Dissertationen zum Thema „Osteoarthritis“

Over 26 million U.S. citizens have a form of arthritis; osteoarthritis (OA) is the most common form. Self-efficacy (SE) is defined as a psychological construct which identifies an individual's confidence when performing a behavior. SE is deemed a vital judge of self-management (SM) in those with OA. The purpose of this evidence-based practice, quality improvement project was to improve SE in OA patients. The identified gap in nursing practice was the lack of SE in OA patients. The project question asked whether a toolkit with information regarding SE in OA can improve SE of management of disease-associated symptoms in adults with OA as evidenced by improved Arthritis Self-Efficacy Scale (ASES) scores pre- to post-program. Concepts and theory used to inform the doctoral project were SE, pain, SM and OA, and Bandura's theory of SE. The sources of evidence were obtained from a variety of peer-reviewed journals related to OA management, and the outcome was measured using the ASES. Thirty-five participants (16 males and 19 females) with a mean age of 62 from a physical medicine and rehabilitation clinic in San Antonio, Texas participated in the project. The National Institute of Arthritis and Musculoskeletal and Skin Disorders 2015 Handout on Health: OA was used as the SE OA toolkit. Mean scores from pre- and post-program were tabulated and compared to determine the outcome. Results showed improved ASES levels by 11.84%. Implications for nursing practice and positive social change include the enhancement of SE levels, which can improve compliance in SM by use of a toolkit and further as policy implementation for OA patients to improve SE and SM abilities.

Osteoarthritis (OA) is a multifactorial disease characterized by a progressive degeneration and eventual failure of the synovial joint functionality. Although it has been traditionally considered as an exclusive disease of the articular cartilage, nowadays it is considered as a whole joint disease. Therefore, the progression of the disease involves articular cartilage degeneration, osteochondral bone sclerosis and synovial membrane hypertrophy. Articular cartilage is a connective tissue resistant to tensile and shears strength that is composed of water (>70%) and a dense extracellular matrix (ECM) that encompasses the unique cellular type of the cartilage, the chondrocytes. The major component of the ECM is the collagen network consisting mainly of type II collagen fibers and type IX and XI collagen macromolecules attached to the surface of the fiber. Non-collagenous components such as GAGs, aggregated proteoglycans (mainly aggrecan) and small leucine rich proteoglycans (SLRP’s) are also binding the collagen fiber. Cartilage is a no innervated and also an avascular tissue, thus gets its nutrients by diffusion from the synovial fluid. Due to this condition, chondrocytes live in a hypoxic environment, and intracellular survival factors, such as hypoxia-inducible factor 1α, are required for maintenance of homeostasis and adaptation to the mechanical environment. Under physiological conditions, the collagen network and proteoglycan content is maintained by the chondrocytes. However, local and systemic risk factors could lead chondrocytes to fail to maintain the ECM and thus the cartilage tissue is progressively degraded. To this point, the three general objectives of this thesis are: 1. Collagenase-3 (COL3) also known as MMP13, is a matrix metalloproteinase abnormally over-expressed in pathological processes. Several COL3 transcripts are expressed in human chondrocytes although their role in OA is still unknown. This study aimed to characterize the presence of two non-canonical COL3 isoforms, named COL3-DEL (deleted form) and COL3-9B (exon 9 added form) in human OA cartilage, and to analyse their proteolytic activity. 2. Opticin (OPTC), a SLRP known to play a role in the assembly of the fibrillar collagens and the structural stability of the extracellular matrix, was previously demonstrated to be produced and degraded in osteoarthritic (OA) human cartilage. Here, we further investigated the OPTC role in OA cartilage by the study of the in vivo effect of OPTC deficiency in mouse model 3. Chondroitin sulfate (CS) is a Symptomatic Slow acting Drug against Osteoarthritis (SySADOA) with anti-inflammatory effects. In this study, we tried to unveil the mechanism of action on osteoarthritic synovial membrane. The general discussion of this thesis is: OA is a heterogeneous disease that encloses multiple phenotypes. In order to develop new diagnostic and prognostic tools and eventually advance in the discovery of successful treatments, clearly defining the different phenotypes of OA is of great importance. In this line, the findings comprised in this thesis reveal two different approaches to identify patient’s subgroups. On one hand, and as described in chapter 1, the presence of a new discovered collagenase-3 (COL3) isoform (COL3-DEL) resulting from a mutation of the canonical COL3, could be used as an indicator of differential extracellular matrix degradation in human articular cartilage. On the other hand, results from chapter 2 suggest that the compositions of the members of SLRP super-family in the human extracellular matrix of the articular cartilage could be applied as a new tool for OA prognosis classification. Finally, the controversy regarding the efficacy of systemic treatment with nutraceuticals – including chondroitin sulfate - may arrive to an end if new tools are used to predict which patients are best suited for a given drug. Importantly, further work remains to be done to understand how to integrate these findings into a final and comprehensive concept that could explain the patient’s heterogeneity and the differential prognosis of OA disease.
L’artrosi (OA) és la malaltia articular més comuna i es caracteritza, principalment, per la destrucció del cartílag articular. El principal paper del cartílag articular és el de suportar les forces de tensió i compressió a les que es troba sotmès i que recau principalment en els seus components: el col·lagen i els proteoglicans. Durant el metabolisme normal del cartílag hi ha un equilibri entre la síntesi i degradació d’aquest components, però a l’artrosi, aquest equilibri es trenca i el metabolisme catabòlic supera l'anabòlic. Durant el desenvolupament de l'OA, els condròcits expressen la proteasa més involucrada en la degradació del cartílag, la MMP-13, que a diferència d’altres MMPs, en humans presenta 3 transcrits diferents. Malauradament, tot i la seva elevada prevalença, l'OA es troba orfe d’una bona tècnica diagnòstica, ja que actualment es realitza per mitjà de tècniques radiològiques que presenten l'inconvenient de que la malaltia només es reflecteix quan l’articulació es troba molt afectada. Actualment, els tractaments farmacològics més utilitzats són analgèsics, AINEs, i els nutracèutics, anomenats SYSADOA per les seves sigles en anglès (Symptomatic Slow Acting Drugs for Osteoarthritis) d’entre els que destaca el sulfat de glucosamina i el condroitin sulfat. Amb aquests antecedents previs, els objectius de la tesis són: 1. Demostrar la presència de 3 isoformes de MMP-13 en humans • Clonació (sistema Bac-to-bac) i purificació de les 3 isoformes en cèl·lules d’insecte (Sf9). • Producció d’anticossos policlonals específics de cada isoforma al Servei de Producció d’Anticossos de la UAB • Cerca de les isoformes en pacients artròsics per WB. • Comprovació de l’activitat de cada isoforma en front a diferents components matricials. 2. Estudiar l'efecte "in vivo" de la deleció del gen de l'Opticina en un model murí d'artrosi. • Confirmar l'expressió de l' OPTC al cartílag articular dels ratolins. • Induïr OA mitjançant el mètode de destabilització del menisc medial (DMM) a ratolins Optc-/- i Optc +/+ de 10 setmanes d'edat. • Analitzar l'efecte de la deficiència de l'OPTC al desenvolupament de l'OA, evaluant la degradació del cartílag i la hipertròfia de la membrana sinovial, deu setmanes després de l'inducció de l'OA per DMM. • Comparar l'expressió de marcadors pro-inflamatoris, catabòlics i anabòlics entre ratolins Optc-/- i Optc+/+. • Analitzar la producció al cartílag de differents SLRPs. • Analitzar l'organització i l'ultraestructura de les fibres de colàgen. 3. Estudiar l'efecte anti-angiogenic del condroitin sulfat en sinoviòcits sota condicions d'hipòxia i d'hipòxia més inflamació. • Mimetitzar condicions inflamatòries i d'hipòxia "in vitro" en sinoviòcits humans. • Estudiar l'expressió i la producció de mediadors angiogènics per sinoviòcits artròsics sota condicions d'hipòxia i inflamació. • Estudiar l'efecte del pretractament del CS en cultius de sinoviòcits humans artròsics • Estudiar l'interacció entre els sinoviòcits artròsics i les cèl·lules endotelials sota hipòxia i inflamació, amb o sense pretractament de CS. L'artrosi és una malaltia que engloba diferents fenotips, però tots comparteixen una sèrie de característiques com la degeneració del cartílag articular, inflamació de la membrana sinovial i esclerosi de l'ós subcondral. Aquestes característiques resulten en un conjunt de símptomes que impedeixen la correcte mobilitat del pacient i creen dolor que pot arribar a ser crònic. Alhora de desenvolupar noves eines de diagnòstic i prognòstic i eventualment avançar en el descobriment de tractaments exitosos, definir clarament els diferents fenotips de l'artrosi és de gran interès. En aquesta línia, els descobriments compresos en aquesta tesis revelen dues possibles maneres d'identificar subgrups de pacients. Per una part, com es descriu al capítol 1, la presència d'una nova isoforma de la colagenasa-3 podria utilitzar-se com a un indicador de diferencies en la degradació matricial del cartíleg humà articular. Per una altre banda, els resultats del capítol 2 suggereixen que la composició dels membres de la famíla de SLRP a la matriu extracel·lular del cartíleg podria aplicar-se com a una nova eina de classificació del prognòstic de la malaltia artròsic. Finalment, la controversia sobre l'eficàcia del tractament amb nutracèutics com el condroitin sulfat podria arribar a solucionar-se si es descobreixen noves eines per predir quins pacients poden respondre millor a un medicament donat. És important tenir en compte que s'ha de continuar investigant per entendre com integrar aquests resultats en un concepte final que pogués explicar l'heterogeneïtat dels pacients i les diferències en el prognòstic de l'artrosi.

Meniscal tears are the commonest knee injury and currently are addressed almost exclusively by arthroscopy. Ian Smillie the late Professor of orthopaedics in Tayside, popularised open total meniscectomy worldwide during the 1950s believing that this was necessary for a functioning fibrocartilage replica to completely occupy the ensuing space. The cohort in this study underwent open total meniscectomy under his care prior to their 19th birthday. It was documented in their then records that no other knee pathology was observed during the operation and that the same post operative regime was followed by all. This presents a unique opportunity to evaluate the long term outcomes of open total knee meniscectomy during adolescence and to further investigate biological markers of osteoarthritis 40 years down the line. Fifty-three patients who underwent radiographic evaluation at the 30 year follow-up were further studied at this 40 year review. All surviving and contactable patients were consented prior to assessment and were evaluated clinically; biochemically, radiologically and subjectively once ethical approval and funding were secured. Standardisation of all methods used for examination, radiographic evaluation, sampling of serum and synovial fluid and patient reported outcome measures (PROMs) was achieved by the use of recognised, validated and credible systems as well as good communication between all involved parties. Such examples include the construction of a wooden apparatus standardising the weight bearing skyline views and the need for a smooth and efficient transition between sampling, preparing, storing and transferring the synovial and serum samples. Once all the data were collected, the first striking finding was the proportion of total knee arthroplasties (TKAs) observed as a hard endpoint in this cohort, which suggested a 132 fold increase when compared to their age and geographically matched population data, as per Scottish Arthroplasty Project. It was important to assess if in this cohort the site of meniscectomy demonstrated a significant difference in terms of tibiofemoral joint (TFJ) osteoarthritis, range of motion (ROM) and PROMs as per our chosen scoring systems. As this proved not to be the case, the operated knee was assessed against the non-operated knee where possible and not as per site of meniscectomy. Also the assessed sagittal laxity between the knees did not demonstrate any significant difference and as such was excluded as a confounding factor in terms of initiators of osteoarthritis. A linear correlation was observed between the chosen scoring systems of TFJ osteoarthritis. The calculated relative risk (RR) of developing osteoarthritis (OA) in the operated vs. non-operated knee was calculated for both the KL & Ahlback grading systems with presumed osteoarthritis as =2 for KL & =1 for Ahlback. This was found to be 4.5 & 4.25 respectively. Decreased ROM between the Index and Non-index knees was observed, with the ROM correlating with PROMs and inversely with TFJ OA. In addition the usually under investigated patellofemoral joint was assessed. Patellofemoral joint osteoarthritis was noted in the index knees as opposed to the non-index knees with an observed RR of 1.8 as per presence of osteophytes. There was no significant difference in the degree of patellofemoral joint (PFJ) osteoarthritis between lateral and medial meniscectomies. There was however significant correlations between the joint space narrowing (JSN) and PROMs, TFJ OA and ROM. Worsening results were observed where the PFJ was <5mm. Malalignment was greater in those knees that underwent medial meniscectomy as opposed to either lateral or medial & lateral meniscectomies. Malalignment demonstrated correlation with ROM and TFJ OA. Serum and synovial fluid was processed and analysed with regards to biomarkers of OA in the form of MMP-3 and GAG. Neither serum nor synovial MMP-3 demonstrated any significant correlation with other measured parameters. GAG on the other hand demonstrated a significant difference between the index and non-index knee as well as a positive correlation to IKDC and an inverse correlation with TFJ OA. Although this is suggesting that synovial GAG as a biomarker for OA may indicate progression of disease and symptoms, the wider spread of values questions this. Two different PROMs were utilised to assess this cohort. Interestingly the KOOS demonstrated that in all its 5 parameters the cohort was symptomatic. Correlations were observed between the KOOS ADL & Sport as well as IKDC with TFJ OA. This is currently the longest follow-up of open total meniscectomy in adolescence worldwide. A >4 fold increased risk of osteoarthritis in the operated knee as compared to the non-operated knee was demonstrated and possibly a 132 fold increase in TKA as compared to their aged matched geographical peers.

Book Summary: Serves the needs of advanced practice nurses because it’s written by nurse practitioners for nurse practitioners, in collaboration with a physician. Organizes content around the Circle of Caring framework for nursing-based knowledge and holistic care. Explores complementary and alternative treatments for each disorder. Covers the broadest range of human disease and disorders using a systems-based approach, presenting both common complaints and common problems to help students narrow down the possible differentials to the most likely diagnosis. Considers interactions of pharmaceuticals with alternative medications and nutraceuticals. Features coverage of pathophysiology and diagnostic reasoning as well as up-to-date guidance on laboratory and diagnostic tests. Emphasizes evidence-based practice with information on evidence levels and more references to primary studies. Integrates discussions of health policy and primary care throughout the text.

Osteoarthritis (OA) is a common, disabling disease of the elderly. It is uncommon in individuals under 45 years of age, and prevalence is higher in females than males. OA is characterised by focal cartilage loss accompanied by osteophytes, sclerosis and often some inflammatory response. A significant genetic component to OA has been demonstrated by twin-pair, sibling risk and segregation studies. Our group previously carried out a two-stage genome-wide linkage scan and identified six chromosomal regions as potentially harbouring OA susceptibility genes. The aim of this project was to examine three of these linkage regions, one on chromosome 4 and two on chromosome 16, in more detail in order to further localise and determine the identity of the OA susceptibility gene(s) within these regions. Initially the three regions were linkage mapped using a staged approach. Linkage mapping of the region on chromosome 4q identified a region showing evidence of linkage to female hip OA with a multipoint LOD (MLS) score of 3.1. For chromosome 16 linkage mapping identified two regions with MLS of 1.7 and 1.9 respectively. The first region was restricted to families with female siblings concordant for hip OA while the second is accounted for by families with affected female siblings. The ADAMTS3 gene was identified as a strong candidate gene within the linkage region on chromosome 4 as a result of its role in procollagen processing. SNPs within the gene were identified and typed in cohorts of cases and controls. There was no evidence for an association with any of these SNPs and OA disease. Within the first linkage region on chromosome 16 the IL4R gene was identified as a strong candidate gene as a result of its proposed role in the normal response of chondrocytes to mechanotransduction. Again SNPs within the gene were identified and typed. Four SNPs show evidence of association with OA and for three of these there is evidence for a relationship between genetic variation and evidence for linkage. It appears that there is a dosage effect with at least two copies of different variant alleles being necessary to increase OA disease susceptibility.

Osteoarthritis develops secondary to the action of hostile biomechanics upon susceptible cartilage. Cam morphology describes a loss of concavity at the femoral head-neck junction resulting in femoral impaction against the acetabular rim within a functional range of movement. This impaction is termed femoroacetabular impingement and can result in pain and cause damage to adjacent articular cartilage that progresses to osteoarthritis. Cam morphology is a target for joint preservation strategies. The first study in this thesis compares hip development in academy football players with controls from local schools. The results provide strong evidence that cam morphology can develop in response to intense sporting activity during adolescence. At present, it is not possible to recommend activity modification as the cardiovascular benefits of exercise are likely to outweigh potentially detrimental effects on hip morphology. Nevertheless, individuals participating in high-level sports during youth may represent a high risk cohort for osteoarthritis warranting surveillance. Diagnostic tools currently available only allow identification of late joint degeneration when disease is irreversible. Advances in the field of disease biomarkers may overcome this challenge. The second study of this thesis explored the prognostic value of compositional MRI. Baseline delayed gadolinium-enhanced MRI of cartilage was able to predict the development of radiographic hip osteoarthritis in asymptomatic individuals within five years. As well as identifying individuals who are likely to benefit from intervention, compositional MRI may allow the evaluation of treatment efficacy within short timeframes. A number of interventions aimed at joint preservation are under investigation for treating cam morphology femoroacetabular impingement. Arthroscopic surgery is increasingly performed to restore the concavity at the femoral head-neck junction and prevent impaction against the acetabular rim. A feasibility study was performed for a proposed randomised controlled study comparing operative and non-operative treatment. The study protocol was developed for the 'Femoroacetabular Impingement Trial' (FAIT).

Introduction: The overall goal of this thesis is to improve understanding of physical activity (PA), one of the most important, modifiable but controversial risk factors in osteoarthritis (OA). OA is the major public health problem in musculoskeletal medicine and leading cause of physical disability in older adults. The ultimate purpose is to provide evidence to inform OA prevention strategies, something not currently available. Objectives: 1) To construct and describe lifetime trajectories of hip and knee joint force from physical activity in a large Canadian sample; 2) To validate self-report measures of medically-diagnosed OA and novel measures of joint vulnerability against clinical criteria; 3) To evaluate the relationship of lifetime joint force and hip and knee OA. Methods: PA data were collected online from 4,269 subjects via a validated PA survey in a national population-based cohort from 2005 to 2007 and subjects ranked and lifetime trajectories plotted in terms of the ‘cumulative peak force index’, a novel joint force measure. Validation studies were conducted in a sub-sample. Population-based multivariable studies examining the relationship between joint force and incident hip and prevalent knee OA were conducted. Results: 1) Overall women had slightly higher lifetime PA-related force then men. Six percent of subjects developed hip OA and seven percent knee OA during follow up. There was no risk from sport/recreational activity. Very high levels of total lifetime force (hip and knee), occupational force in men (knee) and household-related force in women (knee) were associated with an approximate 2-fold increase in risk of OA, as was previous joint injury (5-fold increase hip, 3-fold knee). At the knee, lower limb malalignment but not joint hypermobility, was associated with knee OA. Higher coordination was protective. Conclusions: Taken collectively, the results show that lifelong physical activity-related joint force is generally safe for the hip and knee, and the promotion of exercise as a major public health initiative should continue without concern for increased rates of OA. Very high levels of occupational force in men and household force in women were risk factors for knee OA. Joint injury, lower limb malalignment and lower coordination were associated with OA.

Osteoarthritis (OA) is the most common form of arthritis in the world, and it has been estimated that about one tenth of the world's population, aged over 60, have symptoms that can be attributed to OA. Despite the size of the global impact of OA, there is a significant unmet need for effective treatments. Knee replacement surgery is commonly used in patients with moderate to severe knee OA, in order to reduce pain. However, 10-34% of patients report an unfavourable long-term outcome with persistent pain after surgery. The neural mechanisms for the generation of pain in knee OA are not fully understood. Previous work has shown that around 20% of patients have features of neuropathic pain, and that the underlying mechanism for this may be through central sensitisation. This mechanism-based understanding of pain is important in order to aid targeted intervention, and it may be that this patient group is more likely to have an adverse outcome following surgery. This thesis uses a combination of methods to investigate the neural mechanisms underlying pain experienced by patients with knee OA, across the full spectrum of disease severity. Quantitative sensory testing (QST) was initially used in a community-based cohort to show that pain sensitisation can be detected in early disease, and also contributes to the observed discordance between radiographic structural and symptomatic disease. The clinical relevance of neuropathic pain was then investigated in patients with knee OA, who were awaiting knee replacement surgery. Prior to surgery patients with neuropathic pain had increased sensitivity to experimental pain, as well as higher symptom severity and psychological distress. Functional magnetic resonance imaging (fMRI) was then used to confirm that these features were also associated with central sensitisation in the form of increased descending facilitation as well as reduced descending inhibition prior to surgery. The presence of neuropathic pain prior to surgery was associated with statistically and clinically significantly worse outcome following surgery, compared to those with purely nociceptive pain in the absence of any significant structural differences between the two groups. Taken together, this mechanism-based understanding of the pain provides an opportunity for targeted therapy prior to surgery, which may enhance outcome following surgery.

Loss of chondrocytes along with diminishment of specialised extracellular matrix (ECM) is often the outcome of articular cartilage injury and can progress to the onset of osteoarthritis (OA). OA is typically seen as an age related disease and with the changing world demographic, the fastest growing age category is the elderly population. Cartilage injury, which may result in OA, results in the clinical requirement for replacement of degenerated tissues and with an ageing population the demand for clinical replacement of degenerated tissue is set to dramatically increase posing socio-economic burdens. MicroRNAs are a class of small non-coding single stranded RNA which regulate gene expression post transcriptionally. MicroRNAs could be directly administered to articular cartilage, having direct effect upon resident articular chondrocytes, as a form of treatment for cartilage injury and OA. Exogenous microRNAs could be used in combination with isolated skeletal stem cells and transplanted to defective articular sites to induce articular cartilage regeneration. The identification and function of dysregulated microRNAs in OA and identification of microRNAs which govern chondrogenic differentiation may help the development of novel strategies to enhance cartilage formation and possibly in the treatment of cartilage injury and OA. The work in this thesis has looked to identify if specific microRNAs are dysregulated in OA. Specifically the expression of miR-146b in human OA articular chondrocytes was investigated. MiR-146b shares close sequence homology to miR-146a and was hypothesised to exhibit similar expression in OA chondrocytes and be receptive to IL-1β stimulation. Using TaqMan qPCR, the expression of miR-146b-5p was found to be significantly up-regulated in human articular OA chondrocytes, with a fold change of ~123. Treatment of human chondrocytes with pro-inflammatory IL-1β did not induce miR-146b up-regulation, but did induce miR-146a up-regulation. Specific microRNAs were also hypothesised to be involved with chondrogenic differentiation of human stem cells. Specifically, miR-34a was hypothesised to target specific chondrogenic associated genes in human fetal femur-derived cells and miR-146b was hypothesised to target chondrogenic associated genes in human bone marrow derived skeletal stem cells. Skeletal stem cell chondrogenic differentiation can be conducted in vitro with the use of chemical cues and a three dimensional micromass culture system. Despite the identification that miR-34a was found to be significantly up-regulated during chondrogenic differentiation of fetal femur-derived cells, no effect was observed following modulation of miR-34a levels on the selected predicted mRNA target; TGIF2. MiR-146b was found to be significantly down-regulated during chondrogenic differentiation of human bone marrow derived skeletal stem cells and both SMAD4 and SOX5 were identified as predicted targets of miR-146b. Overexpression of miR-146b resulted in the significant down-regulation of SOX5. SOX5 is required for early chondrogenic differentiation. MiR-146b is suggested to be down-regulated during chondrogenesis to prevent the inhibitory effects upon SOX5 expression. Future studies to modulate anti-chondrogenic miR-146b through stable transfection and future in vivo studies will assess the ability of miR-146b to modulate chondrogenic differentiation in vivo. Identification of miR-146b, which was shown to display dysregulated expression in human OA articular chondrocytes, could be used as a potential therapeutic target in the future, as a potential anti-inflammatory therapeutic. Identification of miR-146b down-regulation during the chondrogenic differentiation of human bone marrow derived skeletal stem cells may enable the potential use of miR-146b modulation in combination with skeletal stem cell therapy to induce articular cartilage regeneration at articular cartilage defect sites, which may prevent to the onset of OA.

Joint damage in osteoarthritis (OA) involves characteristic changes in multiple joint tissue types, with alterations in the shape of subchondral bone and composition of synovial fluid occurring concomitantly with cartilage damage. Systemic risk factors such as obesity and local biomechanical processes that determine joint loading have long been shown to predispose to the disease, and more recent evidence suggests that specific genetic loci and inflammatory processes may make contributions, but the exact mechanisms driving the initiation and progression of the disease are yet to be elucidated. Advances in imaging that allow visualisation of minute changes in structures such as cartilage composition and subchondral bone shape have enabled longitudinal tracking of early disease development. It is hoped that the identification of early imaging biomarkers for later disease will not only assist in clarifying disease pathogenesis, but facilitate the development of disease-modifying interventions by shortening the lead time for assessing their effectiveness. This thesis aimed to clarify the current information on the utility of imaging biomarkers in research into the pathogenesis and treatment of osteoarthritis, and investigate potential imaging markers in the two regions that contribute the most to the disease burden of osteoarthritis; the knee and the hand. It utilises data from three clinical studies; a cross-sectional survey on quality of life indices in patients with arthritis, and the ongoing KANON (Knee Anterior Cruciate Ligament Non-operative vs. Operative Treatment) and COMBO (Combined Conservative Therapies on Clinical Outcomes in Thumb Base Osteoarthritis) trials. The chapters are presented in mixed manuscript form and journal format owing to differing stages of publication, and are designed to be read independently. Chapter one presents an introduction to the current understanding of the pathogenesis of OA and the role of imaging in clinical research. Chapter two presents published data from a cross-sectional survey that included 1039 participants via an online platform and utilised the validated ICOAP (Measure of Intermittent and Constant Osteoarthritis Pain) and EQ5D scores. The data identified the knees and hands as the regions in which pain was the most common, and in which arthropathy was the most detrimental to activities of daily living. This regional distribution differed slightly from studies in which participants were drawn from clinician referral, rather than from a convenience sample, raising points on the potential advantages of online platforms, and framing the subsequent focus on research into imaging biomarkers for OA of the knees and hands. Chapter three provides a further introduction to magnetic resonance imaging (MRI) to provide a background for the subsequent two chapters, and consists of a published book chapter that focuses on MRI as an imaging modality that enables direct visualisation of changes in both structure and composition in different joint tissues through the modification of contrast and sequences. In chapter four, post-traumatic osteoarthritis is discussed in two published manuscripts in terms of its value in investigating disease pathogenesis through imaging. Traumatic joint injury, and anterior cruciate ligament (ACL) rupture in particular, is strongly linked to the subsequent development of osteoarthritis, so provides an opportunity to track pathological changes from a defined starting point, and therefore potentially identify early imaging markers for subsequent disease. Chapter five presents data from serial MRIs in the KANON trial, which included 121 individuals who had sustained an acute anterior cruciate ligament (ACL) rupture to a previously uninjured knee. In the trial, 62 participants were randomised to undergo early ACL reconstruction and structured rehabilitation, and 59 were allocated to undergo structured rehabilitation alone, with an optional delayed ACL reconstruction. MRIs of the knee were obtained for all participants at baseline, two years, and five years, and for a subgroup of 63 participants, additional MRIs were performed at 3, 6, and 12 months. The serial MR imaging has enabled the effects of concomitant injuries such as osteochondral fractures and meniscal tears to be tracked, as well as providing longitudinal measurements of changes in cartilage and subchondral bone that can then be correlated with clinical outcomes. Data on the regional changes in the area of bone covered by cartilage (cAB) as a potential biomarker for disease, and the effects of baseline injuries to other joint structures, are presented. In chapter six, data on radiographic markers in trapeziometacarpal (base of thumb) osteoarthritis are presented from the COMBO trial. It includes two manuscripts – one published and one in the process of review – that present data from the first 100 participants included in the COMBO trial. Radiographic markers have traditionally had a poor correlation with symptomatic and functional outcomes, so generalised estimating equations are used for bilateral data to minimize the influence of interpersonal confounding factors and account for the fact that within-person measurements are not independent. The radial subluxation ratio is investigated in the manuscripts as a marker for structural and functional osteoarthritis progression, and radiographic markers of disease severity are analysed in relation to pain and functional outcomes in symptomatic disease. In summary, magnetic resonance imaging and radiographic markers are investigated in relation to their utility as indicators of osteoarthritis progression, and in acting as outcomes for the assessment of potential interventions in clinical trials.

Osteoarthritis (OA) is a common debilitating disease ofmammalian joints. Canine OA was classically understood to arise secondary to articular diseases, such as elbow dysplasia, hip dysplasia and cranial cruciate ligament rupture which produced a mechanical dYllfunction of the affected joint. However it is now recognised that primary factors, such as genetics, govern the severity ofOA for a given articular disease. In this study, two different aspects ofthe genetic basis to OA were investigated; gene transcription in diseased tissues and gene polymorphism frequencies in populations of dogs with diseases predisposing to OA. Evaluation of the quality of extracted mRNA from canine joint tissues by rnicrofluidic electrophoresis traces revealed that there were no differences in the quality of samples extracted using either an isopropanol or ethanol precipitation method. However, a significant proportion ofRNA samples (32%) were identified as degraded, highlighting the importance of assessing RNA quality before usage. In OA canine hip cartilage, there was an increase in the gene expression of structural matrix molecules (collagens and small leucine rich proteoglycans) and proteinases (matrix metalloproteinase 13, cathepsin -B and -D), with concurrent decreased expression of selected inhibitors or protease activity (tissue inhibitors of metalloproteinase-2 and -4) when compared to normal articular cartilage using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analyses. The general pattern of changes in matrix-associated gene expression was similar to that reported in naturally occurring human OA cartilage. Canine-specific oligonucleotide microarray gene expression profiling of a small sample set of normal and OA articular cartilage samples identified differential expression of a number of genes not previously associated with the disease. However, the high degree of heterogeneity observed in the expression profile data generated hampered subsequent data interpretation, and highlighted the limitations of expression profiling small sample sets with limited phenotype stringency. Quantification of matrix-associated gene expression in OA elbow cartilage by RT-qPCR identified changes which were consistent with those identified in end stage hip OA cartilage, and which correlated with the radiographic severity of elbow OA for a number of genes (such as type I collagen alpha two chain, type III collagen alpha one chain and tissue inhibitor of matrix metalloproteinase 2). Matrix metalloproteinase expression in OA elbow trabecular bone was also identified to be increased when compared to normal trabecular bone, when quantified by RT-qPCR. A general pattern of increased protease and extracellular structural matrix gene expression was identified in ruptured canine cranial cruciate ligaments (CCL) when compared to intact CCLs, with both RTqPCR and oligonucleotide microarray. No significant differences were identified between the gene expression profiles of normal CCLs of a breed predisposed to CCL rupture (Labrador Retriever) when compared to a breed relatively resistant to CCL rupture (Greyhound), although a degree of risk-specific clustering was observed for expression profiles of genes which were differentially expressed in CCL rupture. The expression profiles ofruptured canine CCLs were similar to those previously reported for ruptured human CCLs. A transcriptomic basis to breed specific risk for the development ofcanine CCL rupture was not identified. Microarray data sets generated from normal and OA canine articular cartilage and normal and ruptured CCL were filtered to identify new reference genes for use in RT-qPCR experiments. One of the new reference genes (Mitochondrial ribosomal protein S7 [MRPS7]) demonstrated a high degree of stability across multiple articular tissues from normal and OA canine joints, as determined by multiple, different reference gene stability assessment algorithms, making it a potential universal reference gene for use in canine OA tissue studies. Silica membrane spin columns provided the most consistent recovery of high quantities of genomic DNA (gDNA) from EDTA preserved and clotted blood samples without the co-extraction of PCR inhibitors, when compared to phenol-chloroform or modified salt precipitation methods of DNA extraction. Spectrophotometer quantification of extracted gDNA did not provide an accurate assessment of the functional gDNA quantity with phenol-chloroform extracted samples, because ofprotein contamination. Single nucleotide polymorphisrns (SNPs) were identified in twenty candidate genes and their allele frequencies evaluated in populations of Labrador Retrievers and Golden Retrievers with cruciate ligament disease, populations of Labrador Retrievers with elbow dysplasia and hip dysplasia and compared to general populations of Labradors Retrievers and Golden Retrievers. Significant associations were identified for the minor allele and haplotype frequencies of SNPs in interieukin 12B (ILI2B) in Labrador Retrievers with elbow dysplasia, interieukin 4 (IL4) and interieukin 6 (IL6) in Labrador Retrievers with hip dysplasia, IL4 and ILl2B in Labrador Retrievers with cranial cruciate ligament rupture, and interieukin 10 (ILID) and Ankyrin repeat domain 10 (ANKRDIO) in Golden Retrievers with cranial cruciate ligament rupture. A common genomic risk for the articular disease, or OA, was not identified between the two different breeds of dog evaluated, but common genomic risks were identified for different articular diseases within a single breed. A genetic basis to canine articular disease, or OA, was confirmed.

Osteoarthritis of the hip frequently occurs in the absence of osteoarthritis in other large joints, suggesting that local factors are important in its pathogenesis. Hip morphology has been recognised as a potential local biomechanical risk factor for the development of hip osteoarthritis. There are no adequate studies examining osteoarthritis development in the hip. Historical cohorts are either limited by a short follow up or by small numbers. This thesis explores the natural history of hip osteoarthritis in a large population cohort with particular attention to hip morphology as a predictor of osteoarthritis development. Software was developed which allows objective measurements of hip morphology in a reproducible manner. Hip morphology was then measured in a 1000 subject cohort. A detailed description of hip morphology is presented in this thesis, with interesting observations of wide variation and a bimodal distribution for alpha angle (a measure of cam-type femoroacetabular impingement). This is suggestive of a discrete pathological entity, which was associated with osteoarthritis in the cross-sectional analysis. No significant changes exist in terms of morphology during the course of the study and no significant relationship exists between age and hip morphology. Longitudinal analysis of hip morphology with radiographic osteoarthritis and total hip replacement revealed a significant association between cam-type femoroacetabular impingement and acetabular dysplasia with both outcome measures. Measurements of hip morphology were independently predictive of outcome when controlling for baseline age, BMI and joint space width, and significantly increased our ability to predict osteoarthritis and total hip replacement. Similar associations were seen when considering hip pain and symptomatic osteoarthritis as the outcome measures of interest. Pincer-type femoroacetabular impingement was not significantly associated with any of the outcome measures of interest and pain remains relatively poorly explained by both hip morphology and/or radiographic change. The understanding of hip morphology and its role in the natural history of osteoarthritis is significantly improved by this research. Further research is now required to determine whether these morphological abnormalities represent modifiable risk factors for osteoarthritis progression.

Purpose: Variation in morphology in the proximal femur and pelvis (e.g. acetabular dysplasia, non-spherical femoral head) can biomechanically compromise the hip joint and predispose to hip osteoarthritis (OA). Such morphological variation may in part explain the heritability of hip OA. The objective of this study was to evaluate a range of 2-dimensional morphological measures on standard radiographs to determine: normal range, right: left symmetry, age and gender differences; and to investigate whether they are associated with the risk of hip OA. Methods: A nested case control study was undertaken in 566 unilateral hip OA cases and 1108 controls in the established Nottingham Genetics of Osteoarthritis and Lifestyle (GOAL) database. Unaffected hips of unilateral hip OA cases were compared to the normal controls, under the assumption that similar morphological features would be observed for the affected hips prior to the development of hip OA. Definition of radiographic hip OA was joint space width (JSW) ~ 2.5 mm. Standardized antero-posterior (AP) radiographs of the pelvis were used to measure the morphological features. Measurements were performed by a single observer and the reproducibility was evaluated at baseline, mid and end of the study. Normal values, thresholds (mean±1.96SD) and symmetry of the features were derived from the control subjects. The intra-observer reliability was examined using intra-class correlation coefficient (ICC). Odds ratio (OR) and 95% confidence interval (Cl) were calculated for association. Logistic regression was used to adjust for age, gender and body mass index (BM!). Measurements were divided into tertiles to examine dose response. ii Results: The intra-observer reliability

Osteoarthritis (OA) is a debilitating disease affecting nearly 5 million of Canadians, with aneconomic impact of over $30 billion each year in direct and indirect costs. Pain is the prominent symptom of OA, yet its underlying mechanisms are not completely understood, leading to a lack of effective pain therapies. OA pain is manifested by a hypersensitivity to mechanical stimuli such as joint movement or palpation. Although the mechanical sensitization of pain-sensing nerve fibers, known as nociceptors, innervating the joint has been documented, the molecular and cellular mechanisms underlying this sensitization are currently unknown. The peripheral terminals of nociceptors express mechanosensitive ion channels (MSCs) that convert mechanical stimuli into depolarizing potentials. Our central hypothesis is that the sensitization of nociceptors to mechanical stimuli in OA can be explained by changes in the properties of these MSCs, whereby their activation threshold is reduced during the disease. Using the monoiodoacetate model of OA in mice, we demonstrate that mice develop chronic mechanical allodynia. Furthermore, cell–attached electrophysiological recordings of nociceptors isolated from OA mice indicate an increase in the current elicited by mechanical stimuli, as well as a reduced activation threshold of MSCs, when compared to naïve mice. An understanding of the molecular bases for these changes is presently impossible because the identity of the genes encoding MSCs isunknown. Interestingly, in a previous screen for MSC candidates, we identified five transmembrane proteins of unknown function (TMEMs). Our results indicate these candidates are expressed in sensory neurons and the mRNA of three of them is increased in OA. When expressed in COS-7 cells, two of these candidates, TMEM5B and TMEM1, caused an increase and decrease in cellular mechanosensitivity. This project will lead to a better understanding of the molecular mechanisms underlying arthritis pain.
L'ostéoarthrite (OA) est une maladie débilitante affectant près de 5 millions de Canadiens et ayant un impact économique de plus de 30 milliards de dollars en coûts directs et indirects. Bienque le symptôme principal de l'OA est la douleur, les mécanismes qui en sont responsables ne sont pas compris, et par conséquent, les thérapies effectives contre la douleur sont manquantes. La douleur causée par l'OA est manifestée par une hypersensibilité aux stimuli mécaniques tels le mouvement des jointures ou la palpation. Bien que la sensibilisation mécanique des fibres nerveuses qui détectent la douleur (nocicepteurs) innervant la jointure ait été documentée, les mécanismes moléculaires et cellulaires de base de la sensibilisation ne sont pas compris. Les terminaisons périphériques des nocicepteurs expriment des canaux ioniques mécanosensibles (MSCs) responsables de la conversion des stimuli mécaniques en potentiels dépolarisants. Notrehypothèse centrale est que durant l'OA, la sensibilisation des nocicepteurs aux stimulimécaniques peut être attribuée aux changements des propriétés de base des MSCs, où leur seuil d'activation est réduit lors de la maladie. Nous démontrons, en utilisant le modèle de monoiodoacetate de l'OA chez la souris, que cette dernière développe une allodynie mécaniquechronique. De plus, les enregistrements électrophysiologiques en configuration cellule-attachée faites à partir des nociceptors isolés des souris OA indiquent une augmentation du courant élicitépar les stimuli mécaniques, ainsi qu'une réduction dans le seuil d'activation des MSCs encomparaison aux souris naïves. Une compréhension de la base moléculaire de ces changements est présentement impossible car l'identité des gènes qui encodent les MSCs n'est pas connue. Dans des expériences antérieures, au cours desquelles nous avons fait un criblage différentiel afin d'identifier des candidats de MSC, nous avons identifié cinq protéines transmembranaires de fonctions inconnues (TMEMs). Nos résultats indiquent que ces candidats sont exprimés dans les neurones sensoriels et que l'ARNm de trois de ces derniers augmente durant l'OA. Lorsqu'exprimés dans les cellules COS-7, deux des candidats (TMEM5B et TMEM1) ont causé une augmentation et une diminution de la mécanosensibilité cellulaire. Ce projet nous donnera une meilleure compréhension des mécanismes moléculaires de base de la douleur arthritique.

Osteoarthritis (OA) is a common musculoskeletal disability and represents a major health burden to society. Pathological changes are found in all tissues of the joint; however studies on bone are few. The aims of this thesis were to characterize some of the densitometric, mechanical and compositional properties of cancellous bone from the Superior (Sup) and Inferior (Inf) regions of human femoral heads, and to compare age-selected healthy bones with those from patients with end-stage OA. From coronal sections of the femoral head, bone cores were drilled out along the anterior-posterior axis and non-adherent fatty tissues removed. Measurements of apparent (PA), true (pj) and volumetric bone mineral density (BMDv) were made prior to determining the ultrasound-derived elastic modulus (Eu) and the compression-derived parameters, compressive modulus (Ec), yield strength (CTY) and yield energy (Wy). From aliquots of powdered bone, calcium and hydroxyproline were determined and a minerahcollagen ratio calculated, and osteocalcin, IGF-I, IGF-II and IGFBP-5 measured by RIA. Another aliquot was processed to remove soft tissue, demineralized in EDTA and the resulting collagen matrix digested with trypsin. From these crude tryptic digests (CTDs), divalently and trivalently (ICTP, IIINTP) crosslinked collagen telopeptides and procollagen propeptides of collagens type I and III were analysed by RIA. PA, BMDv, EC, ay, Wy and EU were systematically increased in cores from the Sup region of both Normal and OA groups (all/KO.05) and reflects differences in loading experienced by this region. In OA bone, PA, BMDv and EU were increased in the Inf region (all/KO.05) possibly as a result of adaptation of the bone to altered loading patterns at the hip. Furthermore, the relationship between EC and PA was different to those in Normals (p=0.019) indicating that other factors, such as architecture, may influence the stiffness of cancellous bone. Of the compositional measures, IGFBP-5 was systematically increased in both regions of OA bone (all /?<0.005) and may be the factor responsible for maintenance of bone mass in OA. The minerahcollagen ratio was decreased in the OA Sup region (p=0.008) indicating undermineralization of bone. In the mineralized matrix of OA bone, concentrations of ICTP:collagencTD and type III collagen antigens were increased in both Sup and Inf regions (all/><0.05) suggesting an increase in type I collagen trivalent crosslinking at the Qtelopeptide and an overall increase in type III collagen respectively. Of the relationships between the various properties, inverse correlations were found between mineral.'collagen and ICTPrcollagenciD ratios (rs= -0.36,/?<0.05) in the OA group indicating increased trivalent collagen crosslinking when bone is under mineralized. In the Normal group PA correlated inversely with IGFBP-5 (rs=-0.64 and -0.72, bothp<0.05) and OC (rs=-0.59,/7=0.056 and -0.71, p<0.05) at both Sup and Inf regions respectively, but were lost in the OA group suggesting loss of regulation at this level in OA. hi conclusion, the cancellous bone in OA femoral heads is denser, but not stiffer than that of Normal bone, and has an altered composition with respect to both structural (ICTP) and regulatory (IGFBP-5) factors which may affect the quality of the bone matrix.

This study determined the response of elderly human articular cartilage explants to static, cyclic or absence of mechanical load, with or without the addition of the maintenance factor IGF-1.  It revealed that elderly human articular cartilage does not behave in the same way as young animal tissue, with cyclic and static loading both causing an inhibition in matrix biosynthesis.  In addition, cyclic mechanical load appears to block the effects of IGF-1 in elderly human tissue, in contrast to bovine tissue in which the effects were additive.  Osteoarthritic bone was found to have double the fat content of osteoporotic bone and, within the fatty acid profile, arachidonic acid was present at twice the concentration.  Surprisingly, the addition of arachidonic acid to cartilage explants appeared to have no effect on matrix biosynthesis though, in retrospect, this may have been a methodological problem.

A pilot study using gene arrays revealed factors previously unidentified which may be crucial to the mechanotransduction process in chondrocytes.  The considerable upregulation of transcripts for anabolic factors with no corresponding upregulation of those for matrix macromolecules raises questions about how mechanical processes regulate genes. In summary, this thesis has demonstrated that mechanical and chemokine factors interact in ways different to those found in animal cartilage.  The role of fatty acids in mature human cartilage requires further investigation, but mechanically stimulated transcription of genes for anabolic factors does not appear to result in increased matrix synthesis.

Knee osteoarthritis (OA) is a common cause of pain and disability. Patients often complain that they overload the other limb when they walk, resulting in disease in the unaffected knee. However, it is unknown whether this happens or the mechanism by which it occurs. Data was analysed from an established longitudinal cohort study to examine the development of bilateral knee OA. One hundred and forty-three subjects were examined over a 12 year period with bilateral radiographs. Bilateral knee osteoarthritis was found to be very common over time, and the majority of individuals with unilateral knee OA eventually developed bilateral disease. A gait analysis study was performed on 20 subjects with unilateral knee OA awaiting arthroplasty surgery and 20 healthy age equivalent controls. Abnormal moments and muscle co-contractions were observed in the other knee and hips when they walked due a characteristic slow, cautious, stiff-legged gait pattern. Fifteen subjects re-attended 12 months following their surgery. Whilst moments returned to normal in most of the replaced knees, they remained elevated at the contra-lateral side and co-contraction failed to recover in either knee. A novel study design is presented to examine the effect of gait-derived loading waveforms on fresh human osteochondral plugs. By applying mechano-biology techniques and Finite Element Modelling to fresh human tissue, new observations vi can be made about the relationship between in-vivo loading and cartilage mechano-biology. A characteristic gait pattern was observed in knee OA which is not simply antalgic but tends towards symmetry, with an increase in joint loading bilaterally. The observed gait behaviour does not resolve, despite arthroplasty of the affected joint. This would be expected to contribute to the development of disease in an inherently vulnerable joint. Additional training may have a role to play in restoring normal biomechanics and protecting the other knee from disease.

Pain is the foremost clinical symptom of osteoarthritis (OA), however the processes that lead to pain in the joint are poorly understood. I have used two different murine models of OA, induced by surgical destabilisation of the joint. Upon destabilisation, two episodes of painful behaviour are observed, an initial post-operative phase, lasting a few days, and a late phase that starts between 8-11 weeks post-surgery, detected by differences in weight bearing measured by the Linton incapacitance tester. The later phase is associated with significant cartilage damage, but no overt synovitis. A screen of pain sensitising molecules expressed in the whole joint at the time animals developed late pain was performed. Statistically significantly regulated molecules included nerve growth factor (Ngf), bradykinin receptors 1 and 2 (Bdkrb1 and Bdkrb2), Tachykinin 1 (Tac1) and its receptor (Tacr1), determined by real-time PCR. When a similar analysis was performed in the microdissected tissues of the joint (cartilage, bone and meniscus), all but one (Bdkrb2), of the previously regulated molecules were significantly regulated in the articular cartilage. Ngf, Bdkrb1, Bdkrb2 and Tac1 were also statistically significantly regulated upon in vitro cartilage injury, using the murine hip and porcine metacarpophalangeal explantation models, suggesting that in vivo mechanical damage may be sufficient to drive pain molecules. One mechanism previously described by our group, by which chondrocytes respond to injury, is via release of FGF2 from the pericellular matrix. When in vivo cartilage injury studies were performed in Fgf2-/- tissues, or in the presence of an FGF2 receptor inhibitor, induction of Ngf was significantly blunted, indicating that FGF2 drives Ngf induction upon in vitro cartilage injury. Induction of Bdkrb1, Bdkrb2 and Tac1 were not FGF-dependent in vitro. To test the significance of FGF2-driven Ngf induction in vivo, pain was assessed in Fgf2-/- mice following joint destabilisation. Rather than reducing or delaying the development of pain in these animals, painful behaviour developed earlier than wild type mice, and correlated with cartilage damage scores. Pain could also be significantly neutralised by anti NGF antibody, indicating that FGF2 is unlikely to be driving Ngf expression in vivo. Female mice are relatively protected from OA following joint destabilisation, a phenomenon that may be related to differences in their molecular response to acute joint destabilisation. Despite having little in the way of cartilage damage, they develop painful behaviour at a similar stage as male mice, associated with the same pain sensitising molecules.

Osteoarthritis (OA) is the most common degenerative joint disease affecting more than 40% of people above the age of 65 (Neogi et al., 2013). Obesity is one of the main risk factors of OA and has become a major problem in Western societies. With sedentary lifestyle and the aging of the population, it is estimated that more than 50% of British adults will be obese in 2030 (Wang et al., 2011). So far, the effect of obesity on joint degeneration has primarily been explained by the increased load on the joints. However, a growing number of studies have revealed that adipose tissue can affect cartilage and other joint tissues at a molecular level. The main goal of this thesis was to investigate the role of local knee joint tissues in obese patients with OA. The expression of molecular markers was investigated in local knee tissues: cartilage, synovium, infrapatellar fat pad (IPFP) and subchondral bone collected during Total Knee Replacement (TKR). A range of techniques (RT-PCR, Real Time qPCR, WB, IHC/ICC and ELISA) was used to examine the differences between genes and proteins expression in both lean and obese patients with OA. Further, the local immune cell infiltration was investigated in knee adipose tissue depots (synovium and IPFP) using flow cytometry. In addition, the subchondral bone microstructure was analysed using micro-Computed Tomography (μCT) and IHC techniques. Chondrocytes from OA patients were found to express a range of obesity-related genes. ADIPOR1 was produced significantly higher than ADIPOR2 in OA chondrocytes. Furthermore, CCL2 was produced at higher while PPARγ and visfatin were produced at a lower level in obese patients’ chondrocytes in comparison to lean ones. Synovium and IPFP also expressed a range of obesity-related genes. PPARγ and visfatin expression was lower in obese synovium and IPFP in comparison to lean. Surprisingly, adiponectin was expressed at a significantly lower level in obese patients’ synovium. In contrast, adiponectin was not differently expressed in lean and obese patients’ IPFP. The IPFP was found to be a significantly higher producer of PPARγ and adiponectin in comparison to synovium. Synovium, on the other hand, has an increased expression of VCAM-1, TLR4 and CCL2 in obese patients. An increased number of macrophages (defined by CD45+CD14+ and CD14+CD206+ markers expression) was detected in the synovium and IPFP from obese OA patients. Furthermore, there was an increased number of CD86+CD14+ cells in the synovium from obese patients. Other macrophage-related proteins including HLA-DR, CD36 were also expressed at a higher level in synovium from obese patients. T-lymphocyte detection revealed a higher number of CD3+CD4+ T cells in the synovium (but not IPFP) from obese patients but no change in the CD3+CD8+ population in both the synovium and IPFP. Subchondral bone analysis revealed possible differences in this tissue in obese male patients with OA in comparison to lean patients. μCT examination of subchondral bone showed a significantly lower bone mineral density (BMD) in obese in comparison to lean male OA patients. IHC analysis of bone sections suggested that there was an increased number of bone marrow adipose tissue macrophages. In addition, osteoblasts obtained from obese OA donors expressed a significantly higher level of ADIPOR2 and lower level of PPARγ mRNA in comparison to lean patients’ osteoblasts. The data obtained suggests that there were differences between lean and obese patients with OA at a molecular level. This proposes possible future directions for targeting these diseases. The limitation of the study were as follows: 1) possible different stages of end-stage OA between analysed patients, which could lead to differences in obtained data, 2) no non-OA control samples included in the study. However, the presented study may suggest that all tissues in the knee joint contribute to the interplay between OA and obesity. In addition, the data obtained is the first to suggest that there are differences in gene and protein expression in the synovium and IPFP from the same donor. Furthermore, there are differences in the immune cell populations in local adipose tissue depots (synovium and IPFP) from OA joints, which are linked to obesity. All of this data has helped to increase our understanding of the interaction between obesity and OA.

Articular cartilage is a remarkable tissue with mechanical performance that surpasses engineering standards. Collagen, the most abundant protein in cartilage, plays an important role in this performance. Building on observations of collagen structural changes at the earliest stages of osteoarthritis, we here explore the role of collagen in the disease process. Specifically, we focus on the material properties and organization of collagen fibrils in the cartilage surface, seeking to answer three key questions: • What are the physical changes to collagen in early osteoarthritis? • How do physical changes relate to osteoarthritic progression mechanistically? • Can we detect early, irreversible changes to cartilage intraoperatively to facilitate early treatment? Ten human tibial plateaus were characterised by atomic force microscopy and Raman spectroscopy, with clinical and histological scoring used for comparison. Minimal changes were found in the collagen molecules themselves, yet a marked increase in intra-fibrillar crosslinking was observed. A bottom-up numerical model of the collagen fibril network was then implemented to specifically explore the relationship between fibril interactions and mechanics in osteoarthritis. Simulating two different fibril configurations, we established that high connectivity is critical to the resistance to, and recovery from, loading. Osteoarthritis-like bundled configurations were produced by networks with sparse interactions, and identified as a mechanically irreversible stage in disease progression. Coupling the model with experimental results, we argue that the intra-fibril changes appear to link to disease initiation and it is the inter-fibril interactions that drives the disease. Finally, a pilot study was conducted to explore the potential for quantitative intraoperative imaging to detect irreversible collagen-related changes in the cartilage surface. Based on spectral analysis of the available light from standard arthroscopic equipment, it may be possible to advance arthroscopy from a qualitative to quantitative assessment tool.

Osteoarthritis (OA) is the most common degenerative joint disease. The impact loading on the articular cartilage of the large weight bearing joints (hip, knee, and ankle joints) during distance running might be a potential precipitating factor in OA. The aim of this case-control study was to investigate the relationship between total accumulated running volume and OA in the weightbearing joints. In this study, OA was defined as pain and/or stiffness and/or swelling in the weight-bearing and non-weight-bearing joints (wrists and fingers). The subjects for this study were selected from previous and current runners of the Two Oceans Ultra-marathon (56 km) in Cape Town (South Africa). The database (1356) consisted of all the runners who participated in this race between 1970 and 1983. From this data-base a random group of male runners (n =128) were divided into six 10-year age groups of runners (18 and 79 years). There was a random sample of 25 runners in five of these groups and three in the 70-79 year age group. Runners were age matched with a random sample of past pupils (n=204) of a school who were in their final year between 1923 and 1994. This was the control group. A questionnaire to diagnose OA was designed and validated with a sensitivity of 92% and a specificity of 71 %. The questionnaire was posted to the runners and controls. Incentive prizes were offered to improve the response rate, which was 59%. Completed information was obtained from 76 ultra-distance marathon runners (response rate 59%) and 114 controls (response rate 56%). In the control group there was a group who participated in running. This group was combined with the runners who were then divided into three groups according to their total running volume which was calculated by the following formula; years involved in running x months/year running x 4 x hours/week running. The subjects were thus divided into four groups: 1) controls (non-runners) (n=60), 2) low volume runners (n=43), 3) medium volume runners (n=43), and 4) high volume runners (n=44). Of these, 22 low volume runners, 7 medium runners, and 7 high volume runners stopped running. The prevalence (%) of OA in all groups was compared. The mean age of the control group was significantly higher than the three running groups. The mean height and weight of the medium volume group was significantly higher than the other groups. There was no significant difference in the BMI in each group. The frequency of professional and retired people was significantly higher in the control and each running group. A significantly greater percentage of controls had a history of admission to hospital. There were more controls on long-term medication, compared to runners. A significant number of injuries to the weight-bearing joints (specifically the knee joint) occurred in all groups, due to other sports (p =0.007). There were no significant differences in symptoms suggestive of OA in all groups when not adjusting for age and previous injuries. However, when assessing the odds ratio to determine the risk for OA in the weight-bearing joints, adjusting for age and previous injuries, the low volume group had the highest risk to develop OA (O.R. = 3.2, 95% C.I. = 1.0-10.3); the medium group had the second highest risk (O.R. = 1.7, 95% C.I. = 0.6- 4.8) and the high-volume group (O.R. = 1.1, 95% C.I. = 0.4-3.1) and control groups (O.R. = 1.0) had equally the lowest risk to develop OA. This study confirmed that distance running is unlikely to be a predisposing factor in the development of OA in the weight-bearing joints, even at high running volumes commonly seen in ultra-distance running.

Osteoarthritis (OA) is a common disease characterised by the progressive loss of the articular cartilage of synovial joints. It is multifactorial and polygenic. Candidate genebased association studies and genome-wide association scans (GWAS) have been used to identify OA risk alleles with over 10 regions of the human genome so far reported as harbouring OA susceptibility. Expression quantitative trait loci (eQTLs) demonstrate a correlation between gene expression levels and DNA polymorphism, the assumption being that the polymorphism resides within a regulatory element of the gene. The functional polymorphism can be cis-acting (within or close to the gene) or trans-acting (located a distance from the gene). It has become apparent for many common diseases that the majority of risk alleles are eQTLs rather than polymorphisms that alter amino acid sequences. The aim of this thesis was to investigate whether genes proximal to or harbouring OA-associated polymorphisms identified by GWAS are subject to cisregulation, which could therefore be contributing to the association signal. The expression levels of the genes were analysed in joint tissues from OA patients who had undergone joint replacement. Overall gene expression was measured by quantitative PCR whilst allelic expression was assessed using assays that can distinguish mRNA output from each allele of a transcript SNP. The functional effect of SNPs was tested in vitro using luciferase reporter assays. Expression analysis of genes at the OA-associated chromosome 7q22 locus identified HBP1 as the likely target of the OA susceptibility mapping to this region. Expression analysis of SMAD3 and GNL3 identified eQTLs active in OA joint tissues for both genes, whilst luciferase reporter assays identified two 3´UTR SNPs as potential mediators of the SMAD3 eQTL. Overall, this thesis demonstrates that OA tissues are subject to a range of regulatory elements that can influence disease during development or ageing.

Background: Halofuginone (HF) is a natural product that has been shown to have therapeutic benefits in a variety of pathologic conditions, from cancer to autoimmune diseases. These beneficial effects are mainly through inhibition of pro-inflammatory cytokines. Inflammatory cytokines have been recently shown to exert a crucial role in development of osteoarthritis and one of their most important targets is Matrix metallopeptidase-13 (MMP-13). In this preclinical study, we investigated the effect of HF on the progression of Osteoarthritis. Methods: The effect of adding HF ± IL-1β/TNF-α on mRNA expression of MMP-13 on the C28/I2 Chondrocytes was evaluated with qPCR in vitro. To study the effect of HF in vivo, a mice destabilization of the medial meniscus (DMM) osteoarthritis model was employed and untreated control group were compared with early treatment with HF (starting 48 hours post-surgery for 12 weeks) and late treatment (4 weeks post-surgery for 8 weeks). After sacrificing the animal, joint destruction in the knee tissue was assessed with Safranine O/Fast Green staining and MMP13 expression was evaluated by immunohistochemical staining. Results: In chondrocytes, MMP-13 expression was significantly increased with IL-1β or TNF-α (13.77-fold, p-value<0.05 and 5.8-fold, p-value<0.05, respectively). Addition of HF Significantly reduced MMP13 expression close to baseline levels (1.73-fold, p-value<0.05 when co-incubated with IL-1β and 1.6-fold p-value<0.05 when co-incubated with TNF-α). Injection of HF in the mice osteoarthritis model in vivo, significantly reduced Osteoarthritis progression according to OARSI scoring (3.8 vs. 1.16 vs. 1.07, p-value<0.05) and there was no difference between early vs. late administration of HF. While MMP-13 was overexpressed in the control (DMM surgery without HF treatment) groups in IHC staining, expression of MMP13 was suppressed by injection of HF in both groups. Conclusion: Halofuginone inhibits MMP-13 expression and diminishes joint destruction. These preclinical findings provide supporting data for clinical investigation of HF as a therapeutic target for osteoarthritis.

Osteoarthritis (OA) is a prevalent and disabling joint condition. Despite numerous treatments for OA, the clinical benefits of core treatments such as exercise remain modest. Adherence (the extent to which treatments have been completed as prescribed) is an important factor influencing outcomes in OA. However, understanding of adherence remains poor. This thesis aims to investigate adherence to core treatments for OA. Chapter One provides a background of OA and presents a narrative review of adherence. Chapter Two presents a systematic review of adherence measures of core treatments in knee OA. Chapter Three investigates the relationship between adherence, pain, and function in thumb base OA participants. Those with higher baseline pain were more likely to be adherent to a home management program. Chapter Four investigates predictors of adherence to a step count intervention following total knee replacement (TKR). Younger age, higher patient activation, and higher technology self-efficacy were associated with higher adherence. Chapter Five investigates the correlation between self-reported and objective adherence measurements of step count following TKR. One-third of participants underestimated their step count adherence when comparing self-reported to objective measures. Chapter Six examined the presence of different trajectories of step count adherence following TKR using latent class analyses. Three distinct trajectories were identified. Baseline BMI and pain levels differed between groups. Taken together, the findings of this thesis provide greater insight into adherence in OA. The results of this work may be used to 1) make recommendations for reporting adherence in clinical practice and research; 2) identify those at risk of low adherence; 3) support the use of objective measures of adherence, and the need for valid and reliable tools of adherence measurement; 4) better understand how adherence varies between individuals and how to best support them.

The aim of this thesis was to study two objective methods of osteoarthritis (OA) diagnosis in horses and use them on the assessment of new intra-articular treatments. The studied methods were a new inertial-sensor based system of lameness detection and cartilage biomarkers in serum. It was found that distal limb flexion is significantly correlated to the presence of metacarpo-phalangeal OA in hind limbs and that inertial-sensors are sensitive in detecting asymmetry in these cases. A positive and significant correlation was observed between Coll2-1 concentration in serum and the presence of joint disease in males and young horses. Fib3-2 measurement has good potential to be used since it is not influenced by sex or age. Using an experimental model of OA, adipose stem cells pre-activated with interferon-gamma decreased joint inflammation and radiographic lesions. In clinical cases, a single injection of high-concentrated and high-molecular weight hyaluronic-acid decreased joint inflammation and biomarkers’ concentration; OSTEOARTRITE DO MEMBRO DISTAL NO CAVALO Resumo: A finalidade desta tese foi estudar dois métodos de diagnóstico objetivo de osteoartrite (OA) em equinos e aplicá-los na avaliação de novas terapias intra-articulares. Utilizou-se um sistema de sensores de movimento e foi avaliada a concentração de biomarcadores de cartilagem no soro. Concluiu-se que a flexão distal positiva está correlacionada com OA na articulação metacarpofalângica nos membros posteriores e que os sensores são sensíveis na detecção de assimetria nestes casos. Existe uma correlação positiva e significativa entre as concentrações de Coll2-1 e a presença de doença articular, sobretudo em machos e jovens. A dosagem de Fib3-2 tem utilidade por não ser influenciada pelo sexo nem idade. Num modelo experimental da doença, a terapia à base de células estaminais reduziu a inflamação articular e as lesões radiográficas. Em casos clínicos, o tratamento com ácido-hialurónico de alta concentração e peso molecular provoca uma diminuição da inflamação articular e dos biomarcadores no soro.

This thesis focuses on the role of a bone cell, osteocytes in the progression of osteoarthritis. The biological relevance of this study has opened doors to new possibilities of understanding the pathogenesis of osteoarthritis.

Several lines of research indicate that osteoarthritis (OA) is not only a joint disorder associated with mechanical stress and aging but also a ‘metabolic syndrome’ in which several risk factors work together to contribute to disease initiation and/or development. One such metabolic risk factor could be high cholesterol levels in the body. Even though high cholesterol level is a well-known risk factor for cardiovascular disorders, its possible role in musculoskeletal diseases, particularly OA, is not clear. The authors discuss the fundamental viewpoints on cholesterol involvement in the pathogenesis of OA, stressing the need for understanding the molecular mechanisms behind this association.

L’arthrose est la plus commune des maladies articulaires dans le monde. Elle induit des charges physiques, psychologiques, sociétales et économiques majeures. Selon les Nations Unies, en 2050, 315 millions de personnes souffriront d’arthrose symptomatique et 105 millions d’entre elles seront fortement handicapées par la maladie.Les symptômes cliniques et les techniques d’imagerie constituent les éléments principaux pour le diagnostic et le suivi de l’arthrose aujourd’hui. En dépit de progrès technologiques et des recherches intensives de nouveaux biomarqueurs, aucun outil ne permet de détecter précocement la maladie, de classer les patients selon le pronostic d’évolution de la maladie ou encore de quantifier l'effet des traitements. Ce manque de biomarqueurs sensibles, spécifiques et reproductibles limite les progrès thérapeutiques dans la perspective d’une médecine personnalisée.Dans les articulations synoviales, l’homéostasie est assurée par le maintien de la composition physiologique du liquide synovial, fluide visqueux et lubrifiant. Au cours du processus arthrosique, des changements de composition du liquide synovial interviennent en même temps que la dégradation des tissus articulaires.Dans ce travail, une technique mini-invasive a été mise au point pour caractériser l’état arthrosique du liquide synovial, dans le cadre du suivi de la pathologie et de l’effet de traitements, chez les animaux et les patients. Le dépôt de quelques microlitres de liquide synovial sur une lame en verre forme une goutte circulaire qui, en séchant, concentre dans un anneau périphérique les solutés, dont les caractéristiques tribologiques renseignent sur la présence d’une pathologie articulaire. La spectroscopie Raman fournit des informations précises sur la composition chimique du liquide synovial et permet de suivre les altérations biomoléculaires et structurelles des solutés de l’anneau périphérique de la goutte de liquide synovial séchée. Cette thèse aboutit à la proposition de biomarqueurs synoviaux représentatifs du processus arthrosique articulaire
Osteoarthritis is the most common joint disorder worldwide. Symptomatic osteoarthritis is a painful and debilitating disease which is associated with a large physical, psychological, societal and economic burden. According to the United Nations, by 2050, 315 million people will suffer from symptomatic osteoarthritis worldwide, of whom 105 million will be severely disabled by the disease.Osteoarthritis has traditionally been diagnosed and followed-up based on clinical symptoms and imaging modalities. However, these techniques and molecular biomarker developments are insensitive and identification of patients at risk to disease progression remains a challenge. They also fail to subgroup the heterogenous osteoarthritis population while this would allow a better scrutiny of diagnosis and treatment options. A surrogate biomarker with prognostic and/or diagnostic capabilities able to identify potential responders is much needed in osteoarthritis care and therapy.In diarthrodial joints, homeostasis is based on physiological composition and function of synovial fluid, a viscous fluid that lubricates joints. During osteoarthritis, synovial fluid biochemical properties are modified simultaneously to other joint tissues damages.In this work, a minimally invasive technique has been developed to characterize synovial fluid osteoarthritic state in order to diagnose, predict and follow-up joint diseases in patients and animals. Deposition of only a few microliters of synovial fluid on fused silica slide forms a circular dried drop with a ring-like deposit of concentrated solutes at the droplet edge. Tribological analysis based on the morphology of the dried drops reflects synovial fluid biomolecular and structural modifications. Raman Spectroscopy provides more specific information on chemical composition and monitors biomolecular structural alterations. This thesis leads to the proposal of synovial biomarkers representative of the joint osteoarthritic process

Skeletal muscle quality may play a role in skeletal muscle health affecting its response to muscle strengthening exercises and susceptibility to osteoarthritis (OA). Chapter One of this thesis provides an introduction to the role of skeletal muscle in the incidence, progression and exercise management of knee OA. Chapter Two examines skeletal muscle quality (muscle attenuation and lean muscle volume), knee extensor strength and percentage whole-body fat as mediators in the symptomatic and functional improvements achieved in a diet and exercise (D+E) compared to D (diet) or E (exercise) interventions. Null mediation results were found for skeletal muscle quality and knee extensor strength. However, a reduction in the percentage of whole- body fat significantly partially mediated pain, function, 6-minute walk test and gait speed improvements achieved in the D+E compared to E groups. Chapter Three presents the relationship between a change in lower extremity lean mass (LM) and fat mass (FM) with radiographic knee OA (RKOA) incidence in women longitudinally and concurrently. A 2-year change in LM or FM measures did not predict subsequent RKOA incidence. A 5-year increase of LM percentage was associated with a 10% reduced odds of RKOA incidence (OR 0.90, 95% CI 0.82-0.98, P=0.016]. A 0.25 unit increase in LM:FM ratio over 5 years was associated with a 31% reduced odds of RKOA incidence [OR 0.69, 95% CI 0.49-0.97, P=0.032]. Thigh muscle quality and knee extensor strength did not mediate symptomatic and functional improvements, however, a reduction in percentage whole-body fat partially mediated symptomatic and functional improvements achieved in a D+E compared to E groups. Lower extremity LM and FM changes were significantly associated with RKOA incidence concurrently, but did not precede RKOA incidence longitudinally. Further understanding of skeletal muscle properties would enable a higher treatment effect size to be achieved from rehabilitation programmes in knee OA.

Abstract This work engaged Finnish females affected with osteoarthritis (OA) of the hand to define the role of common sequence variations within the genes of the important structural protein of cartilage, aggrecan (AGC1), and the genes of inflammatory mediators, the interleukin 1 gene cluster and interleukin 6 (IL6), as possible risk factors for the disease. Also, a genome-wide linkage analysis was performed in a sample consisting of Finnish families with multiple individuals affected with hip and knee OA in order to reveal new chromosomal areas that are likely to contain disease associated variations. OA is a chronic disease that leads to the degeneration of articular cartilage in synovial joints. The etiology of the disease is for the most part unknown. Joints of the hand, hip and knee are most commonly affected, and obesity, trauma and excess mechanical stress are known risk factors for the disease. OA also has a significant genetic component. AGC1 carries a variable number of tandem repeats (VNTR) polymorphism, which may be significant for the biomechanical properties of cartilage. It was shown that the most common allele with 27 tandem repeats is protective against hand OA (HOA) (odds ratio 0.46, 95% confidence interval 0.27–0.78). Also, carrying two copies of any of the shorter or longer alleles increased the risk of the disease. Inflammation seems to play a role in the etiology of OA and certain polymorphisms within the interleukin 1 gene cluster and IL6 have been previously shown to increase the transcription of these molecules and to associate with OA. In this study it was shown that the G alleles in three common IL6 promoter single nucleotide polymorphism (SNP) sites are associated with the risk of more severe forms of HOA (p =  0.001 for GGG haplotype). A SNP in IL1B associated with the bilateral form of the disease (p =  0.006) and two IL1B-IL1RN extended haplotype alleles were associated with the same phenotype. Genome-wide and fine mapping linkage analyses recognized chromosomal locus 2q21 with a multipoint LOD score of 3.96. Despite the association analyses of several candidate genes within the locus, no disease-associating sequence variants were identified.

Osteoarthritis is a common joint disease causing pain and inflammation that limits mobility and functionality. Osteoarthritis is a widespread disease, and despite it's prevalence there is no cure. The disease progresses by degrading joint cartilage and synovial fluid. The current work aims to contribute to existing knowledge regarding osteoarthritis through three research projects including a rheological study of novel anti-inflammatory hyaluronic acid derivatives, a case study on the effects of oral glucosamine supplementation on synovial fluid viscosity, and development of a microfluidic rheometer that may be used to study low viscosity fluids such as hyaluronic acid and synovial fluid at high shear rates. The anti-inflammatory hyaluronic acid derivative demonstrated shear thinning and viscoelastic behaviour as expected. This behaviour is common among viscosupplements and is therefore promising for it's potential use as a viscosupplement, however, the viscosity and viscoelasticity were significantly lower than commercial viscosupplements. It is recommended to investigate modulating viscosity and viscoelasticity with techniques such as cross-linking and increasing hyaluronic acid concentration. In a patient discontinuing oral glucosamine supplementation the viscosity was found to be greater prior to stopping treatment at low shear rates and greater after stopping treatment at high shear rates. In a second patient no significant change was observed in viscosity. Future study should investigate the effect of glucosamine on viscoelastic behaviour, and to study the effect of glucosamine on synovial fluid viscosity over an extended period of time. A PDMS microfluidic rheometer was developed using a soft-lithography process. The rheometer was validated with water at room temperature and was found to predict fluid viscosity with a maximum of 6% error at shear rates as high as 30,000 s-¹. It is recommended to investigate possible channel deformation as a cause for decreased accuracy at higher shear rates and resultant operating pressures.
Applied Science, Faculty of
Graduate

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
Medicine, Faculty of
Experimental Medicine, Division of
Medicine, Department of
Graduate