Osteoarthritis (OA) is the most prevalent joint pathology, an incurable and painful condition that is a leading cause of reduction in quality of life among the elderly. Originally considered a cartilage disorder due to lesions observed in this tissue, it is now agreed by the scientific community that OA is a complex ‘organ failure’ involving the whole joint. Notwithstanding the dramatic modifications observed radiographically in subchondral bone, their contribution to the pathology is not fully understood and little is known about bone role in the pathogenesis. The aim of this thesis was to characterize the involvement of subchondral bone using a surgical mouse model. Due to their amenability for genetic studies, murine models are increasingly essential tools to identify new therapeutic targets, but their assessment is limited by time-consuming, qualitative histopathology. This work presents novel high-throughput approaches to automatically quantify subchondral bone, articular cartilage, and osteophytes in mouse tibiae. Cartilage was assessed by novel 3D imaging methodologies that overcame the lack of non-destructive imaging previously available for mouse cartilage and constitute a major technological novelty. Changes in bone mechanical properties were further investigated by nanoindentation testing. Microstructural changes in bone were detected by micro-CT before evident cartilage degradation. Nanomechanical properties were altered only at the late stages of the pathology and not associated with alterations in mineral composition. Furthermore, although changes in bone and cartilage were co-localised, correlation analysis showed that they developed independently over time. In conclusion, this thesis presents a 3D quantitative assessment of changes occurring in bone and cartilage within tibiae during OA progression and demonstrates the key involvement of bone in the pathogenesis. The proposed approaches provide an unprecedented high-throughput solution for analysis in the mouse model and might be useful to improve the understanding of murine OA.

Flattening of the distal femoral condyle is a commonly cited feature of knee OA. However, it is unknown whether this is a result of bone wear, or subchondral bone remodelling. Studies show that subchondral bone turnover is increased and remodelling of new bone is pathologic. Such bone is denser with thicker trabeculae, but is also softer and osteopenic. Could remodelling of pathological subchondral bone be the aetiology of flattened condyles? Unlike the hip, not much is known about the morphological parameters of the knee’s articular surface in influencing OA risk outcome. If flattening is the result of the early OA disease process, mechanical stress to the joint will increase due to numerous causes, which will also be discussed. The common sites of wear in knee OA include the distal femoral condyles, and the anteromedial portion of the medial tibia plateau. This study analysed the morphometry of these segments with the aim of quantifying any morphological changes in early OA. The findings were that the extension facets in early OA are indeed flattened, even before the onset of bone to bone contact. Such flattening is hence part of the pathomorphological positive feedback loop, aggravating mechanical conditions early in the disease process.

Bone marrow lesions (BML) are well described in osteoarthritis (OA) using magnetic resonance imaging (MRI) and are associated with pain, however little is known about their pathological characteristics and gene expression. This study evaluated BMLs using tissue analysis tools to gain a deeper understanding of how they mediate pain. A total of 117 participants were recruited, 74 with advanced OA requiring total knee replacement (TKR), 23 with mild OA, 7 healthy controls and 13 non-OA tissue comparator controls. A multidimensional approach was taken to investigate how clinical, radiological and molecular changes in OA knee joints correlate with pain perception. Pain scoring (Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Pain DETECT) and quantitative sensory testing (QST) were used to characterise the pain experienced by participant. To assess structural changes MR images were scored using MRI Osteoarthritis Knee Score (MOAKS) by two independent scorers. The MRI scans were used to localise BMLs for whole transcriptomic analysis, qPCR for array validation, histological evaluation, scanning electron microscopy and quantitative protein expression. Serum samples obtained at baseline visits were used for biomarker investigation. The result demonstrates that BMLs are regions of increased cellular and metabolic activity and correlated with increased pain and sensitisation. The findings contribute to the understanding of OA pathogenesis and could help lead to the development of new diagnostic tools and future therapies for the improved management of this increasingly prevalent condition.

Introduction: Despite OA heritability estimated at 40-65%, fewer than 20 robustly-associated genetic loci explaining ~10% of heritability have been identified. Genome-wide association studies (GWAS) have been limited by modest sample sizes and the employment of simple dichotomous descriptions of joint involvement. Risk loci associated with hip shape variation, a heritable risk factor for the development of OA, is also largely unknown. Aims: We aimed to determine whether case stratification into precise phenotypic sub-categories is a critical parameter in signal detection in GWAS of hip and knee OA, and hip shape variation. This has not previously been tested systematically. Method: This thesis describes several nested studies from the arcOGEN resource. We initially describe several GWAS in 2,118 cases with radiographic hip OA, stratified by site of maximal joint space narrowing and bone remodelling response, versus 6,500 population-based controls (PBC). We also performed several GWAS’ of 2,010 cases with radiographic knee OA, stratified by dominant compartment involvement, versus 6,500 PBC. Finally, we describe several GWAS with radiographically-derived indices of acetabular and proximal femoral shape. Results: We identified suggestive evidence (p < 9.9 x 10-6) of associations at 6 and 10 variants with hip and knee OA endophenotypes respectively. All would have been missed by a broad phenotype definition approach. The signal yield was increased beyond that associated with chance. Compartmental stratification at the knee also increased the disease heritability estimate. We identified 4 loci (p < 9.9 x 10-6) associated with hip shape variation, including associations with genes regulating myogenesis and skeletal development. Conclusion: We show that increased phenotypic definition in nested case/control cohorts of OA leads to substantial increases in signal detection for susceptibility loci. We report novel associations that were entirely obscured by the heterogeneity of the disease, indicating that the use of narrower OA phenotype definitions can lead to a comprehensive account of variants affecting risk.

The Intraosseous Transcutaneous Amputation Prosthesis (ITAP) may improve quality of life for amputees by avoiding soft tissue complications associated with socket prostheses and by improving sensory feedback and function. ITAP relies on a soft tissue seal forming at the skin-implant interface in order to prevent epithelial downgrowth and infection. A successful soft tissue seal is dependent on soft tissue cells winning the ‘race for the surface’ against bacteria. The current ITAP design includes a flange with drilled holes to promote soft tissue attachment. Despite this, infection remains a significant risk. This thesis aimed to investigate the effect of a fully porous titanium alloy (PT) flange with interconnected pores and fibronectin (Fn)-functionalised hydroxyapatite (HA) coatings on soft tissue integration. Silver (Ag) was incorporated into coatings for its antimicrobial properties. In vitro fibroblast viability and bacterial colonisation on HAAgFn coatings was studied. HAAgFn was applied to PT flanges of transcutaneous pins implanted into sheep tibiae. A histological assessment of soft tissue integration was undertaken and bacterial colonisation within the soft tissues and on the flange was quantified. The key original contributions to knowledge from this thesis are that firstly, HAAgFn coatings have antibacterial activity and are cytocompatible after serum- preconditioning in vitro. Secondly, in vivo, PT significantly reduces epithelial downgrowth, increases soft tissue integration and reduces bacterial colonisation compared with the current ITAP drilled-hole flange. Overall, the addition of coatings did not enhance these effects in vivo. HA reduced the favourable effects of PT. Fn and Ag counteracted some of the negative effects of HA suggesting that using these coatings without HA could improve results. In conclusion, a PT flange has the potential to reduce the susceptibility of ITAP to infection compared with the current ITAP design. It is hoped that this finding will be translated into clinical practice.

Following the observation that individuals with hemiplegia or poliomyelitis resist the development of rheumatoid arthritis in the affected limb the sensory nervous system has been implicated in the aetiology of joint inflammation. Sensory neuropeptides, produced in dorsal root ganglia (DRG), released from central and peripheral terminals of sensory nerves upon noxious stimulation, are implicated in central and peripheral events underlying inflammation. Previous work has explored neuropeptide gene expression in DRG neurones in the Freund's Complete Adjuvant (FCA) model of unilateral tibio-tarsal joint inflammation. Within 8h of FCA injection, expression of mRNAs from the β preprotachykinin-A (β PPT-A) gene, encoding the sensory neuropeptides substance P and neurokinin A, and the α-calcitonin gene-related peptide (CGRP) encoding gene increase in small diameter innervating DRG neurones. Small diameter sensory nerves include the class of nerve implicated in the transmission of noxious stimuli. No changes occur in contralateral DRG neurones. A recent report of mRNAs encoding various transcription factors in innervating over the first 8h after FCA injection members showed that AP-2 mRNA increased in small diameter neurones within 1h of adjuvant injection, returning to control levels at subsequent time points. Despite members of the AP-1 transcription factor group been implicated in the control of PPT -A <I>in vitro </I>mRNAs encoding members of AP-1 family were not detected or showed no changes in expression. The same was found for NGF1-A and NGFI-B. The aim of this thesis was to utilise in-situ hybridisation to explore the early changes in βPPT-A and α-CGRP mRNAs, and the possible role of AP-2 in their regulation over the first 8h after FCA injection. Subsequent investigations were designed to investigate molecular mechanisms underlying changes in neuropeptide gene expression.

<I>Objectives. </I> To examine the effects of high intensity physical exercise and menstrual irregularity on the bone mineral density (BMD) of female athletes aged 16 - 65 years. <I>Subjects. </I> Group A comprised 50 middle or long-distance runners aged 16 to 35 years who ran at least 25 miles per week and trained for at least 3 hours per week. The remaining 74 veteran athletes aged 40 years or over were required to have run at least 15 miles per week or trained for at least 3 hours per week for the last three years. They were divided into premenopausal (Group B, n=50) and postmenopausal (Group C, n=24). <I>Results. </I> Group A was subdivided into athletes who were amenorrhoeic (0-3 menstrual cycles per year, AM, n=24), oligomenorrhoeic (4-9 cycles per year, OL, n=9) and eumenorrhoeic (10-13 cycles per year, EU, n=17). Bone mineral density was low in the AM group at all sites measured when compared to the EU group (p<0.0001), and compared to the age-matched European reference range (p<0.05 - p<0.001). BMD increased in a stepwise fashion with menstrual group and was high in the proximal femur in EU athletes compared to the European reference range (p<0.005). Group B was subdivided into athletes who had trained continuously since leaving school (All-timers, AT, n=24) and those who had started after the age of 30 years (late-starters, LS, n=27). <I>Conclusions. </I> Bone density at important fracture sites was substantially greater than expected in women undertaking long-term exercise in the oestrogenized state whether started in the 'teens or the thirties. When endogenous oestrogen levels are low the apparent beneficial effects of exercise are lost. In postmenopausal women this may be due to low body weight and body fat in those who are the most highly trained. Amenorrhoea in young adult life may have persistent effects on non weight-bearing sites. Individual circumstances must be assessed before exercise can be used as a prescription for the prevention of osteoporosis.

Sjögren’s syndrome (SS) is a systemic, chronic, autoimmune inflammatory disease that affects the exocrine glands. The absence of early diagnostic markers contributes to delays in its diagnosis. Furthermore, SS patients have an increased incidence of lymphoma and there is a need for biomarkers to identify its development. To enable a better understanding of disease activity and progression, the following aims were undertaken; 1. Investigate the potential use of the salivary gland assessment tests (whole flow rates (WFR), parotid flow rates (PFR), clinical oral dryness score (CODS) and ultrasound score (USS)) in discriminating SS and non- SS sicca patients as well as to differentiate between the different subgroups of SS. 2. Determine the diagnostic accuracy of USS. 3. Complete a longitudinal study on dry mouth patients at 5 and 10-year time-points. 4. Identify salivary markers of disease activity and progression in SS; MALT-L risk and MALT-L subgroups. Methodology: Clinical parameters (WFR, PFR, CODS and USS) of 244 patients involved in the cross- sectional study were recorded for SS of different subgroups as well as for disease controls and ROC curves were constructed to determine an optimal cut- off USS. Five-year follow- up of 80 patients was done while only 16 were involved in the 10-year follow- up. Salivary (parotid) proteomic analysis was done followed with initial candidate biomarker selection (S100A8/A9) and verification via immunoassay (ELISA) involving SS of different subgroups, disease and healthy controls (n=83). Salivary (parotid) cytokine array analysis was done followed by further screening of cytokines via multiplex bead- based assay on 76 samples and verification of the cytokines with significantly altered levels via a more sensitive multiplex bead- based performance assay on 82 samples. Results: 1. All parameters (WFR, PFR, USS and CODS) of the overall SS group showed a significant difference when compared to the disease control group. This was attributed mainly to the advanced subgroups of SS (SS at risk and MALT-L groups) (p < 0.0001 for all parameters). 2. USS would be an ideal non-invasive test to differentiate and monitor SS patients in general; an optimal cut- off of 4 yielded 81% sensitivity and 94% specificity and odds ratio of 70.5% with a negative predictive value of 66% while the positive predictive value was 97%. 3. The follow- up of patients over 5 and 10 years has proven that in patients with a longer disease duration SS is a slowly progressing disease where all the parameters remained relatively stable, although some individuals may display flare ups and remissions as in any other rheumatic diseases. S100A8/A9 ELISA analysis of parotid saliva showed significant differences between the overall SS group and both disease and healthy controls (p=0.001 and 0.031 respectively). SS at risk of MALT-L and those with MALT-L also demonstrated increased levels when compared to healthy controls (p=0.019 and 0.014). IL-1α, -4, -6, and MCP-1 were significantly different in the overall SS group when compared to the disease control group but only IL-6 was increased when compared to the healthy control group. Further verification via more sensitive assays revealed that IL-6 and IL -4 continued to show significance compared to the disease control group and IL-6 compared to the healthy group. Conclusion: the results of the above-mentioned studies suggest that several clinical parameters can aid in differentiating between SS and non- SS sicca especially the subgroups of SS (MALT-L risk and MALT-L sub groups) with attention to USS as a valuable diagnostic tool. Salivary biomarkers showed differences between SS and the other groups as well.

Achilles tendinopathy (AT) is a debilitating condition affecting active and inactive subjects. The aetiology of AT is contentious but most theoretical models are underpinned by the same core elements: increased tendon stress that exceeds the ability of the tendon to repair, eventually leading to tissue degradation. Proposed risk factors affect tissue load or the ability of the tendon to repair, unfortunately many proposed risk factors lack biological plausibility. This thesis reports a mixed methods approach to analyse risk factors for AT development. A Delphi study (using world tendon experts) was used to develop a consensus on risk factors for AT which were then used in an epidemiological study of UK runners – a high risk group for tendinopathy who have not previously been examined. Plantarflexor strength was identified as a potential contributor in AT development and this led to the development of a theory on why current treatment modalities work. This was further examined using a systematic review to determine whether changes occur in the muscle or tendon with the most common rehabilitation method (eccentric exercise). This review highlighted evidence for changes in plantaflexor power, amongst other factors. These findings were used to inform two studies examining how plantarflexor power was affected by AT and ultimately how rehabilitation altered these elements. The thesis reports multiple novel findings related to risk factors, the mechanism by which eccentric exercise may work, the effect of AT on plantarflexor function and how rehabilitation affects these deficits. The experimental studies contained in this thesis are some of the largest studies in the area, these studies identify significant torque and endurance deficits of the plantarflexors highlighting that Soleus appears to be most affected, whilst the later study reported how an eccentric rehabilitation regime improved these deficits but failed to fully resolve them. This offers an important clinical target but also highlights that rehabilitation needs to be for a longer duration with further modifications to resolve these identified plantarflexor deficits.

Chronic pain is a complex condition with a significant social and economic impact and a better understanding of the factors affecting improvement is required to inform best practice in the management process. Few studies have considered the effect of Weinberger et al.’s personality types in the management of pain. The four personality types are suggested to respond differently to threatening information such as pain, because of their attentional and interpretive biases. Using a variety of research methods, the global aim of this programme of research was to determine whether the management of chronic pain would be enhanced through the use of treatment stratified on the basis of personality type. This thesis highlights important differences in the response to pain and pain management between the personality types, which are masked if the population is analysed homogenously. Defensive high-anxious individuals were more prevalent in the patient population compared to the asymptomatic control group and attended to pain related information more than the other groups. Defensive high-anxious individuals reported greater improvement for both pain and disability and showed stronger links between improvements in pain and disability and baseline psychological factors than the non-extreme individuals. The findings suggest that current treatments are more effective for defensive high-anxious patients. Furthermore, the high proportion of defensive high-anxious individuals highlights the need for psychologically based interventions to be delivered earlier. Stratifying the population may allow for more targeted interventions, which could be more cost effective and reduce the number of patients remaining in the care system.