimental group of mice underwent treadmill running at a speed of 14 cm/second for 20 minutes on alternate days for a period of four weeks. This was determined with the same formula as for high intensity treadmill running. Thus, low intensity aerobic exercise for the mouse at 50% vO2 is achieved at 14 cm/sec (Baker and Gleeson, 1999). Treadmill exercising was performed at the same time each day for all groups. It represented a significant increase in physical activity for each animal because it was performed during the day when mice are usually sleeping, resting, or showing minimal physical activity. Following the four week exercise period, the mice were injected with TGF! 1 as described previously and then underwent 13 days of either cage activity or high intensity treadmill running as described previously. Controls for these groups were taken through the same exercise intervention then maintained with cage activity or high intensity running, but did not receive TGF! 1 injections. A daily record was kept for each animal. It included: duration of the run (minutes), distance run (calculated from the run time and speed of the treadmill), 136

PAGE 151

and number and time of interactions with the shock grid. In addition, treadmill performance was measured by the amount of time each mouse stayed on the front half of the running treadmill. In addition, to ensure that the effects of inducing this model were the same for 16 week old mice as they were for 12 week old mice, 16 week old male mice were divided into 3 groups: Cage + TGF! 1 Day 5, Cage + TGF! 1 Day 18, and Mechanical Overuse + TGF! 1 Day 5. These mice received TGF! 1 injections as described previously followed by either cage activity or mechanical overuse via high intensity treadmill running. Data Evaluation: Statistical Parameters Statistical analyses were performed using Statistical Package for Social Sciences (SPSS) version 15.0 for Windows. Cartil age scores for all four quadrants were analyzed individually then summed to obtain the overall cartilage score for each cartilage surface (right femur, left femur, right tibia, and left tibia). In addition, quadrants 1 and 2 were summed for the anterior surface score, quadrants 3 and 4 for the posterior score. Finally quadrants 1 and 3 were summed for the lateral cartilage score and quadrants 2 and 4 for the medial cartilage score. Data was analyzed for descriptive statistics (means, medians, standard deviations) and fixed effect one way analysis of variance (ANOVA) with Tukey post hoc testing and level of significance at p value less than 0.05. Group comparisons were made for overall cartilage scores, total scores for each 137

PAGE 152

cartilage surface (right femur, left femur, right tibia, and left tibia), anterior, posterior, medial and lateral areas of each cartilage surface, and each quadrant (anteromedial, anterolateral, posteromedial, and posterolateral) on each cartilage surface. Power analysis based on the 4 point grading system and with the assumption based on preliminary studies of a between group mean difference of at least one point revealed a sample size of 3 in control groups and 6 in treatment groups per time point sufficient for power > 80%. In addition Repeated Measures ANOVA with Tukey post hoc testing and level of significance at p value less than 0.05 was used to analyze data on treadmill performance data. The time each mouse spent on the front half of the treadmill each day was expressed as a percentage of the total time and data were analyzed for descriptive statistics and differences within and between groups and within and between days. 138

PAGE 153

Results and Analysis Effect of Exercise Intervention on Joint Pathology There was a statistically significant difference between cartilage scores for the groups receiving exercise as an intervention prior to inducing OA-like changes through TGF-1 and mechanical over use and those that did not with the exercised mice having lower scores. This difference was apparent on both the femur (Figure 36) and tibia (Figure 37) cartilage surfaces at day 18. Cartilage scores for the femoral condyle cartilage surfaces were higher than those for the tibial plateau cartilage surfaces indicating the involvement of a prominent biomechanical component in the pathway leading to cartilage damage. No statistically significant difference existed between exercise groups and controls. However, scores trended slightly higher in the exercise groups than in the controls. 139

PAGE 154

Figure 36: Mean cartilage scores for right femurs by quadrant after exercise intervention (1 = exercise; 2 = TGF! 1; 3 = exercise + TGF! 1) denotes statistically significant difference from controls and exercise groups at p< 0.05; ** denotes statistically significant difference from controls and exercise groups at p< 0.005 Postero-Medial Antero-Medial Antero-Lateral Day 5 Cage Day 18 Cage Day 18 Mechanical Overuse Postero-Lateral * * * * * * 140

PAGE 155

Figure 37: Mean cartilage scores for right tibias by quadrant after exercise intervention (1 = exercise; 2 = TGF! 1; 3 = exercise + TGF! 1) denotes statistically significant difference from controls and exercise groups at p< 0.05; ** denotes statistically significant difference from controls and exercise groups at p< 0.01; Day 5 Cage Day 18 Cage Day 18 Mechanical Overuse Postero-Lateral Postero-Medial Antero-Lateral Antero-Medial * * * * 141

PAGE 156

Treadmill Performance There was a statistically significant difference between groups in treadmill performance (p< 0.05) with the exercised mice spending more time on the front half of the treadmill compared to the non-exercised, TGF! 1 injected mice. This difference was apparent in overall average daily times as well as daily times on days 8 through 13 (Figure 38). Mice in the exercise intervention groups averaged above 90% on a daily basis as did the mice undergoing mechanical use only in the absence of TGF! 1 injection. However, mice in the mechanical overuse + TGF! 1 group showed a steady decline in treadmill performance beginning day 5 and progressively worsening through day 13 when they spent less than 75% of the time on the front half of the treadmill. 142

PAGE 157

Figure 38: Percentage of time spent on front half of treadmill days 3 through 13 by group for mice in the mechanical overuse, mechanical overuse + TGF! 1, and mechanical overuse + TGF! 1 + exercise groups 143

PAGE 158

Discussion and Conclusions A four week period of alternate day, low intensity treadmill running minimized cartilage damage from TGF! 1 injections and overuse in our mouse model of OA. While cartilage damage was not entirely eliminated, the mice that exercised via low intensity treadmill running showed significantly less cartilage damage than those mice who did not exercise. Mice in exercise groups scored minimal cartilage fibrillation or less on femurs and tibias at day 18 of both cage activity and overuse compared to mice in non-exercise groups that scored overt cartilage erosion to ulceration (Grades 1 and 3, respectively; See Figure.12). The effect of treadmill exercise on OA pathogenesis in mice has not been studied. The findings reported here, that pathological effects of joint inflammation (synovitis, capsular fibrosis) did not proc eed to induction of cartilage lesions in exercised mice, are also supported by another study showing that voluntary wheel running slowed the progression of spontaneous OA in mice expressing a col IIa mutation (Lapvetalainen et al., 2002). Possible mechanisms for this protective effect warrant further investigation, but may include biochemical and/or biomechanical pathways. Although strengthening is not a primary outcome of aerobic exercise, it is possible that the four week exercise program resulted in increased hind limb muscle strength. Studies in humans have found that increased quadriceps strength (in the presence of normal joint biomechanics) results in a decreased risk of OA (Hootman et al., 2004; Slemenda et al., 1998). By improving muscle 144

PAGE 159

balance and strength, stress and structural trauma to the cartilage may be reduced. Another possible mechanism may be related to the increased production and deposition of aggrecan in the cartilage itself. In a study of exercise in humans, Roos et al. found aerobic exercise improved cartilage quality as evidenced by increased GAG content measured by dGEMRIC (Roos et al., 2005). While total proteoglycan contents (as chondroitin sulfate by FACE, see Chapter 3) was not analyzed for the mice in the exercise intervention, FACE analysis on the mice undergoing high intensity aerobic exercise in the absence of TGF! 1 injections showed increased chondroitin sulfate content of the cartilage. It is reasonable to hypothesize the mice in the exercise intervention groups may also have increased chondroitin sulfate content. Mice undergoing exercise without TGF! 1injections scored slightly (but not significantly; p > 0.70) higher than controls for cartilage damage as graded by India ink stain. This may be in part a result of the age of mice in the exercise group being four weeks older than the model controls. C57Bl mice spontaneously develop OA with increasing incidence and severity with age. At eight weeks, 20% have some evidence of OA on histology. This increases to 80% at 64 weeks (Yamamoto et al., 2005). It is possible the additional four weeks resulting in some increased cartilage fibrillation. However, no difference was noted in cartilage grading when comparing 16 and 12 week old mice. In addition to structural changes noted with inducement of this OA model and remediated with interventions of HA injections and exercise, there appeared to be functional 145

PAGE 160

consequences as well in the form of altered treadmill performance. Mice in the exercise intervention group spent greater than 90% of the time on the front half of the treadmill as compared to mice in the non-exercise group which spent < 90% of the time on the front half of the treadmill beginning at day 8 and worsened to <75% by day 13 of the mechanical overuse protocol. This would seem to indicate the exercised mice had a better ability or desire to run. Studies on the effects of mechanical perturbation on degenerative and reparative actions of chondrocytes have been performed ex vivo (Fitzgerald et al.; 2006, Lee et al, 2005; Sah et al., 1989). In addition, low magnitude mechanical strain on cartilage has been shown to suppress IL-1 and TNF" (proinflammatory cytokines) and up regulate proteoglycan and collagen synthesis while high magnitude mechanical strain was detrimental in vitro (Deschner et al., 2003). This model may provide a means of examining cellular and biochemical events involved in the complex function of joint movement and determining doseresponse relationships of mechanical forc es and cartilage health as well as responses to different types of force (s hear, compression, friction, combination). The data reported represents the first time report of aerobic treadmill exercise in mice as a possible intervention for minimizing the development of OA in the knee joint. The non-surgical mouse model developed here will allow further mechanistic studies of aerobic exercise as well as other types of exercise, such as resistance exercise. In addition it will be useful for determining dose-response relationships of exercise and the effects of timing of exercise on OA development and progression. 146

PAGE 161

Chapter 6 Summary and Conclusions Significance and Implications Comprehensive OA-like changes in mouse knee joints can be induced by anabolic stimulation of joint tissues through intra-articular injections of TGF! 1 followed by mechanical overuse of the activated joint. Specific changes noted initially following TGF! 1 injection include synovial hyperplasia, cellular infiltration, and granulation tissue formation. Within two weeks these changes progressed to soft tissue fibrosis, chondrophyte/ osteophyte development, and degeneration of the articular cartilage and menisci. This pathology was enhanced after mechanical overuse through daily high intensity treadmill running. Functional consequences of the pathology in this model also occurred, and was detected by poorer treadmill performance of the mice injected with TGF! 1 compared to untreated mice.. Development of this non-surgical mouse model of OA provides a tool for studying the development and progression of OA as the knee is not destabilized by surgical procedures or the introducti on of noxious substances in the joint space. While in the past spontaneous age-related or genetic mouse models were used, they require housing for an extended period of time significantly to the cost of research. Genetic manipulation models are often not amenable to interventions as a result of the genetic alteration (Bendele, 2002). The rapidity of 147

PAGE 162

the mouse model of OA presented in this research makes it an option for studying OA development. Further, the presence of pathological joint changes in an otherwise intact knee joint makes it amenable for pharmacological and nonsurgical intervention studies. Utilization of this model has shown that intra-articular injections of HA may have a disease-modifying effect on the development of OA. Mice injected with HA immediately following anabolic stimulation of the joint with TGF! 1 still exhibited the initial synovial hyperplasia but did not continue to develop fibrosis, chondrophyte/osteophyte formation, or cartilage and meniscal degeneration even after mechanical overuse of the activated joint. Further, treadmill performance of mice injected with HA remained the same as that displayed by normal mice. HA injections are used in humans to treat the symptoms of OA, primarily pain. However, data obtained in this study indicate HA injections may be able to actually alter the disease process and impact the functional consequences of OA. The model developed here was also used to demonstrate a preventative effect of aerobic exercise on disease development in OA in mice. The mice that were subjected to four weeks of alternate day, low intensity treadmill running prior to the inducement of OA developed less cartilage damage than nonexercised mice. In addition, this intervention also appears to have positively impacted function as treadmill performance in OA induced exercised mice did not show the progressive decline seen in OA induced, non-exercised mice. This is the first study investigating the effects on OA in mice of aerobic exercise through 148

PAGE 163

low intensity treadmill running, and findings support those of Lapvetalainen et al., in a study of lifelong voluntary wheel running in mice (Lapvetalainen et al., 2002). Further, this research indicates aerobic exercise may have a disease-modifying effect and warrants more investigation. In addition it was shown that 13 days of mechanical overuse alone was not sufficient to cause significant cartilage degeneration and other joint changes. This supports findings of studies in human OA that repetitive, high intensity exercise in the absence of altered joint biomechanics does not result in increased OA development (Bennell and Hinman, 2005; Cymet, 2006). Limitations While the data presented here suggest OA-like changes can be induced in mouse knees through TGF! 1 injections followed by mechanical overuse, and both HA injections and aerobic exercise intervention decrease OA development, limitations do exist. Translation of these results into treatment of human disease needs to be further investigated. Mouse models are commonly utilized to test an intervention effect and to determine disease and intervention mechanisms. However, studies of this kind need to be repeated in larger animals before introduction to human patients. A large variability was noted in baseline concentrations of serum HA during the HA ELISA. This large variability requires a much larger sample size than that used here in order to draw more definite conclusions. A small sample 149

PAGE 164

size was also used for the serum TGF! 1 ELISA. Therefore, the data reported for both the HA and TGF! 1 ELISAs should be treated as preliminary data. The data suggests that mechanica l overuse in the absence of TGF! 1 activation does not result in OA development. However, it must be pointed out that the mechanical overuse was only for 13 days. While this represents approximately 2% of a mouse’s life (or approximately 1 years for humans), whether a more extended period of mechanical overuse would still result in little or no joint disease remains to be seen. The duration of the chondroprotective effects of the HA intervention and the aerobic exercise intervention is uncertain. The results reported here show effects at 14 days post TGF! 1 injection. However, whether the inhibition of cartilage degeneration persists beyond two weeks is not known. Future Directions While the specific aims for this dissertation were addressed, many questions remain unanswered and provide opportunity for further research. The mechanism of OA development in this non-surgical model is not completely understood. It is evident that the synovial hyperplasia and cellular infiltration occurs, but it is less clear what cells are present and from where they originate. Immunohistochemistry and other biochemical techniques may assist in determining this. Histological assessment of TGF! 1 injected knees indicates there may be disruption of the menisci and ligaments that stabilize the knee joint. If so, faulty 150

PAGE 165

joint biomechanics may be partially responsible for the joint damage reported here. Ligamentous integrity should be investigated to determine what role, if any, the ligaments play in the development and progression of OA in this model. ADAMTS 5 appears to be involved, at least in part, in this non-surgical mouse model of OA. Further studies with ADAMTS 5 knockout mice are warranted to investigate the extent of involvement and specific role of ADAMTS 5. In addition, other knockout strains should be utilized to investigate the role of other proteases. The duration of chondroprotection seen with intra-articular HA injections warrants more investigation, as does the actual onset of this inhibition of cartilage damage. It is clear that synovial hyperplasia still occurs at day 5 even in the presence of HA. However, it is not know if the same cell types are present. Protection of the cartilage surfaces is evi dent at day 18, but what occurs between day 5 and day 18 of this model remains to be studied. Further, the importance of the timing of HA injection and the effect of multiple injections at various time points should be investigated. This research shows injection of HA one day after inducing OA minimizes OA development, but it is not known if HA injected later in the disease process continues to offer protection and/or has a reparative role. The disease-modifying effects of exercise on OA have not been well studied. The data reported here show a preventative bout of aerobic exercise prior to the inducement of OA provides chondroprotection. The mechanism(s) of this chondroprotection warrant further investigation via histological, biochemical, and biomechanical assessment. Future exercise studies should be undertaken 151

PAGE 166

with this model to determine what type (aerobic, resistance, water-based), dose (intensity, frequency, duration), and timing (preventative, early disease, mid disease, late disease) of exercise provides optimal joint benefits. The mouse model developed as part of this dissertation provides a means to answer these and other questions. Fu rther, the development of procedures for intra-articular HA injections, treadmill exercise, and rapid data analysis methods facilitate investigation of the development and progression of OA and the effects of other pharmacological and non-pharmacological interventions. 152

PAGE 167

References Abel K, Reneland R, Kammerer S, Mah S, Hoyal C, Cantor CR, Nelson MR, Braun A. (2006). Genome-wide SNP association: identification of susceptibility alleles for osteoarthritis. Autoimmun Rev 5, 4, 258-263. Adams ME. (1989). Cartilage hypertrophy following canine anterior cruciate ligament transaction differs among different areas of the joint. J Rheumatol 16, 6, 818-824. Adams ME, Billingham ME, Muir H. (1983). The glycosaminoglycans in menisci in experimental and natural osteoarthritis. Arthritis Rheum 26, 1, 69-76. Adams ME, Brandt KD. (1991). Hypertr ophic repair of canine articular cartilage in osteoarthritis after anterior cruciate ligament transection. J Rheumatol 18, 3, 428-435. Altman R, Asch E, Bloch D, Bole G, Borenstein D, et al. (1986). Development of criteria for the classification and reporting of OA. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum 29, 1039-1049. Altman R, Brandt K, Hochberg M, Moskowitz R. (1996). Design and conduct of clinical trials in patients with osteoarthritis: recommendations from a task force of the Osteoarthritis Research Society. Osteoarthr Cart 4, 217-243. Altman RD, Hochberg MC, Moskowitz RW, Schnitzer TJ. (2000). Recommendations for the medical management of osteoarthritis of the hip and knee. American College of Rheumatology Subcommittee on Osteoarthritis Guidelines. Arthr Rheum 43, 1905-1915. Altman RD, Moskowitz R. (1998). Intraarticular sodium hyaluronate (Hyalgan) in the treatment of patients with osteoarthritis of the knee: a randomized clinical trial. J Rheumatol 25, 2203-2212. Amadio PJ and Cummings DM. (1983). Evaluation of acetaminophen in the management of osteoarthritis of the knee. Curr Ther Res Clin Exp, 34, 59-66. American College of Rheumatology Subcommittee on Osteoarthritis Guidelines (2000). Recommendations for the medical management of osteoarthritis of the hip and knee. Arthritis Rheum 43, 1905-1915. American Geriatrics Society Panel on Exercise and Osteoarthritis. (2001). Exercise prescription for older adults with osteoarthritis pain: consensus practice recommendations. J Am Geriatr Soc 49, 808-823. 153

PAGE 168

American Physical Therapy Association. (2003). Guide to Physical Therapy Practice. Ameye LG, Young MF. (2006). Animal models of osteoarthritis: lessons learned while seeking the “Holy Grail”. Curr Opin Rheumatol 18, 537-547. Amiel D, Toyoguchi T, Kobayashi K, Bowden K, Amiel ME, Healey RM. (2003). Long-term effect of sodium hyaluronate (Hyalgan) on osteoarthritis progression in a rabbit model. Osteoarthr Cart 11, 636-643. Anderson-Mackenzie JM, Billingham ME, Bailey AJ. (1999). Collagen remodeling in the anterior cruciate ligament associated with developing spontaneous murine osteoarthritis. Biochem Biophys Res Commun 258, 753767. Arrich J, Piribauer F, Mad P, Schmid D, Klaushofer K, Mullner M. (2005). Intra-articular hyaluronic acid for the treatment of osteoarthritis of the knee: systematic review and meta-analysis. CMAJ, 172, 1039–1043. Arroll B, Goodyear-Smith F. (2004). Corticosteroid injections for osteoarthritis of the knee: meta-analysis. B Med J, 328, 869. Arshinoff SA, Albiani DA, Taylor-Laporte J. (2002). Intraocular pressure after bilateral cataract surgery using Healon, Healon5 and Healon GV. J Cataract Refract Surg 28, 4, 617-625. Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B. (1990). CD44 is the principal cell surface receptor for hyaluronate. Cell 61, 1303-1313. Ashworth MA, Brule C, Day A. (2002). The Development of an orthopedic waiting list algorithm for elective total hip and total knee replacement surgery. Ottawa, Ontario, Canada: Canadian Health Services Research Foundation. Ayral X, Dougados M, Listrat V, Bonvarlet JP, Simonnet J, Amor B. (1996). Arthroscopic evaluation of chondropathy in osteoarthritis of the knee. J Rheumatol 23, 4, 698-706. Bailey AJ, Mansell JP, Sims TJ, Banse X. (2004). Biochemical and mechanical properties of subchondral bone in osteoarthritis. Biorheolog, 41, 3-4, 349-358. Baker EJ, Gleeson TT. (1999 ). The effects of intensity on the energetics of brief locomotor activity J Exper Bio 202, 3081-3087. 154

PAGE 169

Bakker AC, van de Loo FA, van Beuningen HM, Sime P, van Lent PL, et al. (2001). Overexpression of active TGF! 1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation. Osteoarthr Cart 9, 128-136. Balazs EA, Denlinger JL. (1993). Viscosupplementation: a new concept in the treatment of osteoarthritis. J Rheumatol Suppl 39, 3-9. Bartz RL, Laudicina L. (2005). Osteoarthritis after sports knee injuries. Clin Sports Med 24, 1, 39-45. Bateman JF. (2005). Genetic aspects of osteoarthritis. Semin Arthritis Rheum 34, 6, Suppl 2, 15-18. Bauer DC, Hunter DJ, Abramson SB, Attur M, Corr M, Felson D, et al. (2006). Osteoarthritis Biomarkers Network. Classification of osteoarthritis biomarkers: a proposed approach. Osteoarthr Cart 14, 8, 723-727. Bauer S, Jendro MC, Wadle A, Kleber S, Stenner F, et al. (2006). Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes. Arthr Res Ther 8, 6, R171. Bellamy N, Campbell J, Robinson V, Gee T, Bourne R, Wells G. (2006). Viscosupplementation for the treatment of osteoarthritis of the knee (Review). The Cochrane Review issue 4, Chichester, UK: John Wiley and Sons, pp 1-625. Bendele AM. (2001). Animal models of osteoarthritis. J Musculoskel Neuron Interact 1(4), 363-376. Bendele AM. (2002). Animal models of osteoarthritis in an era of molecular biology. J Muscluloskel Neuron Interact, 2(6), 501-503. Bendele AM, Hulman JF, Harvey AK, Hrubey PS, Chandrasekhar S. (1990). Passive role of articular chondrocytes in quinolone-induced arthropathy in guinea pigs. Toxicol Pathol 18, 304-312. Benito MJ, Veale DJ, FitzGerald O, van den Berg WB, Bresnihan B. Synovial tissue inflammation in early and late osteoarthritis. Ann Rheum Dis 64, 1263-1267. Bennell K, Hinman R. (2005). Exercise as a treatment for osteoarthritis. Curr Opin Rheum, 17, 634-640. 155

PAGE 170

Berumen-Nafarrate E, Leal-Berumen I, Luevano E, Solis FJ, Munoz-Esteves E. (2002). Synovial tissue and synovial fluid. J Knee Surg 15, 1, 46-48. Billat VL, Mouisel E, Roblot N, Melki J. (2005). Interand intrastrain variation in mouse critical running speed. J Appl Physiol, 98, 1258-1263. Blaney Davidson EN, Scharstuhl A, Vitters EL, van der Kraan PM, van den Berg WB. (2005). Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity. Arthr Res Ther 7, R13381347. Blaney Davidson EN, van der Kraan PM, van den Berg WB. (2007a). TGF! and osteoarthritis. Osteoarthr Cart 15, 597-604. Blaney Davidson EN, Vitters EL, van Beuningen HM, van de Loo FAJ, van den Berg WB, van der Kraan PM. (2007). Resemblance of osteophytes in experimental osteoarthritis to transforming growth factor! induced osteophytes. Arthr Rheum 56, 12, 4065-4073. Blom AB, van Lent PL, Holthuysen AE, van der Kraan PM, Roth J, et al. (2004) Synovial lining macrophages mediate osteophyte formation during experimental osteoarthritis. Osteoarthr Cart 12, 8, 627-635. Boegard T, Rudling O, Peterson IF, Jonsson K. (1998). Correlation between radiographically diagnosed osteophytes and magnetic resonance detected cartilage defects in the patellofemoral joint. Ann Rheum Dis 57, 7, 395400. Bondeson J, Wainwright SD, Lauder S, Amos N, Hughes CE. (2006). The role of synovial macrophages and macrophage-produced cytokines in driving aggrecanases, matrix metalloproteinases, and other destructive and inflammatory responses in osteoarthritis. Arthritis Res Ther 8, 6, R187. Bos PK, van Osch GJ, Frenz DA, Verhaar JA, Verwoerd-Verhoef HL. (2001). Growth factor expression n cartilage wound healing: temporal and spatial immunolocalization in a rabbit auricular cartilage wound model. Osteoarthr Cart 9, 4, 382-389. Botter SM, van Osch GJ, Waarsing JH, van der Linden JC, Verhaar JA, et al. (2007). Cartilage damage pattern in relation to subchondral plate thickness in a collagenase-induced model of osteoarthritis. Osteoarthr Cart doi:10.1016/j.joca.2007.08.005. 156

PAGE 171

Bove SE, Calcaterera SL, Brooker RM, Huber CM, Guzman RE, et al. (2003). Weight bearing as a measure of disease progression and efficacy of antiinflammatory compounds in a model of monosodium iodoacetate-induced osteoarthritis. Osteoarthr Cart 11, 821-830. Brandt KD, Smith GN, Simon LS. (2000). Intraarticular injection of hyaluronan as treatment for knee osteoart hritis. Arthr Rheum, 43, 6, 1192-1203. Brittberg M, Lindahl A, Nilsson A, Olsson C, Isaksson, et al. (1994). Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 331, 14, 889-895. Brosseau L, Yonge KA, Robinson V, Marchand S, Judd M, et al. (2004). Thermotherapy for treatment of osteoarthritis. The Cochrane Library issue 3, Chichester, UK: John Wiley and Sons, pp1-21. Brouwer RW, Raaij van TM, Bierma-Zeinstra SM, Verhagen AP, Jakma TS, Verhaar JA. (2007). Osteotomy for treating knee osteoarthritis. Cochrane Database Syst Rev, 18, 3, CD004019. Buckwalter JA, Mankin HJ. (1997). Degeneration and osteoarthrosis, repair, regeneration, and transplantation. J Bone Joint Surg 79A, 612-632. Buijs JT, Henriuez NV, van Overveld PG,van der Horst G, Ten Dijke P, van der Pluijm G. (2007). TGF-beta and BMP7 interactions in tumor progression and bone metastasis. Clin Exp Metastasis 24, 8, 609-617. Burke JP, Mulsow JJ, O’Keane C, Docherty NG, Watson RW, O’Connell PR. (2007). Fibrogenesis in Crohn’s disease. Am J Gastroenterol 102, 2, 439448. Burstein D and Gray ML. (2006). Is MRI fulfilling its promise for molecular imaging of cartilage in arthritis? Osteoarthr Cart 14, 11, 1087-1090. Buss A, Pech K, Kakulas BA, Martin D, Schoenen J, et al. (2007) TGFbeta1and TGF-beta2 expression after traumatic human spinal cord injury. Spinal Cord doi:10.1038/sj.sc.3102148. Cahue S, Dunlop D, Hayes K, Song J, Torres L, Sharma L (2004). Varus-valgus alignment in the progression of patellofemoral osteoarthritis. Arthr Rheum 50, 7, 2184-2190. Cake MA, Read RA, Appleyard RC. (2004). The nitric oxide donor glyceryl trinitrate increases subchondral bone sclerosis and cartilage degeneration following ovine meniscectomy. Osteoarthr Cart 11, 872-878. 157

PAGE 172

Calabro A, Midura R, Wang A, West L, Plaas A, Hascall VC. (2001). Fluorophore-assisted carbohydrate electrophoresis (FACE) of glycosaminoglycans. Osteoarthr Cart 9, Suppl A, S16-S22. Carter DR, Wong M. (2003). Modelling cartilage mechanobiology. Philos Trans R Soc Lond B Biol Sci 358, 1437, 1461-1471. Cawston TE, Wilson AJ. (2006). Understanding the role of tissue degrading enzymes and their inhibitors in development and disease. Best Pract Res Clin Rheumatol, 20, 5, 983-1002. Cerejo R, Dunlop DD, Cahue S, Channin D, Song J, Sharma L. (2002) The influence of alighment on risk of knee osteoarthritis progression according to baseline stage of disease. Arthr Rheum 46, 2632-2636. Chang A, Hayes K, Dunlop D, Hurwitz D, Song J, et al. (2004). Thrust during ambulation and the progression of knee osteoarthritis. Arthritis Rheum 50, 12, 3897-3903. Chang DG, Iverson EP, Schinagl RM, Sonoda M, Amiel D, Coutts RD, Sah RL. (1997). Quantitation and localization of cartilage degeneration following the induction of osteoarthritis in the rabbit knee. Osteoarthr Cart 5, 357-372. Chang RW, Falconer J, Stulberg SD, Aernold WJ, Manheim LM, Dyer AR. (1993). A randomized, controlled trial of arthroscopic surgery vs closed needle joint lavage for patients with osteoarthritis of the knee. Arthr Rheum, 36, 289-295. Christ W, Lehnert, Ulbrich B. (1988). Specific toxicologic aspects of the quinolones. Rev Infect Dis, 10, Suppl 1, S141-146. Clements DN, Carter SD, Innes JF, Ollier WE. (2006). Genetic basis of secondary osteoarthritis in dogs with joint dysplasia. Am J Vet Res 67, 5, 909918. Clements KM, Price JS, Chambers MG, Visco DM, Poole AR, Mason RM. (2003). Gene deletion of either interleukin-1beta, interleukin-1betaconverting enzyme, inducible nitric oxide synthase, or stromelysin 1 accelerates the development of knee osteoarthritis in mice after surgical transaction of the medial collateral ligament and partial medial meniscectomy. Arthr Rehum 48(12), 3452-3463. Cochrane T, Davey RC, Matthes Edwards SM. (2005). Randomised controlled trial of the cost-effectiveness of water-based therapy for lower limb osteoarthritis. Health Tech Assess 9(31), 1-127. 158

PAGE 173

Collins DH. (1949). Osteoarthritis, The Pathology of Articular and Spinal Disease Edward Arnold & Co, London, 74-115. Colwell CWJ, D’Lima DD, Hoenecke HR, Fronek J, Pulido P, et al. (2001). In vivo changes after mechanical injury. Clin Orthop 391(Suppl), S116S123. Cooper C, Snow S, McAlindon TE, Kellingray S, Stuart B, et al. (2000). Risk factors for the incidence and progression of radiographic knee osteoarthritis. Arthr Rheum 43, 995-1000. Corrado EM, Peluso GF, Gigliotti S, DeDurante C, Palmieri D, et al., (1995). The effects of intra-articular administration of hyaluronic acid on osteoarthritis of the knee: a clinical study with immunological and biochemical evaluations. Eur J Rheumatol Inflamm 15, 47-56. Creamer P, Lethbridge_Cejku M, Costa P, Tobin JB, Herbst JH, Hochberg MC. (1999). The relationship of anxiety and depression with selfreported knee pain in the community: data from the Baltimore Longitudinal Study of Aging. Arthritis Care Res 12, 3-7. Cresci B, Tesi F, La Ferlita T, Ricca V, Ravaldi C, et al. (2007). Group versus individual cognitive –behavioral treatment for obesity: results after 36 months. Eat Weight Disord 12, 4, 147-153. Crichton MH, Puppione AA. (2006). Geriatric neutrophils: implications for older adults. Sem Onc Nurs 22(1), 3-9. Criscione LG, Elliott AL Stabler T, Jordan JM, Pieper CF, et al. (2005). Variation of serum hyaluronan with activity in individuals with knee osteoarthritis. Osteoarthr Cart 13, 837-840. Cushnaghan J, McCarthy C, Dieppe P. (1994). Taping the patella medially: a new treatment for osteoarthritis of the knee joint? Brit Med J 308, 753-755. Cymet TC, Sinkov V. (2006). Does long-distance running cause osteoarthritis? J Am Osteopath Assoc 106, 6, 342-345. Dahl LB, Dahl IMS, Engstrom A, Granath K. (1985). Concentration and molecular weight of sodium hyaluronate in synovial fluid from patients with rheumatoid arthritis and other arthropathies. Ann Rheum Dis 44, 817-822. 159

PAGE 174

Darling EM, Athanasiou KA. (2005). Growth factor impact on articular cartilage subpopulations. Cell Tissue Res 322, 3, 463-467. Davis MA, Ettinger WH, Neuhaus JM, Barclay JD, Segal MR. (1992). Correlates of knee pain among US adults with and without radiographic knee osteoarthritis. J Rheumatol 19, 1943-1949. Davoli MA, Lamplugh L, Beauchemin A, Chan K, Mordier S, et al. Enzymes active in the areas undergoing cartilage resoption during the development of the secondary ossification center in the tibiae of rats aged 0-21 days Dev Dyn 222, 1, 71-88. Day JS, Van Der Linden JC, Bank RA, Ding M, Hvid I, Sumner DR, Weinans H. (2004). Adaptation of subchondral bone in osteoarthritis. Biorheology, 41, 3-4, 359-368. De Bari C. (2006). Cell-based approaches to joint surface repair: from bench to bedside. Eur Cell Mat 12, Suppl 1, 38. Deed R, Kumar S, Freemont AJ, Smith J, Norton JD, et al. (1997). Early response gene signaling is induced by angiogenic oligosaccharides of hyaluronan in endothelial cells. Int J Cancer 10, 251-256. de Hooge ASK, van de Loo FAJ, Bennink MB, Arntz OJ, de Hooge P, et al. (2005). Male IL-6 gene knock out mice developed more advanced osteoarthritis upon aging. Osteoarthr Cart, 13, 66-73. Delamater AM, Jacobson AM, Anderson B, Cox D, Fisher L, et al. (2001). Psychosocial therapies in diabetes: report of the Psychosocial Therapies Working Group. Diabetes Care 24, 7, 1286-1292. Deschner J, Hofman CR, Piesco NP, Agarwal S (2003) Signal transduction by mechanical strain in chondrocytes. Curr Opin Clin Nutr Metab Care, 6, 289-293. Dieppe PA, Lohmander LS. (2005). Pathogenesis and managementof pain in osteoarthritis. Lancet 365, 9463, 965-973. Diracoglu D, Aydin R, Bakent A, Celik A. (2005). Effects of kinesthesia and balance exercises in knee osteoarthritis. J Clin Rheum 11(6), 303-310. Divine JG, Zazulak BT, Hewett TE. (2006). A systematic review of viscosupplementation for knee osteoarthritis. Clin Orthop Rel Res 455, 113-122. 160

PAGE 175

Dougados M, Nguyen M, Listrat V, Amor B. (1993). High molecular weigh sodium hyaluronate (hyalectin) in osteoarthritis of the knee: a 1 year placebo-controlled trial. Osteoarthr Cart 1, 97-103. Eccles M, Freemantle N, Mason J. (1998). North of England evidence based guideline development project: summary guideline for non-steroidal antiinflammatory drugs vs basic analgesia in treating the pain of degenerative arthritis. B Med J, 317, 526-530. Elliott AL, Kraus VB, Luta G, Stable T, Renner JB,e t al. (2005). Serum hyaluronan levels and radiographic knee and hip osteoarthritis in African Americans and Caucasians in the Johnston County Osteoarthritis Project. Arthr Rheum 52, 1, 105-111. Ellis I, Schor SL. (1996). Differential effectsof TGF! 1 on hyaluronan synthesis by fetal and adult skin finbbroblasts: implications for cell migration and wound healing. Exp Cell Res 228, 326-333. Emery CF, Keefe FJ, France CR, Affleck G, Waters S, Fondow MD, McKee DC, France JL, Hackshaw KV, Caldwell DS, Stainbrook D. (2006). Effects of a brief coping skills training intervention on nociceptive flexion reflex threshold in patients having osteoarthritic knee pain: a preliminary laboratory study of sex differences. J Pain Symptom Manage, 31, 3, 262-9. Ettinger WH Jr, Burns R, Messier SP, Applegate W, Rejeski WJ, Morgan T, et al. (1997). A randomized trial comparing aerobic exercise and resistance exercise with a health education program in older adults with knee osteoarthritis. The Fitness Arthritis and Seniors Trial (FAST). J Am Med Assoc, 277, 1, 25-31. Evans RG, Collins C, Miller P, Ponsford FM, Elson CJ. (1994). Radiological scoring of osteoarthritis progression in STR/ORT mice. Osteoarthr Cart 2, 2, 103-109. Eyigor S, Hepguler S, Capaci K. (2004). A comparison of muscle training methods in patients with knee osteoarthritis. Clin Rheumatol, 23, 2, 109-115. Fang MA, Taylor CE, Nouvong A, Masih S, Kao KC, Perell KL. (2006). Effects of footwear on medial compartment knee osteoarthritis. J Rehabil Res Dev, 43, 4, 427-34. Farr J, Mont MA, Garland D, Caldwell JR, Zizic TM. (2006). Pulsed electrical stimulation in patients with osteoarthritis of the knee: follow up in 288 patients who had failed non-operative therapy. Surg Technol Int,15, 227-33. 161

PAGE 176

Felson DT. (1990). The epidemiology of knee osteoarthritis: results from the Framingham Osteoarthritis Study. Semin Arthritis Rheum 20, 42-50. Felson DT, Anderson JI, Naimark A, Walker AM, Meenan RF. (1988). Obesity and knee osteoarthritis. Ann Intern Med ,109,18-24. Felson DT, Gale DR, Elon Gale M, Niu J, Hunter DJ, et al. (2005). Osteophtyes and progression of knee osteoarthritis. Rheumatol 44, 1, 100-104. Felson DT, Goggins J, Niu J, Zhang Y, Hunter DJ. (2004). The effect of body weight on progression of knee osteoarthritis is dependent on alignment. Arthr Rheum 50, 3904-3909. Felson DT, Lawrence RC, Dieppe PA, Hirsch R, Helmick CG, et al. (2000). Osteoarthritis: new insights. Part 1: the disease and its risk factors. Ann Intern Med, 133, 635-646. Felson DT, Lawrence RC, Hochberg MC, McAlindron T, Dieppe PA, et al. (2000). Osteoarthritis: new insights. Part 2: treatment approaches. Ann Intern Med 133, 9, 726-737. Fiedler J, RodererG, Gunterh KP, Brenner RE. (2002). BMP-2, BMP-4, and PDGF-bb stimulate chemotactic migrationof primary human mesenchymal progenitor cells. J Cell Biochem 87, 3, 305-312. Fitzgerald JB, Jin M, Grodzinsky AJ. (2006). Shear and compression differentially regulate clusters of functionally related temporal transcription patterns in cartilage tissue. J Biol Chem 281, 34, 24095-24103. Foley A, Halbert J, Hewitt T, Crotty M. (2003). Does hydrotherapy improve strength and physical function in patients with osteoarthritis—a randomized controlled trial comparing a gym based and a hydrotherapy based strengthening programme. Ann Rheum Dis, 62, 12, 1162-1167. Forssblad M, Jacobson E, Weidenheilm L. (2004). Knee arthroscopy with different anesthesia methods: a comparison of efficacy and cost. Knee Surg Sports Traumatol Arthrosc 12, 5, 344-349. Fortin PR, Penrod JR, Clarke AE St.Pierre Y, Joseph L, et al. (2002). Timing of total joint replacement affects clinical outcomes among patients with osteoarthritis of the hip or knee. Arthr Rheum, 46, 3327-3330. Franceschi C, Bonafe M, valensin S, Olivieri F, De Luca M et al. (2000). Inflamm-aging. An evolutionary perspective on immunosenescence. Ann NY Acad Sci 908:244-254. 162

PAGE 177

Fraser JR, Gibson PR. (2005). Mechanisms by which food intake elevates circulating levels of hyaluronan inmhumans. J Intern Med 258, 5, 460-466. Fukui N, Zhu Y, Maloney WJ, Clohisy J, Sandell LJ. (2003). Stimulation fo BMP-2 expression by pro-inflammatory cytokines IL-1 and TNF-alpha in normal and osteoarthritic chondrocytes. J Bone Joint Surg Am 85-A, Suppl, 3, 59-66. Fulop T Jr, Fouquet C, Allaire P, et al. (1997). Changes in apoptosis of human polymorphonuclear granulocytes with aging. Mech Ageing Dev 96, 1534. Furman BD, Olson SA, Guilak F. (2006). The development of posttraumatic arthritis after articular fracture. J Orthop Trauma 20, 10, 719-725. Gaines JM, Metter EJ, Talbot LA. (2004).The effect of neuromuscular electrical stimulation on arthritis knee pain in older adults with osteoarthritis of the knee. Appl Nurs Res, 17, 3, 201-6. Gao G, Plaas A, Thompso VP, Jin S, Zyi F, Sandy JD. (2004). ADAMTS4 (aggrecanase-1) activation on the cell surface involves C-terminal cleavage by glycosylphosphatidyl inositol-anchored membrane type 4-matrix metalloproteinase and binding of the activated proteinase to chondroitin sulfate and heparin sulfate on syndecan-1. J Biol Chem 279, 11, 10042-10051. Garland D, Holt P, Harrington JT, Caldwell J, Zizic T, Cholewczynski J. (2007). A 3-month, randomized, double-blind, placebo-controlled study to evaluate the safety and efficacy of a highly optimized, capacitively coupled, pulsed electrical stimulator in patients with osteoarthritis of the knee. Osteoarthr Cart, 15, 6, 630-7. Ghosh P. (1994). The role of hyaluronic acid (hyaluronan) in health and disease: interactions with cells, cartilage and components of the synovial fluid. Clin Exp Rheumatol 12, 75-82. Giancotti FG, Ruoslahti E. (1999). Integrin signaling. Science 285, 1028-1032. Giles JT, Mease P, Boers M, Bresnihan B, Conaghan PG, et al.(2007). Assessing single joints in arthritis clinical trials. J Rheumatol 34, 3, 641-647. 163

PAGE 178

Girling SL, Bell SC, Whitelock RG, Rayward RM, Thomson DG, et al. (2006). Use of biochemical markers of osteoarthritis to investigate the potential disease-modifying effect of tibial plateau levelling osteotomy. J Small Anim Pract, 47, 12, 708-714. Glass GG. (2006). Osteoarthritis. Dis Mon 52, 343-362. Glasson SS, Blanchet TJ, Morris EA. (2007). The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthr Cart, 15, 1061-1069. Goldberg VM, Buckwalter JA. (2005). Hyaluronans in the treatment of osteoarthritis of the knee: evidence for disease-modifying activity. Osteoarthr Cart 13, 216-224. Gomez Cr, Boehmer ED, Kovacs EJ. (2005). The aging innate immune system. Curr Opin Immun 17, 457-462. Gtzsche PC. (2005). Musculoskeletal disorders. Non-steroidal antiinflammatory drugs Clin Evid, 14, 1498-1505. Gough AW, Kasali OB, Sigler RE, Baragi V. (1992). Quinolone arthropathy-acute toxicity to immature articular cartilage. Toxicol Pathol 20, 436450. Gray ML, Pizzanelli AM, Lee RC, Grodzinsky AJ, Swann DA. (1989). Kinetics of the chondrocyte biosynthetic response to compressive load and release. Biochim Biophys Acta, 991, 3, 415-425 Gross DE, Brenner SL, Esformes I, Gross ML. (1991). Arthroscopic treatment of degenerative joint disease of the knee. Orthop, 14, 1317-1321. Guingamp C, Gegout-Pottie P, Phillippe L, Terlain B, Netter P, et al. (1997). Mono-iodoacetate-induced experimental osteoarthritis: a dose-response study of loss of mobility, morphology and biochemistry. Arthr Rheum 40, 16701679. Gur H, Cakin N, Akova B, Okay E, Kucukoglu S. (2002). Concentric versus combined concentric-eccentric isokinetic training: effects on functional capacity and symptoms in patients with osteoarthrosis of the knee. Arch Phys Med Rehabil 83, 308-316 Hakim AJ, Sahota A. (2006). Joint hypermobility and skin elasticity: the hereditary disorders of connective tissue. Clin Dermatol 24, 6, 521-533. 164

PAGE 179

Hannan MT, Felson DT, Andrews, JJ, Naimark A. (1993). Habitual physical activity is not associated with knee osteoarthritis: the Framingham Study. J Rheumatol 20, 204-209. Hansen PA, Reed K. (2006). Common musculoskeletal problems in the performing artist. Phys Med Rehabil Clin N Am 17, 4, 789-801. Hayami T, Pickarski M, Zhuo Y, et al. (2006). Characterization of articular cartilage and subchondral bone changes in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthrits. Bone 38, 234-243. Hecht PJ, Backmann S, Booth RE, Rothman RH. (1983). Effects of Thermal Therapy on Rehabilitation after Total Knee Arthroplasty : A Prospective Randomized Study. Clin Orthop Rel Res, 178, 198–201. Heino TJ, Hentunen TA, Vaananen HK. (2002). Ostoecytes inhibit osteoclastic bone resorption through transforming growth factor-beta: enhancement by estrogen. J Cell Biochem 85, 185-197. Heuts PH, de Bie R, Drietelaar M, Aretz K, Hopman-Rock M, Bastiaenen CH, Metsemakers JF, van Weel C, van Schayck O. (2005). Self-management in osteoarthritis of hip or knee: a randomized clinical trial in a primary healthcare setting. J Rheumatol, 32, 3, 543-9. Hootman J, Fitzgerald S, Macera C, et al. (2004). Lower extremity muscle strength and risk of self-reported hip or knee osteoarthritis. J Phys Activity Health 1, 321-330. Hrobjartsson A & Gotzsche PC. (2001). Is the placebo powerless? An analysis of clinical trials comparing placebo with no treatment. N E J Med, 344, 1594-1602. Hunt TR. (2006). Degenerative and post-traumatic arthritis affecting the carpometacarpal joints of the fingers. Hand Clin 22, 2, 221-228. Huskisson EC, Donnelly S. (1999). Hyaluronic acid in the treatment of osteoarthritis of the knee. Rheumatology 38, 602-607. Huysse WCJ, Verstraete KL. (2009). Heath technology assessment of magnetic resonance imaging of the knee. Eur J Radiol 65, 2, 190-193. Iannone F, Lapadula G (2003). The pathophysiology of osteoarthritis. Aging Clin Exp Res ,15, 364-372. 165

PAGE 180

Inoue A, Takahashi KA, Arai Y, et al. (2006). The therapeutic effects of basic fibroblast growth factor contained in gelatin hydrogel microspheres on experimental osteoarthritis in the rabbit knee. Arthr Rheum 54, 264-270. Jacenko O, Olsen BR. (1995). Transgenic mouse models in studies of skeletal disorders. J Rhuematol Suppl 43, 39-41. Janusz MJ, Hookfin EB, Heitmeyer SA, Woessner JF, Freemont AJ, et al. (2001). Moderateion of iodoacetate-induced experimental osteoarthritis in rats by matrix metalloproteinase inhibitors. Osteoarthr Cart 9, 751-760. Jinks C, Jordan K, Croft P. (2006). Disabling knee pain—another consequence of obesity: results from a prospective cohort study. BMC Pub Health 6, 258. Johnson LA, Prevo R, Clasper S, Jackson DG. (2007). Inflammationinduced uptake and degradation of the lymphatic endothelial hyaluronan receptor LYVE-1. J Biol Chem 282, 46, 33671-33680. Jordan KM, Arden NK, Doherty M, Bannwarth B, Bijlsma JW, et al. (2003). EULAR Recommendations 2003: an evidence based approach to the management of knee osteoarthritis: Report of a task force of the standing committee for international clinical studies including therapeutic trials (ESCISIT). Ann Rheum Dis 62, 12, 1145-1155. Jordan JM, Luta G, Renner JB, Dragomir A, Hochberg MC, et al. (1996). Ethnic differences in self-reported functional status in the rural South: the Johnston County Osteoarthritis Project. Arthritis Care Res 9, 483-491. Julovi SM, Yasuda T, Shimizu M, Hiramitsu T, Nakamura T. (2004). Inhibition of interleukin-1beta-stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage. Arthritis Rheum 50, 516525. Ju¨ni P, Dieppe P. (2004). Older people should NOT be prescribed ‘coxibs’ in place of conventional NSAIDs. Age Ageing, 33, 100-104. Ju¨ni P, Dieppe P, Egger M. (2002). Risk of myocardial infarction associated with selective COX-2 inhibitors: questions remain. Arch Int Med, 162, 2639-2642. Kamekura S, Hoshi K, Shimoaka T, Chung U, Chikuda H, et al. (2005). Osteoarthritis development in novel ex perimental mouse models induced by knee joint instability. Osteoarthr Cart, 13, 632-641. 166

PAGE 181

Kerin A, Patwari P, Kuettner K, Cole A, Grodzinsky A.(2002). Molecular basis of osteoarthritis: biomechanical aspects. Cell Mol Life Sci 59, 1, 27-35. Khan R, Sheppard R. (2006). Fibrosis in heart disease: understanding the role of transforming growth factor-beta in cardiomyopathy, valvular disease and arrhythmia. Immunology 118, 1, 10-24. Kim H, Lo M, Pillarisetty R. (2002). Chondrocyte apoptosis following intrarticular fracture in humans. Osteoarthr Cart ,10, 747-749. Kim YJ, Bonassar LJ, Grodzinsky AJ. (1995). The role of cartilage streaming potential, fluid flow and pressure in the stimulation of chondrocyte biosynthesis during dynamic compression. J Biomech, 28, 9, 1055-1066. Kirkinezos IG, Hernandez D, Bradley WG, Moraes CT. (2003). Regular exercise is beneficial to a mouse model of amyotrophic lateral sclerosis. Ann Neurol 53, 804-807. Kloppenburg M, Stamm T, Watt I, Kainberger F, Caston TE, et al. (2007). Research in hand osteoarthritis: time for reappraisal and demand for new strategies. Ann Rheum Dis, 66, 9, 1157-1161. Kloth LC. Electrical stimulation for wound healing: a review of evidence from in vitro studies, animal experiments, and clinical trials. (2005). Int J Low Ext Wounds 4, 1, 23-44. Kobayashi H, Terao T. (1997). Hyaluronic acid specific regulation of cytokines by human uterine fibroblasts. Am J Physiol 376, C1151-1159. Kobayashi K, Amiel M, Harwood FL, Healey RM, Sonoda M, et al. (2000). The long-term effects of hyaluronan during development of osteoarthritis following partial meniscectomy in a rabbit model. Osteoarthr Cart 8, 359-365. Kobayashi K, Mishima H, Harwood F, Hashimoto S, Toyoguchi T, Goomer R, et al. (2002). The suppressive effect of hyaluronan on nitric oxide production and cell apoptosis in the central region of meniscus following partial meniscectomy. Iowa Orthop J 22, 39-41. Koh J, Dietz J. (2005). Osteoarthritis in other joints (hip, elbow, foot, ankle, toes, wrist) after sports injuries. Clin Sports Med 24, 1, 57-70. Koli K, Myllamiemi M, Keski-Oja J, Kinnula VL. (2008). Transforming growth factor-beta activation in the lung: focus on fibrosis and reactive oxygen species. Antioxid Redox Signal 10, 2, 333-342. 167

PAGE 182

Kopec JA, Rahman MM, Berthelot JM, Le Petit C, Aghajanian J, et al. (2007). Descriptive epidemiology of osteoarthritis. J Rheumatol 34, 2, 386-393. Kotz R, Kolarz G. (1999). Intra-articular hyaluronic acid: duration of effect and results of repeated treatment cycles. Am J Orthop 28, 11 suppl, 5-7. Kovar PA, Allegrante JP, MacKenzie CR, Peterson MG, Gutin B, Charlson ME. (1992). Supervised fitness walking in patients with osteoarthritis of the knee. A randomized, controlled trial. Ann Intern Med, 116, 7, 598-599. Kraus VB. (2006). Do biochemical markers have a role in osteoarthritis diagnosis and treatment? Best Pract Res Clin Rheumatol 20, 1, 69-80. Lane NE, Lian K, Nevitt MC, Zmuda JM, Lui L, et al. (2006). Frizzledrelated protein variants are risk factors for hiop osteoarthritis. Arthr Rheum 54, 1246-1254. Langworthy MJ, Nelson FRT, Coutts RD. (2004). Basic science. In Articular Cartilage Lesions: A Practical Guide to Assessment and Treatment. jCole BJ, Malek MM. Springer: New York. Lapvetelainen T, Hyttinen MM, Saamanen AM, Langsjo T, Sahlman J, et al. (2002). Lifelong voluntary joint loading increases osteoarthritis in mice housing a deletion mutation in type II procollagen gene, and slightly also in non transgenic mice. Ann Rheum Dis 61, 810-817. Laurent TC, Laurent UBG, Fraser JRE. (1996). The structure of hyaluronan: an overview. Immunol Cell Biol 74, A1-A7. Laurent D, O’Byrne E, Wasvary J, Pellas TC. (2006). In vivo MRI of cartilage pathogenesis in surgical models of osteoarthritis. Skeletal Radiol 35, 555-564. Lawrence RC, Helmick CG, Arnett FC, Deyo RA, Felson DT, et al. (1998). Estimates of the prevalence of arthritis and selected musculoskeletal disorders in the United States. Arthritis Rheum 41, 778-799. Leardini G, Franceschini M, Mattara L, Bruno R, Perbellini A. Intraarticular sodium hyualuronate (Hyalgan) in gonarthrosis. Clin Trial J 24, 341350. Lee CL, Huang MH, Chai CY, Chen CH, Su JY, Tien YC. (2007). The validity of in vivo ultrasonographic grading of osteoarthritis femoral condylar cartilage: a comparison with in vitro ultrasonographic and histologic gradings. Osteoarthr Cart Article in press, doi:10:1016/j.joca.2007.07.013. 168

PAGE 183

Lee JH, Fitzgerald JB, Dimicco MA, Grodzinsky AJ. (2005). Mechanical injury of cartilage explants causes specific time-dependent changes in chondrocyte gene expression. Arthritis Rheum 52, 8, 2386-2395. Lewis CW, Williamson AK, Chen AC, Bae WC, Temple MM, et al. (2005). Evaluation of subchondral bone mineral density associated with articular cartilage structure and integrity in healthy equine joints with different functional demands. Am J Vet Res 6, 10, 1823-1829. Li X-Q, Thonar EJ-MA, Knuedson W. (1989). Accumulation of hyaluronate in human lung carcinoma as measured by a new hyaluronate ELISA. Connect Tissue Res 19, 243-253. Li Y, Olsen BR. (1997). Murine models of human genetic skeletal disorders. Matrix Biol 16, 49-52. Lievense AM, Bierma-Zienstra SM, Verhagen AP, van Baar ME, Verhaar JA, et al. (2002). Influence of obesity on the development of osteoarthritis of the hip: a systematic review. Rhematology, 41, 1155-1162. Lightfoot TJ, Turner MJ, Debate KA, Kleeberger SR. (2001). Interstrain variation in murine aerobic capacity. Med Sci Sports Exerc 33, 12, 2053-2057. Lin DA, Lin YF, Chai HM, Han YC, Jan MH. (2007) Comparison of proprioceptive functions between computerized proprioception facilitation exercise and closed kinetic chain exercise in patients with knee osteoarthritis. Clin Rheum 26, 4, 520-528. Lohmander LS, Eyre DR. (2005). From biomarker to surrogate outcome to osteoarthritis--what are the challenges? J Rheumatol 32, 6, 1142-1143. Lopponen T, Korkko J, Lundan T, Seppanen U, Ignatius J, Kaariainen H. (2004). Childhood-onset osteoarthritis, tall stature, and sensorineural hearing loss associated with Arg75-Cys mutation in procollagen type II gene (COL2A1). Arthr Rheum 51, 6, 925-932. Lord JM, Butcher S, Killampali V, et al. (2001). Neutrophil ageing and immunosenescence. Mech Ageing Dev 122, 1521-1535. Lorig K. Ritter PL, Plant K. (2005). A disease specific self-help program compared with a generalized chronic disease self-help program for arthritis patients. Arthr Rheum 53, 6, 950-957. 169

PAGE 184

Lorig KR, Sobel DS, Stewart B, Brown WJ, Bandura A, et al (1999). Evidence suggesting that a chronic disease self-management program can improve health status while reducing hospitalization: a randomized trial. Med Care 37, 5-14. Lunn RA, Sumar N, Bansal AS, Treleaven J. (2005). Cytokine profiles in stem cell transplantation: possible uses as a predictor of graft-versus-host disease. Hematology 10, 2, 107-114. Mackay HE, Cope MR, Pilling D, Bruce CE. (2006). Can CT determine the site of traumatic osteochondral defects in the paediatric knee? Injury 37, 6, 513-515. Madsen SJ, Patterson MS, Wilson BC. (1992). The use of India ink as an optical absorber in tissue-simulating phantoms. Phys Med Biol 37, 985-993. Mahoney DJ, Rodriguez C, DeVries M, Yasuda N, Tarnopolsky MA. (2004). Effects of high-intensity endurance exercise training in the G93A mouse model of amyothrophic lateral sclerosis. Muscle Nerve, 29, 656-662. Mallen CD, Peat G,Thomas E, Lacey R, Croft P. (2007). Predicting poor functional outcome in community-dwelling older adults with knee pain: prognostic value of generic indicators. Ann Rheum Dis 66, 11, 1456-1461. Mangani I, Cesari M, Kritchevsky SB, Maraldi C, Carter CS, Atkinson HH, et al. (2006). Physical exercise and comorbidity. Results from the Fitness and Arthritis in Seniors Trial (FAST). Aging Clin Exp Res, 18, 5, 374-380. Mangione KK, McCully K, Gloviak A, Lefebvre I, Hoffman M, Craik R. (1999). The effect of high-intensity and low-intensity cycle ergometry in older adults with knee osteoarthritis. J Gerontol 54-A, 4, M184-M190. Marlovits S, Zeller P, Singer P, Resinger C, Vecsei V. (2005). Cartilage repair: Generations of autolougous chondrocyte transplantation. Eur J Radiol 57, 1, 24-31. Maroudas A. (1979). Physico-chemical properties of articular cartilage. In: Freeman MAR, editor. Adult articular cartilage, 2nd ed. England: Tunbridge Wells, pp 215-290. Marsh JL. (2004). Postraumatic arthritis: What is the burder of disease? In: Schemitsch EM ed. Orthopedic Trauma AssociationBasic Science Forum, pp 93. 170

PAGE 185

Martin JA, Scherb MB, Lembke LA, Buckwalter JA. (2000). Damage control mechanisms in articular cartilage: the role of the insulin-like growth factor I axis. Iowa Orthop J 20, 1-10. Martin K, Nicklas B, Dennis K, Goldberg A, Hochberg M. (1996). Weight loss and exercise walking improve pain and physical functioning in overweight women with knee pain. Arthritis Rheum, 39, 9S. Marziali E, Donahue P. (2006). Caring for others: internet videoconferencing group intervention for family caregivers of older adults withneurodegenerative disease. Gerontologist 46, 3, 398-403. Mason RM, Chambers MG, Flannelly J, Gaffen JD, Dudhia J, et al. (2001). The STR/ort mouse and its use as a model of osteoarthritis. Osteoarthr Cart, 9, 85-91. Massett MP, Berk BC. (2005). Strain-dependent differences in responses to exercise training in inbred and hybrid mice. Am J Physiol Regul Integr Comp Physiol 288, R1006-R1013. Mast BA, Haynes JH, Krummel TM, Diegelmann RF, Cohen IK. (1992). In vivo degradation of fetal wound hyaluronic acid results in increased fibroplasia, collagen deposition, and neovascularization. Plast Reconstr Surg, 89, 503-509. Mastenbergen SC, Marijnissen AC, Vianen ME, et al. (2006). The canine “groove” model of osteoarthritis is more than simply the expression of surgically applied damage. Osteoarthr Cart 14, 39-46. Mazzuca SA, Brandt KD, German NC, Buckwalter KA, Lane KA, et al. (2003). Development of radiographic changes of osteoarthritis in the “Chingford knee” reflects progressionof disease or non-standardised positioning of the joint rather thanincident disease. Ann Rheum Dis 62, 11, 1061-1065. McAlindon TE, Cooper C, Kirwan JR, Dieppe PA. (1992). Knee pain and disability in the community. Br J Rheumatol 31, 189-192. McEldowney AJ, Weiker GG. (1995). Open-knee Magnuson debridement as conservative treatment for degenerative osteoarthritis of the knee. J Arthroplasty, 10, 805-809. McLaren AC, Blokker CP, Fowler PJ, Roth JN, Rock MGl. (1991). Arthroscopic debridement of the knee for osteoarthrosis. Can J Surg, 34, 595598. 171

PAGE 186

McMillan G, Nichols L. (2005). Osteoarthritis and meniscus disorders of the knee as occupational diseases of miners. Occup Environ Med 62, 8, 567575. Meachim G. (1972). Light microscopy of India ink preparations of fibrillated cartilage. Ann Rheum Dis 31, 457-464. Messier S, Loeser R, Mitchell M, Valle G, Morgan TP, et al. (2000). Exercise and weight loss in obese older adults with knee osteoarthritis: a preliminary study. J Am Geriatr Soc, 48, 1062–72. Messier SP, Loeser RF, Miller GD, Morgan TM, Rejeski WJ, et al. (2004). Exercise and dietary weight loss in overweight and obese older adults with knee osteoarthritis: the Arthritis, Diet, and Activity Promotion Trial. Arthritis Rheum 50, 1501-1510. Miceli-Richard C, Le Bars M, Schmidely N, Dougados M. (2004). Paracetamol in osteoarthritis of the knee. Ann Rheum Dis 63, 923-930. Minor MA, Hewett JE, Webel RR, Anderson SK, Kay DR. (1989). Efficacy of physical conditioning exercise in patients with rheumatoid arthritis and osteoarthritis. Arthritis Rheum 32, 11, 1396-1405. Mistry D, Oue Y, Chambers MG, Kayser MV, Mason RM. (2004). Chondrocyte death during murine osteoarthritis. Osteoarthr Cart 12, 131-141. Miyaguchi M, Kobayashi A, Kadoya Y, Ohashi H, yamano Y, et al. (2003). Biochemical change in joint fluid after isometric quadriceps exercise for patients with osteoarthritis of the knee. Osteoarthr Cart, 11, 252-259. Modawal A, Ferrer M, Choi HK, Castle JA. (2005). Hyaluronic acid injections relieve knee pain. J Fam Pract 54, 758–767. Moore EE, Bendele AM, Thompson DL, et al. (2005). Fibroblast growth factor-1B stimulates chondrogenesis and cartilage repair in a rat model of injury induced osteoarthritis. Osteoarthr Cart 13, 623-631. Moseley JB, O’Malley K, Petersen NJ, Menke TJ, Brody BA, et al. (2002). A controlled trial of arthroscopic surgery for osteoarthritis of the knee. N Engl J Med 347, 2, 81-88. Muraoka T, Hagino H, Okano T, Enokida M, Teshima R. (2007). Role of subchondral bone in osteoarthritis development: a coparative study of two strains of guinea pigs withand withut spontaneously occurring osteoarthritis. Arthr Rheum 56, 10, 3366-3374. 172

PAGE 187

Murphy EA, Davis JM, Brown AS, Carmichael MD, Mayer EP, Ghaffar A. (2004). Effects of moderate exercise and oat-glucan on lung tumor metastases and macrophage antitumor cytotoxicity. J Appl Physiol 97, 955-959. Mutlu M, Argun M, Kilic E, saraymen R, Yazar S. (2007). Magneium, zinc, and copper status in osteoporotic, osteopenic and normal post-menopausal women. J Int Med Res 35, 5, 692-695. Nagase H, Kashiwagi M. (2003). Aggrecanases and cartilage matrix degradation. Arthritis Res Ther 5, 2, 94-10 3 National Centers for Health Statistics, 2004. Neame R, Zhang W, Doherty M. (2004). A historic issue of the Annals: three papers examine paracetamol in osteoarthritis. Ann Rheum Dis, 63, 897-900. NehlsV, Hayen W. (2000). Are hyaluronan receptors involvedin threedimensional cell migration? Histol Histopathol 14, 2, 629-636. Nemirovskiy OV, Dufield DR, Sunyer T, Aggarwal P, Welsch DJ, Mathews WR. (2007). Discovery and development of a type II collagen neoepitope (TIINE) biomarker for matrix metalloproteinase activity: from in vitro to in vivo. Anal Biochem 361, 1, 93-101. Nevitt MC, Felson DT. (1996). Sex hormones and the risk of osteoarthritis in women: epidemiological evidence. Ann Rheum Dis, 55, 673–6. Noble PW, Lake FR, Henson PM, Riches DW. (1993). Hyaluronate activation of CD44 induces insulin-like growth factor-1 expression by a tumor necrosis factor-alpha-dependent mechanism in murine macrophages. J Cln Invest, 91, 2368-2377. Nuez M, Nuez E, Segur JM, Macule F, Quinto L, Hernandez MV, Vilalta C. (2006). The effect of an educational program to improve health-related quality of life in patients with osteoarthritis on waiting list for total knee replacement: a randomized study. Osteoarthr Cart, 14, 3, 279-85. Oegema TR, Visco D. (1999). Animal models of osteoarthritis. In Animal Models in Orhtopeaedic Research An YH, Friedman RJ, eds., New York: CRC Press. pp 349-367. 173

PAGE 188

Okazaki K, Jinguishi S, Ikenoue T, Urabe K, Sakai H, et al. (1999). Expression of insulin-like growth factor I messenger ribonucleic acid in developing osteophytes in murine experimental osteoarthritis and in rats inoculated with growth hormone-secreting tumor. Endocrinology 140, 10, 48214830. Olsen BR. (1997). Collagen IX. Int J Biochem Cell Biol 29, 555-558. Olson SA, Marsh JL. (2004). Posttraumatic osteoarthritis. Clin Orthop Relat Res 423, 2. Olson TP, Dengel DR, Leon AS, Schmitz KH. (2007). Changes in inflammatory biomarkers following one-year of moderate resistance training in overweight women. Int J Obes online publication February 13, 2007, e –pub ahead of print, accessed Mar 1, 2007. O’Reilly SC, Muir KR, Doherty M. (1998). Knee pain and disability in the Nottingham community assocication with poor health status andd psychological distress. Br J Rheumatol 37, 870-873. Ostergaard K, Petersen J, Andersen CB, B:endtzen K, Salter DM. (1997). Histologic/histochemical grading system for osteoarthritic articular cartilage. Reproducibility and validity. Arthr Rheum 40, 10, 1766-1771. Pannu J, Gardner H, Shearstone JR, Smith E, Trojanowska M. (2006). Increased levels of transforming growth factor beta receptor type I and upregulation of matrix gene program: a model of scleroderma. Arthritis Rheum 54, 9, 3011-3021. Patel S, Adams MR. (2008). Preventionof cardiac disease: lifestyle modification or pharmacotherapy? Intern Med J 38, 3, 199-203. Pavolich RI, Lubowitz J. (2008). Current concepts in synovial tissue of the knee joint. Orthopedics, 31, 160-164. Pennix BW, Abbas H, Ambrosius W, Nicklas BJ, Davis C, et al. (2004). Inflammatory markers and physical function among older adults with knee osteoarthritis. J Rheumatol, 31(10), 2027-2031. Penninx BW, Rejeski WJ, Pandya J, Miller ME, Di Bari M, Applegate WB, Pahor M. (2002). Exercise and depressive symptoms: a comparison of aerobic and resistance exercise effects on emotional and physical function in older persons with high and low depressive symptomatology. J Gerontol B Psychol Sci Soc Sci, 57, 2, P124-132. 174

PAGE 189

Petersson I. (1996). Occurrence of osteoarthritis of the peripheral joints in European populations. Ann Rheum Dis 55, 659-661. Pham T, Maillefert JF, Hudry C, Kieffert P, Bourgeois P, Lechevalier D, Dougados M. (2004). Laterally elevated wedged insoles in the treatment of medial knee osteoarthritis. A two-year prospective randomized controlled study. Osteoarthr Cart, 12, 1, 46-55. Pincus T, Koch G, Lei H, Mangal B, Sokka T, et al. (2004). Patient preference for placebo, acetaminophen (paracetamol) or celecoxib efficacy studies (PACES): two randomised, double blind, placebo controlled, crossover clinical trials in patients with knee or hip osteoarthritis. Ann Rheum Dis, 63, 931939. Plaas A, Osborn B, Yoshihara Y, Bai Y, Bloom T, et al. (2007). Aggrecanolysis in human osteoarthritis: confocal localization and biochemical characterization of ADAMTS5-hyaluraonan complexes in articular cartilages. Osteoarthr Cart 15, 11, 1318-1325. Plaas AHK, West LA, Midura RJ. (2001). Keratan sulfate disaccharide composition determined by FACE analysis of keratanase II and endo! galactosidase digestion products. Glycobiology 11, 10, 779-790. Plackett TP, Boehmer ED, Faunce DE, Kovacs EJ. (2004). Aging and the innate immune cells. J Leukoc Biol 76, 76, 292-299. Polyzois VD, Papakostas I, Zgonis T, Polyzois GD, Soucacos PN. (2006). Current concepts and techniques in posttraumatic arthritis. Clin Podiatr Med Surg 23, 2, 455-465. Pratta MA, Yao W, Decicco C, Tortorella MD, Liu RQ, et al. Aggrecan protects cartilage collagen from proteolytic cleavage. J Biol Chem 278, 46, 45539-45545. Presti D, Scott JE. (1994). Hyaluraonan mediated protective effect against cell damage caused by enzymatically genereated hydroxyl radicals is dependent onhyaluroanan molecular mass. Cell Biochem Funct 12, 281-288. Pritzker KPH, Gay S, Jimenez SA, Ostergaard K,Pelletier JP et al. (2006). Osteoarthrits artilage histopathology: grading and staging. Osteoarthr Cart 14, 1, 13-29. Puhl W, Bernau A, Greiling H, Kopcke W, Pforringer Q, et al. (1993). Intra-articular sodium hyaluronate in osteoarthritis of the knee: a multicenter, double-blind study. Ostoearthr Cart 1, 233-241. 175

PAGE 190

Pujol JP, Galera P, Redini F, Mauviel A, Loyau G. (1991). Role of cytokines ini osteoarthritis: comparative e ffects of interleukin 1 and transforming growth factor-beta on cultured rabbit articular chondrocytes. J Rheumatol 27, Suppl, 76-79. Radin EL. (1999). Subchondral bone changes and cartilage damage. Equine Vet J 31, 2, 94-95. Rappolee DA,, Werb Z. (1992). Macrophage-derived growth factors. Curr Top Microbiol Immunol 181, 87-140. Ravinal RC, Costa RS, Coimbra TM, Dantas M, Dos Reis MA. (2005). Mast cells, TGF-beta1 and myofibroblasts expression in lupus nephritis outcome. Lupus 14, 10, 814-821. Rayan V, Thonar EJ-MA, Chen L-M, Lenz ME, Williams JM. (1998). Regional diffeences in the rise in blood levels of antigenic keratin sulfate and hyaluronan after chymopapain induced knee joint injury. J Rheuamtol 25, 521526. Regan E, Flannelly J, Bowler R, Tran K, Nicks M, et al. (2005). Extracellular superoxide dismutase and oxidant damage in osteoarthritis. Arthr Rheum 52(11), 3479-3491. Rejeski WJ, Ettinger WH Jr, Martin K, Morgan T. (1998). Treating disability in knee osteoarthritis with exercise therapy: a central role for self-efficacy and pain. Arthr Care Res, 11, 2, 94-101. Rejeski WJ, Focht BC, Messier SP, Morgan T, Pahor M, et al. (2002). Obese, older adults with knee osteoarthritis: weight loss, exercise, and quality of life. Health Psychol 21(5), 419-426. Richy F, Bruyere O, Ethgen O, Rabenda V, Bouvenot G, et al. (2004). Time dependent risk of gastrointestinal complications induced by non-steroidal anti-inflammatory drug use: a consensus statement using a meta-analytic approach. Ann Rheum Dis, 63, 759-766. Richter W. (2007). Cell-based cartilage repair: illusion or solution for osteoarthritis. Curr Opin Rheumatol 19, 5, 451-456. Roddy E, Zhang W, Doherty M, Arden NK, Barlow J, et al. (2005). Evidence-based recommendations fo the role f exercise in the management of osteoarthritis of the hip or knee—the MOVE consensus. Rheumatology 44, 6773. 176

PAGE 191

Roos EM. (2005). Joint injury causes knee osteoarthritis in young adults. Curr Opin Rheumatol 17, 2, 195-200. Roos EM, Dahlberg L (2005) Positive effects of moderate exercise on glycosaminoglycan content in knee cartilage. Arthr Rheum 52(11), 3507-3514. Roughley P, Martens D, Rantakokko J, Alini M, Mwale F, Antoniou J. (2006). The involvement of aggrecan polymorphism in degeneration of human intervertebral disc and articular cartilage. Eur Cell Mater 11, 1-7. Saamanen AM, Hyttinen M, Vuorio E. (2007). Analysis of arthritic lesions in the Del1 mouse: a model for osteoarthritis. Methods Mol Med 136, 283-302. Saamanen AM, Salminen H, de Crombrugghe, Vuorio EI, Metsaranta MP. (2000). Osteoarthritis-like lesions in transgenic mice harboring a small deletion mutation in type II collagen gene. Osteoarthr Cart 8, 248-257. Sabaratnam S, Mason RM, Levick JR. (2003). Molecular sieving of hyaluronan by synovial interstitial matrix and lymphatic capillary endothelium evaluated by lymph analysis n rabbits. Microvasc Res 66, 3, 227-236. Sah RL, Kim YJ, Doong JY, Grodzinsky AJ, Plaas AH, Sandy JD. (1989). Biosynthetic response of cartilage explants to dynamic compression. J Orthop Res, 7, 5, 619-636. Sandy JD. (2006). A contentious issue finds some clarity: on the independent and complementary roles of aggrecanase activity and MMP activity in human joint aggrecanolysis. Osteoarthr Cart, 14, 2, 95-100. Sandy JD, Verscharen C. (2001). Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activityis required for C-terminal processing in vivo. Biochem J 358, 615-626. Schilke JM, Johnson GO, Housh TJ, O’Dell JR. (1996). Effects of musclestrength training on the functional status of patients with osteoarthritis of the knee joint. Nurs Res, 45, 2, 68-72. Schmidt TA, Sah RL. (2007). Effect of synovial fluid on boundary lubricationof articular cartilage. Osteoarthr Cart 15, 1, 35-47. 177

PAGE 192

Schrier I. (2004). Muscle dysfunction versus wear and tear as a cause of exercise related osteoarthritis: an epidemiological update. Br J Sports Med 38, 526-535. Schouten JS, van den Ouweland FA, Valkenburg HA. (1992). Prognostic factors of progression of knee osteoarthritis: 25 year follow up study in the general population on prognostic factors of cartilage loss in osteoarthritis of the knee. Ann Rheum Dis 51, 932-937. Scott D, Smith C, Lohmander S, Chard JA. (2003). Osteoarthritis. Clin Evid, 10, 1402-1430. Scott WW Jr, Beall DP, Eng J, Matthiesen CL, Prater S, Enis J. (2007). Improved demonstrationof cartilage narrowingint eh knee joint using standing PA flexed radiographs. J Okla Stat Med Assoc 100, 12, 469-472. Seki E, DeMinicis S, Osterreicher CH, Kluwe J, Osawa Y, et al. (2007). TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nat Med 13, 11, 1324-1332. Senchina DS, Kohut ML. (2007). Immunological outcomes of exercise in older adults. Clin Interv Aging 2, 1, 3-16. Sharma L, Dunlop D, Cahue S, Song J, Hayes KW. (2003). Quadriceps strength and osteoarthritis progression in malaligned and lax knees. Ann Intern Med 138, 613-619. Sharma L, Song J, Felson DT, Cahue S, Shamiyeh E, Dunlop DD. (2001). The role of knee alignment in disease progression and functional decline in knee osteoarthritis. J Am Med Assoc 286, 188-195. Shirasawa S, Sekiya I, Sakaguchi Y, Yagishita K, Ichinose S, Muneta T. (2006). In vitro chondrogenesis of hum an synovium-derived mesenchymal stem cells: optimal condition and comparison with bone marrow derived cells. J Cell Biochem, 97, 1, 84-97. Silberberg M, Silberberg R. (1960). Osteoarthrosis in mice fed diets enriched with animal or vegetable fat. Arch Pathol 70,385-390. Simonin MA, Gegout-Pottie P, Minn A, Gillet P, Netter P, Terlain B. (1999). Proteoglycan and collagen biochemical variations during fluoroquinolonge-induced chondrotoxicity inmice. Antimicrob Agents Chemother 43, 12, 2915-2921. 178

PAGE 193

Slemenda C, Heilman D, Brandt K, Katz B, Mazucca SA, et al. (1998). Reduced quadriceps relative to body weight: a risk factor for knee osteoarthritis? Arthr Rheum 41, 1951-1959. Slevin M, Krupinski J, Gaffney J, Matou S, West D, et al. (2007). Hyaluronan-mediated angiogenesis in vascular disease: Uncovering RHAMM and CD44 receptor signaling pathways. Matrix Biol 26, 1, 58-68. Smith GN Jr. (2006). The role of collagenolytic matrix metalloproteinases in the loss of articularcartilage in osteoarthritis. Front Biosci 11, 3081-3095 Smith MD, Barg E, Weedon H. (2003). Microarchitecture and protective mechanism in syovial tissue from clinically and arthroscopically normal knee joints. Ann Rheum Dis 62, 4, 303-307. Sniekers YH, Intema F, Lafeber FP, van Osch GJ, van Leeuwen JP, et al., A role for subchondral bone changes in the process of osteoarthritis; a microCT study of two canine models. BMC Musculoskelet Disord 9, 1, 20. Sokoloff L, Jay G Jr. (1956). Natural history of degenerative joint disease in small laboratory animals. II. Epiphyseal maturation of OA of the knee of mice of inbred strains. Arch Pathol 62, 129-135. Song RH, Tortorella MD, Malfait AM, Alston JT, Yang Z, et al. (2007). Aggrecan degradation inhuman articular cartilage explants is mediated by both ADAMTS-4 and ADAMTS-5. Arthr Rheum 56, 2, 575-585. Spector TD, Hart DJ, Doyle DV. (1994). Incidence and progression of osteoarthritis in women with unilateral knee disease in the general population: the effect of obesity. Ann Rheum Dis, 53, 565–8. Stack RJ, Sullivan MT. (1992). Electrophoretic resolution and fluorescence detection of N-linked glycoprot ein oligosaccharides after reductive amination with 8animonaphthalene-1,2,3-trisulphonicacid. Glycobiology 2, 8592. Stanton H, Rogerson FM, East CJ, Golub SB, Lawlor KE, et al. (2005). ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature, 434, 648-652. Stoker AM, Cook JL, Kuroki K, Fox DB. (2006). Site-specific analysis of gene expression in early osteoarthritis using the Pond-Nuki model in dogs. J Orthop Surg, 1:8. 179

PAGE 194

Stout RD, Suttles J. (2005). Imunosenescence and macrophage functional plasticity: dysregulation of macrophage function by age-associated mircoenvironmental changes. Immunol Rev 205, 60-71. Sullivan T, Allegrante JP, Peterson MG, Kovar PA, MacKenzie CR (1998). One-year Follow up of patients with osteoarthritis of the knee who participated in a program of supervised fitness walking and supportive patient education. Arthritis Care Res 11, 228-233. Sumer EU, Schaller S, Sondergaard BC, Tanko LB, Qvist P. (2006). Application of biomarkers in the clinical development of new drugs for chondroprotection in destructive joint diseases: a review. Biomarkers 11, 6, 485506. Sutton S, Clutterbuck A, Harris P, Gent T, Freeman S, et al. (2007). The contibutionof the synovium, synovial derived inflammatory cytokines, and neuropeptides to the pathogenesis of osteoarthritis. Vet J, doi:10.1016/j.t vjl.2007.08.013. Swain DP, Parrott JA, Bennett AR, Branch JD, Dowling EA. (2004) Validation of a new method for estimating VO2max based on VO2 reserve. Med Sci Sports Exerc 36, 8, 1421-1426. Takahashi K, Goomer RS, Harwood F, Kubo T, Hirawawa Y, Amiel D. (1999). The effects of hyaluronan on matrix metalloproteinase-3 (MMP-3), interleukin-1b, and tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression during the development of osteoarthritis. Ostoarthr Cart 7, 182-190. Takahashi K, Hashimoto S, Kubo T, Hirasawa Y, Lots M, Amiel D. (2000). Effect of hyaluronan on chondrocyte apoptosis and hitric oxide production in experimentally induced osteoarthritis. Rheumatol 27, 7, 1712-1720. Takahashi T, Tominaga K, Takano H, Ariyoshi W, Habu M, et al., (2004). A decrease in the molecular weight of hyaluronic acid in synovial fluid from patients with temproamandibular disorders. J Oral Pathol Med 33, 224-229. Tameem HZ, Selva LE, Sinha US. (2007). Morphological atlases of knee cartilage: shape indices to analyze cartilage degradation in osteoarthritic and non-osteoarthritic population. Conf Proc IEEE Eng Med Biol Soc 1, 1310-1313. Tammi R, Rilla K, Pienimaki JP, MacCallum DK, Hogg M, et al., (2001). Hyaluronan enters keratinocytes by a novel endocytic rout for catabolism. J Biol Chem 276, 37, 35111-35122. 180

PAGE 195

Tardif G, Reboul P, Pelletier JP, Martel-Pelletier J. (2004). Ten years in the life of an enzyme: the story of the human MMP-13 (collagenase-3). Mod Rheumatol, 14, 3, 197-204. Tetlow LC, Adlam DJ, Woolley DE. (2001). Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations withdegenrative changes. Arthr Rheum, 44, 3, 585-594. Thonar EJ, Shinmei M, Lohmander LS. (1993). Body fluid markers of cartilage changes in osteoarthritis. Rheum Dis Clin North Am 19, 3, 635-657. Thumboo J, Chew LH, Lewin-Koh SC. (2002). Socioeconomic and psychosocial factors influence pain or physical function in Asian patients with knee or hip osteoarthritis. Ann Rehum Dis 61, 11, 1017-1020. Tiderius CJ, Tjornstrand J, Akeson P, Sodersten K, Dahlberg L, Leander P. (2004). Delayed gadolinium-enhanced MRI of cartilage (dGEMRIC): intraand interobserver variability in standardized drawing of regions of interest. Acta Radiol 45, 6, 628-634. Toda Y, Segal N. (2002). Usefulness of an insole with subtalar strapping for analgesia in patiens with medial compartment osteoarthritis of the knee. Arthritis Rheum 47, 5, 466-473. Toda Y, Segal N, Kato A, Yamamoto S, Irie M. (2002). Correlation between body composition and efficacy of lateral wedged insoles for medial compartment osteoarthritis of the knee. J Rheumatol 29, 541-545. Topp, R, Boardley D, Morgan AL, Fahlman M, McNevin N. (2005). Exercise and functional tasks among adults who are functionally limited. West J Nurs Res 27, 3, 252-270. Topp R, Woolley S, Hornyak J 3rd, Khuder S, Kahaleh B. (2002). The effect of dynamic versus isometric resistance training on pain and functioning among adults with osteoarthritis of the knee. Arch Phys Med Rehabil 83, 9, 1187-1195. Tortorella MD, Malfait AM. (2008). Will the real aggrecanse(s) step up: evaluating the criteria that define aggrecanase activityin osteoarthritis. Curr Pharm Biotechnol 9, 1, 26-23. Tsai SW, Fang JF, Yang CL, Chen JH, Su LT, Jan SH. (2005). Preparation and evaluation of a hyaluronate-collagen film for preventing postsurgical adhesion. J Int Med Res 33, 1, 68-71. 181

PAGE 196

Tuominen U, Blom M, Hirvonen J, Seitsalo S, Lehto M, et al., (2007). The effect of co-morbidities on health-related quality of life in patients placed on the waiting list for total joint replacement. Health Qual Lif Outcomes 5, 16. Turan Y, Bal S, Gurgan A, Topac H, Koseoglu M. (2007). Serum hyaluronan levels in patients with knee osteoarthritis. Clin Rheum E-pub ahead of print, accessed Mar 2, 2007. Uebelhart D, Williams JM. (1999). Effect s of hyaluronic acid on cartilage degradation. Curr Opin Rheumatol 11, 427-435. Urman B, Gomel V, Jetha N. (1991). Effect of hyaluronic acid on postoperative intraperitoneal adhesion formation in the rat model. Fertil Steril 56, 563-567. Valdes AM, Hart DJ, Jones KA, Surdulescu G, Swarbrick P, et al. (2004). Association study of candidate genes for the prevalence and progression of knee osteoarthritis. Arthr Rheum 50, 2497-2507. Van Beuningen HM, Glansbeek HL, van der Kraan PM, van den Berg WB. (2000). Ostoearthritis-like changes in the murine knee joint resulting from intra-articular transforming growth factor-beta injections. Osteoarthr Cart 8, 2533. Van Beuningen HM, van der Kraan PM, Arntz OJ, fan den Berg WB. (1994). Transfroming growth factor-beta 1 stimulates articular chondrocyte proteoglycan synthesis and induces osteophyte formation in the murine knee joint. Lab Invest 71, 2, 279-290. van de Loo FAJ, Joosten LAB, van Lent PLEM, Arntz OJ, van den Berg WB. (1997). Interleukin 6 reduces cartilage destruction during experimental arthritis. A study in interleukin 6 deficient mice. Am J Pathol 151, 177-191. Van der Kraan PM, van den Berg WB (2007). Osteophytes: relevance and biology. Osteoarthr Cart E pub ahead of print, accessed Mar 2, 2007. van Lent PLEM, Blom AB, van der Kraan, Holthuysen AEM, Vitters E, et al. (2004). Crucial role of synovial lining macrophages in the promotion of transforming growth factor beta-mediated osteophyte formation. Arthr Rheum 50(1), 103-111. Verbrugge LM, Gates DM, Ike RW. (1991). Risk factors for disability among US adults with arthritis. J Clin Epidemiol, 44, 167–82. 182

PAGE 197

Vikkula M, Metsaranta M, Ala-Kokko L. (1994). Type II collagen mutations in rare and common cartilage diseases. Ann Med, 26, 107–14. Waddell DD, Kolomytkin OV, Dunn S, Marino AA. (2007). Hyaluronan Suppresses IL-1beta-induced Metalloproteinase Activity from Synovial Tissue. Clin Orthop Relat Res 465, 241-248. Walton M. (1979). Patella displacement and osteoarthrosis of the knee joint in mice. J Pathol 127, 4, 165-172. Wang A, Ziyadeh FN, Lee EY, Pyagay PE, Sung Sh, et al. (2007). Interference with TGF-beta signaling by Smad3-knockout in mice limits diabetic glomerulosclerosis without affecting albuminuria. Am J Physiol Renal Physiol 293, 5, F1657-F1665. Wang CT, Lin J, Chang CJ, Lin YT, Hou SM. (2004). Therapeutic effects of hyaluronic acid on osteoarthritis of the knee: a meta-analysis of randomized controlled trials. J Bone Joint Surg Am 86, 538–545. Wang CT, Lin YT, Chiang BL, Lin YH, Hou SM. (2006). High molecular weight hyaluronic acid down-regulates the gene expression of osteoarthritisassociated cytokines and enzymes in fibroblast-like synoviocytes from patients with early osteoarthritis. Osteoarthr Cart 14, 1237-1247. Wang VM, Flatow EL. (2005). Pathomechanicics of acquired shoulder instability: a basic science perspective. J Shoulder Elbow Surg 14, 1 Suppl, 2S11S. Weidow J, Cederlund CG, Ranstam J, Karrholm J. (2006). Ahlbck grading of osteoarthritis of the knee: poor reproducibility and validity based on visual inspection of the joint. Acta Orthop 77, 2, 262-266. Wenisch C, Patruta S, Daxbock F, et al. (2000). Effect of age on human neutrophil function. J Leukocy Biol 67, 40-45. Williams CJ, Jimenez SA. (2003) Skeletal dysplasias and the osteoarthritic phenotype. Best Pract Res Clin Rheumatol 17, 6, 1005-1018. Wisniewski HG, Hua JC, Pepper DM, Naime D, Vilcek J, et al. (1996). TNF/IL-1-inducible protein TSG-6 potentiates plasmin inhibition by inter" inhibitor and exerts a strong anti-inflammatory effect in vivo. J Immunol 156, 1609-1615. 183

PAGE 198

Wobig M, Dickhut A, Maier R, Vetter G. (1998). Viscosupplementation with hylan G-F 20: a 26 week controlled trial of efficacy and safety in the osteoarthritic knee. Clin Ther 20, 410-423. Wright GD, Hughes AE, Regan M, Doherty M. (1996). Association of two loci on chromosome 2q with nodal osteoarthritis. Ann Rheum Dis, 42, 2356-2364. Xu L, Peng H, Wu D, Hu K, Goldring MB, et al. (2005). Activation of the discoidin domain receptor 2 induces expression of matrix metalloproteinase 13 associated with osteoarthritis in mice. J Bio Chem 280(1), 548-555. Yamada K, Healey R, Amiel D, Lotz M, Coutts R. (2002). Subchondral bone of the human knee joint in aging and osteoarthritis. Osteoarthr Cart 10, 5, 360-369. Yamada H, Koshino T, Sakai N, Saito T. (2001). Hip adductor muscle strength in patients with varus deformed knee. Clin Orthop 386, 179-185. Yamamoto K, Shishido T, Masaoka T, Imakiire A. (2005). Morphological studies on the ageing and osteoarthritis of the articular cartilage in C57 black mice. J Orthop Surg 13(1), 8-18. Yan T, Riggs BL, Boyle WJ, Khosla S. (2001). Regulation of osteoclastogensesis and RANK expression by TGF-beta1. J Cell Biochem 83, 320-325. Yasui T, Adatsuka M,Tobetto K, Hayaishi M, Anto T. (1992). The effect of hyaluronan on interleukin-1-a-induced prostaglandin E2 production in human osteoarthris synovial cells. Agents Actions 37, 155-156. You L, Temiyasathit S, Lee P, Kim CH, Tummala P, et al. (2008). Bone 42, 1, 172-179. Youn J, Kim HY, Park JH, Hwang, SH, Lee SY, et al. (2002). Regulation of TNF-alpha-mediated hyperplasia through TNF receptors, TRAFs, and NFkappaB in synoviocytes obtained from patients with rheumatoid arthritis. Immunol Lett 83, 2, 85-93. Yurtkuran M, Kocagil T. (1999). TENS, Electroacupuncture and IceMassage: Comparison of Treatment for Osteoarthritis of the Knee. Am J Acupuncture, 27,133–140. Zemmyo M, Meharra EJ, Kuhn K, Creighton-Achermann L, Lotz M. (2003). Accelerated, aging dependent development of osteoarthritis in alpha1 integrin-deficient mice. Arthr Rheum 48(10), 2873-2880. 184

PAGE 199

Zhang W, Doherty M. (2005). How important are genetic factors in osteoarthritis? Contributions from family studies. J Rheumatol 32, 6, 1139-1142. Zhang W, Jones A, Doherty M. (2004). Does paracetamol (acetaminophen) reduce the pain of osteoarthritis? A meta-analysis of randomised controlled trials. Ann Rheum Dis, 63, 901-907. Zhang Y, McAlindon TE, Hannan MT, Chaisson CE, Klein R, et al. (1998). Estrogen replacement and worsening of radiographic knee osteoarthritis: the Framingham Study. Arthritis Rheum 41, 1867-1873. Zoppi M, Peretti G & Boccard E. (1995). Placebo-controlled study of the analgesic efficacy of an effervescent formulation of 500 mg paracetamol in arthritis of the knee or hip. Eur J Pain, 16, 42-48. 185

PAGE 200

Bibliography Akinbo SR, Aiyejusunle CB, Akinyemi OA, Adesegun SA, Danesi MA. (2007). Comparison of the therapeutic efficacy of phonophoresis and iontophoresis using dexamethasone sodium phosphate in the management of patients with knee osteoarthritis. Niger Postgrad Med J, 14, 3, 190-4. Bancroft JD, Gamble M. (2002). Theory and Practice of Histological Techniques London: Churchill Livingstone. Bevilacqua M, Pober J, Wheeler M, Cotran RS, Gimbrone, MA Jr. (1985). Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte celllines. J Clin Invest, 76, 2003-20. Brosseau L, Pelland L, Wells G, et al. (2004). Efficacy of aerobic exercises for osteoarthritis (Part I): a meta-analysis. Phys Ther Res, 9, 125-145. Chapman K, Mustafa Z, Irven C, Carr AJ, Clipsham K, et al. (1999). Osteoarthritis-susceptibility locus on chromosome 11q, detected by linkage. Am J Hum Genet 65, 167-174. Coleman EA, Buchner DM, Cress ME, Chan BK, de Lateur BJ. (1996). The relationship of joint symptoms with exercise performance in older adults. J Am Geriatr Soc, 44, 14-21. Devos-Comby L. (2006). Do exercise and self-management interventions benefit patients with osteoarthritis of the knee? A metaanalytic review. J Rheum 33(4), 744-756. D’Lima DD, Hashimoto S, Chen PC, Colwell CWJ, Lotz M. (2001) Human chondrocyte apoptosis in response to mechanical injury. Osteoarthr Cart 9, 712-719. D’Lima DD, Hashimoto S, Chen PC, Colwell CWJ, Lotz M. (2001) Human chondrocyte apoptosis in response to mechanical injury. Osteoarthr Cart 9, 712-719. 186

PAGE 201

Eyre DT. (2004). Collagens and collagen matrix homeostasis. Clin Orthop Related Res 427 Suppl, S118-S122. Ezzo J, Hadhazy V, Birch S, et al (2001). Acupuncture for osteoarthritis of the knee: a systematic review. Arthritis Rheum 44, 819-825. Felson DT. (2004). Risk factors for osteoarthritis: understanding joint vulnerability. Clin Orthop Relat Res 427, Suppl, S16-21. Felson DT, Zhang Y, Anthony JM, Naimark A, Anderson N. (1992). Weight loss reduces the risk for symptomatic knee osteoarthritis in women. Ann Intern Med, 116, 535-9. Fernihough J, Gentry C, Malcangio M, Fox A, Rediske J, et al. (2004). Pain related behaviour in two models of osteoarthritis in the rat knee. Pain 112, 83-93. Fischer DC, Siebertz B, Schiwy-Bochat KH, Graeve L, Heinrich PC, et al. (1999). Induction of alpha 1-antitrypsin synthesis in human articular chondrocytes by interleukin 6 type cytoki nes: evidence for a local acute phase response in the joint. Arthr Rheum 42, 1936-1945. Fransen M, McConnell S, Nell M. (2002). Therapeutic exercise for people with osteoarthritis of the hip or knee. A systematic review. J Rhematol 29, 1737-1745. Frenette J, Chbinou N, Godbout C, Marsolais D, Frenette PS. Macrophages, not neutrophils, infiltrate skeletal muscle in mice deficient in P/E selectins after mechanical reloading. Am J Physiol Regul Integr Comp Physiol 285, R727-732. Gaffen JD, Bayliss MT, Mason RM. (1997). Elevated aggrecan mRNA in early murine osteoarthritis. Osteoarthr Cart 5, 227-233. Garnero P. (2006). Use of biochemical markers to study and follow patients with osteoarthritis. Curr Rheumatol Rep 8, 1, 37-44. Garofalo S, Metsaranta M, Ellard J, et al. (1993). Assembly of cartilage collagen fibrils is disrupted by overexpression of normal type II collagen in transgenic mice. Proc Natl Acad Sci USA 90, 3825-3829. Hughes SL, Seymour RB, Campbell RT, Huber G, Pollak N, et al. (2006). Long-term impact of Fit and Strong! on older adults with osteoarthritis. Gerontologist 46, 6, 801-14. 187

PAGE 202

Hulme J, Robinson V, DeBie R, (2004). Electromagnetic fields for the treatment of osteoarthritis. The Cochrane Library issue 3, Chichester UK: John Wiley and Sons, pp 1-34. Hyttinen MM, Toyras J, Lapvetalainen T, et al. (2001). Inactivation of one allele of the type II collagen gene alters the collagen network in murine articular cartilage and makes cartilage softer. Ann Rheum Dis 60, 262-268. Ju¨ni P, Nartey L, Reichenbach S, Sterchi R, Dieppe PA, Egger M. (2004). Risk of cardiovascular events and rofecoxib: cumulative metaanalysis. Lancet, 364, 2021-2029. Ju¨ni P, Rutjes AW, Dieppe PA. (2002). Are selective COX 2 inhibitors superior to traditional non steroidal anti-inflammatory drugs? B Med J, 324, 12871288. Kelley WN, Ruddy S, Harris ED Jr., Sledge CB, eds. (1997) Textbook of Rheumatology. Vol 1. 5th ed. Philadelphia, PA: W.B. Saunders Company. Knudson W, Aguiar DJ, Hua Q, Knudson CB. (1996). CD44-anchored hyaluronan-rich pericellular matrices: an ultrastructural andbiochemical analysis. Exp Cell Res 228, 216-228. Link TM, Stahl R, Woertler K.(2007). Cartilage imaging: motivation, techniques, current and future significance. Eur Radiol 17, 5, 1135-1146. Lo GH, LaValley M, McAlindon T, Felson DT. (2003). Intra-articular hyaluronic acid in treatment of knee osteoarthritis: a meta-analysis. J Am Med Assoc, 290, 3115–3121. Lorenz H, Richter W. (2006). Osteoarthrits: cellular and molecuolar changes in degenerating cartilage. Prog Histochem Cytochem 40, 135-163. Lorig K, Lubeck d, KIraines RG, Seleznick M, Holman HR. (1985). Outcomes of self-help education for patients with arthritis. Arthr Rheum 28, 680685. Maillefert JF, Hudry C, Baron G, Keiffert P, Bourgeois P et al. (2001). Laterally elevated wedged insoles in the treatment of medial knee osteoarthritis: a prospective randomized controlled study. Osteoarthr Cart 9, 738-745. Marko JP, Soderstrom M, Saamanen AMK, Salminen HJ, Vuorio EI. (2004). Up regualtion of cathepsin K in articular chondrocytes in a transgenic mouse model for osteoarthritis. Ann Rheum Dis 63, 649-655. 188

PAGE 203

Martel-Pelletier J (2004). Pathophysiology of osteoarthritis. Osteoarthr Cart 12, Suppl A, S31-S33. Martin JA, Buckwalter JA. (2006).Post-traumatic osteoarthritis: the role of stress induced chondrocyte damage. Biorheology 43, 3-4, 517-521. Moskowitz RW, Kelly MA, Lewallen DG (2004). Understanding osteoarthritis of the knee—causes and effects. Am Jl Orthop suppl, 5-9. Neuhold LA, Killar L, Zhao W, et al. (2001). Postnatal expression in hyaline cartilage of constitutively active collagenase-3(MMP-13) induces osteoarthritis in mice. J Clin Invest 107, 35-44. Oliveria SA, Felson DT, Cirillo PA, Reed JI, Walker AM. (1999). Body weight, body mass index, and incident symptomatic osteoarthritis of the hand, hip, and knee. Epidemiology, 10, 161–6. Oonuki Y, Yoshida Y, Uchiyama Y, Asari A. (2005). Application of fluorophore-assisted carbohydrate electrophoresis to analysis of disaccharides and olilgosaccharides derived from glycosaminoglycans. Anal Biochem 343, 212-222. Pelland L, Brosseau L, Wells G, et al. (2004). Efficacy of strengthening exercises for osteoarthritis (Part I): a meta-analysis. Phys Ther Rev, 9, 77-108. Penninx BW, Messier SP, Rejeski WJ, Williamson JD, DiBari M, Cavaszzini C, Applegate WB, Pahor M. (2001). Physical exercise and the prevention of disability in activities of daily living in older persons with osteoarthritis. Arch Int Med, 161, 19, 2309-2316. Pincus T, Koch GG, Sokka T, Lefkowith J, Wolfe F, et al. (2001). A randomized, double-blind, crossover clinical trial of diclofenac plus misoprostol vs acetaminophen in patients with osteoarthritis of the hip or knee. Arthr Rheum, 44, 1587-1598. Punzi L. (2001). The complexity of the mechanisms of action of hyaluronan in joint diseases. Clin Exp Rheumatol 19, 242-246. Sandell LJ, Aigner T. (2001). Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis Res 3, 107-113. Sinkov V, Cymet T (2003). Osteoarthritis: understanding the pathophysiology, genetics, and treatments. J Natl Med Assoc 95, 475-482. 189

PAGE 204

Sonoda M, Harwood FL, Amiel ME, Moriya H, Temple M, Chang DG, et al. (2000). The effects of hyaluronan on tissue healing after meniscus injury and repair in a rabbit model. Am J Sports Med 28, 90-97. Superio-Cabuslay E, Ward MM, Long KR (1996). Patient education interventions in osteoarthritis and rheumatoid arthritis: a meta-analytic comparison with nonsteroidal anti-inflammatory drug treatment. Arthritis Care Res 9, 292-301. Terlain B, Dumond H, Presle N, Mainard D, Bianchi A, et al. (2005). Is leptin the missing link between osteoarthritis and obesity? Ann Pharm Fr, 63, 3, 186-193. Verrecchia F Mauviel A. (2007). Transforming growth factor! and fibrosis. World J Gastroenterol 13, 22, 3056-3062. Watanabe H, Kimata K, Line S, et al. (1994). Mouse cartilage matrix deficiency (cmd) caused by a 7 bp deletion in the aggrecan gene. Nature Genet 7, 154-157. Westby MD. (2001). A health professional’s guide to exercise prescription for persons with arthritis: a review of aerobic fitness activities. Arthr Care Res 45, 501-511. White A, Foster N, Cummings M, Barlas P. (2006). The effectivenessof acupuncture for osteoarthritis of the knee—a systematic review. Acupunt Med 24, Suppl, S40-S48. White JA, Wright V, Hudson AM. (1993). Relationship between habitual physical activity and osteoarthrosis in ageing women. Pub Health 107, 459-470. Witte M, Barbul A. (2002). Role of nitric oxide in wound repair. Am J Surg, 183, 406-41. Wittekind D. (2003). Traditional staining for routine diagnostic pathology including the role of tannic acid. 1. Value and limitations of the hematoxylin-eosin stain. Biotech Histochem 78, 5, 261-270. 190

PAGE 205

Appendices Appendix 1: Abbreviations used ACL anterior cruciate ligament ACR American College of Rheumatology ADAMTS a disintegrin and metalloproteinase with thrombospondin motifs AGS American Geriatric Society AIMS Arthritis Impact Measurement Scale ANOVA Analysis of variance BAC bovine articular cartilage BMI body mass index BMP bone morphogenetic protein BSA bovine serum albumin C Celcius CES-D Scale Center for Epidemiological Studies—Depression Scale CILP Cartilage Intermediate Layer Protein CRP C-reactive protein CT computed tomography Da dalton dGEMRIC delayed gadolinium-enhanced magnetic resonance imaging DI De-ionized DJD degenerative joint disease ECM extracellular matrix EDTA Ethylenediaminetetraacetic acid ELISA enzyme linked immunoassay ETOH ethyl alcohol EULAR European League Against Rheumatism FACE Fluorophore-assisted Carbohydrate Electrophoresis FITC fluorescein isothiocyanate FRZB frizzled related protein g gram G gauge GAG glycosaminoglycan H/E hematoxylin/eosin HA hyaluronan hr hour IGF-1 insulin-like growth factor 1 IL interleukin iNOS Inductible nitric oxide synthetase kg kilogram km kilometer KO Knock out 191

PAGE 206

KOOS Knee Osteoarthritis Outcomes Survey LCL lateral collateral ligament LYVE-1 lym phatic vessel endothelial HA receptor m meter M molar mA milli amperage MCL medial collateral ligament mg milligram ml milliliter MMP matrix metalloproteinase MOVE MRI magnetic resonance imaging mRNA messenger ribonucleic acid NFR nociceptive flexion reflex ng nanogram nm nanometer NSAID non-steroidal anti-inflammatory OA osteoarthritis OARSI Osteoarthritis Research Society International OASI Osteoarthritis Severity Index PCL posterior cruciate ligament PDGF platelet derived growth factor PG proteoglycan RPE rating of perceived exertion rpm revolutions per minute sec second SF-36 Short Form Health Survey General Questionnaire SPP supr apatella pouch SPSS Statistical Package for Social Sciences sR soluble receptor TEFR Therapeutic Education and Functional Readaptation TGF! 1 transforming growth factor-beta 1 TNFAIP tumor necrosis factor-alpha induced protein TNF" tumor necrosis factor-alpha US ultrasonography V volt VO2 max maximal voluntary oxygen uptake WOMAC Western Ontario and MacMaster’s University Osteoarthritis Index l microliter Appendix 2 (continued): Abbreviations 192

PAGE 207

Appendix 2: Reported outcomes of exercise in persons with OA Study Sample Size Type of Exercise Outcomes Mangione et al. (1999) n=39 Aerobic Improved timed chair rise Improved six minute walk test Improved walking speed Decreased overall pain Improved aerobic capacity Kovar et al. (19992) n=102 Aerobic Decreased pain Less medication use Increased walking distance Increased function (AIMS) Minor et al. (1989) n=120 Aerobic Improved aerobic capacity Improved 50 feet walking time Decreased depression Decreased anxiety Improved physical activity Minor et al. (1989) n=120 Waterbased Improved aerobic capacity Improved 50 feet walking time Decreased depression Decreased anxiety Improved physical activity Ettinger et al. (1997) n=439 Aerobic Increased walking speed Improved pain Improved disability score Penninx et al. (2002) n=439 Aerobic Decreased depressive symptoms Rejeski et al. (1998) n=439 Aerobic Increased self-efficacy for stair climbing Schilke et al. (1996) n=20 ResistanceDecreased pain Decreased stiffness Decrased arthritis activity (OASI; AIMS) Increased strength Increased mobility Topp et al. (2005) n=131 ResistanceIncreased function 193

PAGE 208

Study Sample Size Type of Exercise Outcomes Ettinger et al. (1997) n=439 ResistanceImproved disability score Improved strength Improved pain Improved six minute walk test Penninx et al. (2002) n=439 ResistanceDecreased depressive symptoms Topp et al. (2002) n= 64 ResistanceDecreased time to ascend and descend stairs Decreased knee pain Improved WOMAC score Improve time to stand up from floor Gur et al. (2002) n= 23 ResistanceDecreased pain, improved chair rise Improved walking Improved stair climb Increased strength Eyigor et al. (2004) n= 44 ResistanceImproved strength Decreased disease severity Decreased pain Improved walking time Improved WOMAC score Improved SF-36 score Improved AIMS2 score Foley et al. (2003) n=105 Waterbased Improved function Improved walking distance Improved physical component on SF-12 Cochrane et al. (2005) n= 106 Waterbased Improved pain Improved physical function Improved patient satisfaction Diracoglu et al. (2005) n=66 Functional Decreased time for ADL Improved strength Improved WOMAC score Improved proprioception Improved stair climb Improved 10 meter walk time Diracoglu et al. (2005) n=66 ResistanceDecreased time for ADL Improved strength Improved WOMAC score Improved proprioception Appendix 2 (continued): Reported outcomes of exercise in persons with OA 194

PAGE 209

Study Sample Size Type of Exercise Outcomes Lin et al. (2005) n=81 Functional Improved joint position sense Improved WOMAC functional score Improved walking speed Improved muscle strength Appendix 2 (continued): Reported outcomes of exercise in persons with OA 195

PAGE 210

Appendix 3: Mouse models used to study OA Mechanism Reference Spontaneous STR/ort unidentified Mason et al., 2001 C57/Bl unidentified Yamamoto et al., 2005 Manipulation of Cartilage Specific Genes Cho/+ deletion in the Col XIa1 gene Li and Olsen., 1997 Dmm/+ deletion in the Col IIa1 gene Li and Olsen., 1997 Cmd/+ deletion in the aggrecan gene Watanabe et al., 1994 Del 1 Type II collagen deficiency Morko et al., 2006 ADAMTS5 -/ADAMTS5 deficiency Glasson et al., 2005 Col2a1 +/Col2a1 deficiency Hyttinen et al., 2001 Col 2a 1 overexpression of Col2a1 gene Garafolo et al., 1993 Surgical Models ACL-T Instability via transection of ACL Kamekura et al., 2005 PMM Instability via removal of part of the medial meniscus Clements et al., 2003 MM Instability via removal of the medial meniscus Kamekura et al., 2005 MCL-T + PMM Instability via transection of ACL and part of medial meniscus Clements et al., 2003 Chemical Induced Models Quinolone Intra-articular injection of quinolone antibiotics Bendele et al., 2001 MIA Intra-articular injection of monosodium iodoacetate Blom et al., 2004 Collagenase Intra-articular injection of collagenase Fernihough et al., 2004 TGF! 1 Intra-articular injection of TGF! 1 Van Beuningen et al., 2000 196

PAGE 211

Appendix 4: Poster presented at the International Conference on Preclinical Models of Osteoarthritis, May 2006 197

PAGE 212

Appendix 5: Poster Presented at the Annual Meeting of the Orthopedic Research Society, February 2007 198

PAGE 213

Appendix 6: Poster presented at the American College of Rheumatology Annual Scientific Meeting, November 2007 199

PAGE 214

Appendix 7: Poster presented at the Annual Meeting of the Orthopedic Research Society, March 2008 200

PAGE 215

About the Author Wendy K. Anemaet received Bachelor’s Degrees in Biology, Pre-Med, and Athletic Training from Mount Vernon Nazarene University in 1987 and a Master’s in Physical Therapy from the University of Southern California in 1989. She is a geriatric certified specialist, certified wound specialist, and certified weight trainer. In addition, she is certified in utilization review in Florida and has a geriatric training certificate and certificate of OASIS specialist—clinical. Ms. Anemaet has served as issue editor for Topics in Geriatric Rehabilitation and as a monthly columnist for ADVANCE for Physical Therapists. Publications include numerous physical therapy related articles (published in Topics in Geriatric Rehabilitation, Advance for Physical Therapists, Advance for Rehabilitation Managers, and Home Health Care Nurse) and two books: The User Friendly Home Care Handbook (Learn Publications) and Home Rehabilitation: Guide to Clincial Practice (Mosby Inc).