Parathyroid hormone (PTH) and growth hormone (GH) are known to decline with ageing and have been associated with the process of apoptosis in the osteoblast and osteocyte cell populations. The presence of viable osteocytes in bone is an important parameter in maintaining bone quality while osteocyte death by apoptosis during ageing has been associated with loss of bone strength. PTH (1-34) administration as part of hormone replacement therapy (HRT) has been shown to exert positive effects via the stimulation of bone formation and improved bone microarchitecture, mainly through the inhibition of osteoblast and osteocyte apoptosis. GH on the other hand has been shown to exert positive effects on bone formation, but due to financial cost not many clinical trials have been perfonned. However in vivo studies have demonstrated the positive effects of GH on osteoblasts and osteocytes with a direct or indirect manner, possibly through the stimulation of growth factors and mainly IGF-I. Although their potential use as anabolic treatments has long been recognised and their effects on osteoblasts and osteocytes have been studied for many years, their effects on the maintenance of the osteocytic population in vivo are less clear. Work in this thesis has investigated whether recombinant human parathyroid hormone (rhPTH) and recombinant human growth hormone (rhGH) can inhibit osteocyte apoptosis, either due to ageing or damage as well as the potential of these hormones to trigger specific gene response on the osteoblast in the ageing rat skeleton. An in vitro study in this thesis has indicated the protective effects of rhPTH and rhGH in osteocyte cultures following physical injury in vitro. These data have provided evidence for the first time of effects of these hormones in the repair of cell membranes following disruption. In this study, physical membrane disruption to MLO-Y4 cells in vitro was taken to represent physical injury-which is one aspect of microdamage to osteocytes in bone. These findings may provide a new understanding of how osteocytes sense and respond to injury and the potential of therapeutical compounds to maintain osteocyte viability in disease and old age. rhPTH and rhGH treatment was shown to reduced osteocyte apoptosis in aged female rat bone, while improved trabecular architecture was observed in response to rhPTH treatment but not to rhGH. These data point to the potential of these hormones to be used as anabolic treatments. However the molecular mechanisms by which rhPTH and rhGH maintain the osteocytic population and lead to bone fonnation are not well understood. The effects of mechanical loading on the maintenance of the osteocytic population were investigated in human cancellous bone explants ex vivo in the presence or absence of rhPTH. Osteocyte viability was increased and a significant reduction in osteocyte apoptosis was observed compared to unloading conditions after 7 days in culture. Furthermore alkaline phosphatase a marker of an osteogenic repsonse was increased by mechanical stimulation in the presence or absence of rhPTH compared to unloading conditions, indicating a stimulatory effect on osteoblasts. Moreover SOST gene expression was decreased in the presence or absence of mechanical stimulation following administration of rhPTH. These data pointed to important anabolic effects induced by mechanical stimulation in the presence or absence of rhPTH. Finally a laser microdisection study using the tibias of old female rats presented in this thesis provides the basis for future studies aimed to identify specific osteoblastic genes regulated by rhPTH and rhGH at different anatomical sites of bone. Such data would provide the necessary infonnation to elucidate the molecular mechanisms that regulate rhPTH and rhGH and their effects on bone. In this study identification of the anti-apoptotic and protective effects of rhPTH and rhGH on osteocytes in vivo, in vitro and ex vivo highlighted important infonnation on their bioactivity in vivo. Moreover an attempt to elucidate the molecular mechanisms behind rhPTH and rhGH effects on osteoblasts in vivo has been presented and provides the basis for future studies that may prove useful in identifying specific drug targets for the treatment of pathological bone.

Osteoporosis affects bone mass and bone micro-architecture, reducing mechanical strength. SDF-1 and its ligand CXCR4 play significant roles in the migration and engraftment of mesenchymal stem cells (MSCs). The aim of this study was to investigate the effects of CXCR4 transfected MSCs on bone formation in osteopenic rats. The hypothesis was that MSCs genetically modified to over-express CXCR4, would enhance migration of stem cells from osteopenic rats and when injected intravenously in ovariectomised (OVX) rats, would improve bone formation. MSCs were harvested from femora of young, OVX and adult control rats. The differentiation, CXCR4 expression, in vitro migration and phenotypic characteristics of the cells were compared. Although the phenotypic characteristics of cells from all groups of rats were the same, their differentiation capability, CXCR4 expression and migration was significantly different. MSCs were genetically modified to over-express CXCR4 and in vitro migration investigated. It was found that although young MSCs had the highest migration capability (2x more than their uninfected counterparts, p=0.006), the OVX MSCs when transfected with CXCR4 had the most significant migration from their un-transfected counterpart cells(5x more, p=0.025). Additionally, differentiating MSCs to osteoblasts reduced their CXCR4 expression as well as their migration towards SDF1. The CXCR4 transfected MSCs were administered intravenously in OVX rats. Fluorescent labelled cells were tracked after 1 week and were located in the blood vessels of the femur. 11-weeks post-injection, OVX rats injected with young-CXCR4 MSCs had significantly higher BMD(694.0±80.1mg/cm3) (p < 0.05) in comparison to rats injected with saline. Rats injected with OVX-CXCR4 MSCs(645.4±79.3mg/ccm) had a higher BMD in comparison to those injected with OVX MSCs(631.4±69.5mg/ccm) and saline(563.4±82.9mg/ccm). The L4 vertebral stiffness was also higher in rats treated with young-CXCR4 MSCs in comparison to those treated with saline. CXCR4 genetically modified MSCs from young and OVX patients may help in boosting bone formation in osteoporosis.

BACKGROUND: The results of surgery for tears of the rotator cuff are variable, with failure occurring in up to 94% of cases. Demineralised bone matrix (DBM) consists of a collagen scaffold containing multiple growth factors and has been used successfully to improve tendon-bone healing. By combining DBM with stem cells its effects may be enhanced given that many of the growth factors it contains are able to direct stem cell differentiation down tenogenic, chondrogenic, and osteogenic lineages. These cell lines produce elements essential to the formation of a naturally graded enthesis. AIM: To investigate the effect of DBM on regeneration of the tendon-bone interface, and whether its function can be enhanced by mesenchymal stem cells (MSCs). HYPOTHESIS: DBM will improve tendon-bone healing in an enthesis defect model, and its effect may be further enhanced by the incorporation of MSCs. METHODS: The following experiments were undertaken in order to investigate the hypothesis: 1. Tensile testing of allogenic and xenogenic cortical/cancellous DBM. 2. Evaluating the effect of allogenic and xenogenic DBM, incorporated with MSCs, on regeneration of the enthesis in a large animal model of severe tendon retraction. 3. Development of a chronic rotator cuff tear model. 4. Investigation of tendon-bone healing using DBM in a chronic rotator cuff tear model. 5. Investigation of tendon-bone healing using DBM and MSCs in a chronic rotator cuff tear model. RESULTS: Allogenic cortical DBM possessed the greatest tensile strength and was used in vivo to examine tendon-bone healing complicated by retraction. In this, DBM regenerated a direct enthesis characterised by fibrocartilage. A similar effect was noted in a chronic rotator cuff tear model with no additional effect conferred by the stem cells. CONCLUSION: This thesis has shown that DBM can regenerate a fibrocartilaginous enthesis in models of tendon retraction and chronic rotator cuff degeneration.

Overview: Whilst each section of the thesis is required to stand alone, it is presented in the following order so that the thesis has a logical flow. Literature review: Presents an overview of the rheumatic diseases and their impact upon parenting and families. We note the provision of patient education for parents, but not for their children. The comprehensive but selective narrative review focuses upon how adults and children conceptualise and understand illness, and explores the concept of normalisation as a potential family management strategy. Service Evaluation: We sought to establish whether parents/grandparents would welcome the provision of appropriate patient education about rheumatic disease being made available to their children/grandchildren. Using a cross-sectional study design, a questionnaire was distributed to adult patients attending the local rheumatology service and members of four UK national rheumatology charities. Participants were strongly in favour of developmentally appropriate patient education for their children/grandchildren. Suggestions were made for content, format, timing and method of delivery. Main Report: We sought to understand how the diagnosis and impact of parental rheumatic disease has been understood, talked about, and managed within families who have young children. Again, we asked for views about providing patient education for children. We were particularly keen to give children a voice in determining whether, and how, any resources designed for them should develop. Utilising a qualitative design informed by an interpretivist framework, we employed semi-structured interviews and visual data collection methods. Eleven families with children aged between seven and 11were recruited from the local rheumatology service. Interviews and visual data were analysed using thematic analysis. The results are discussed within the concept of 'normalization'. Implications for clinical practice and further research are highlighted. Critical Appraisal: Contains a critique of both the research process and the methodology used.

Joint kinematics has been suggested to play an important role in the initiation and progression of a number of musculoskeletal pathologies. Thus, investigation of joint kinematics can help clinicians and researchers to better understand musculoskeletal conditions such as osteoarthritis and trochanteric bursitis. Existing motion capture technologies used in clinical settings suffer from various limitations, including soft tissue artefact. These limitations result in a lengthy examination process, an incomplete movement description, or an inaccurate representation of the real bony landmarks. The recent introduction of a motion analysis with ultrasound (MAUS) system aimed to provide a less constrained approach to detect actual bony structures and describe joint kinematics in three dimensional (3D) space. However, the accuracy of the original MAUS system was highly dependent on the operator's experience. In this doctoral thesis, a computer-aided tracking and motion analysis with ultrasound (CAT & MAUS) system is developed to track underlying bony landmarks and describe hip joint kinematics during gait. The key contribution of this thesis is to combine state-of-the-art computer vision approaches with the original MAUS system to improve the speed, accuracy and repeatability of joint kinematics examination for clinical measurement and diagnosis. Firstly, a comprehensive review of gait analysis and relevant clinical diagnostic modalities is presented. Then, an augmented MAUS system architecture is presented, which is more flexible than the previous MAUS system of the data acquisition. It combines an optoelectronic motion analysis (MA) system with a 2D ultrasound (US) device to build up a 3D representation of the bony structure of interest. A novel calibration box with multiple functions for the augmented MAUS system is designed to spatially and temporally match US images to the motion analysis data. The average Euclidean distance error of the spatial calibration is found to be 0.34 mm and the accuracy of the temporal calibration is found to be within half of the frame acquisition interval. The augmented MAUS system with its more accurate calibration procedures can accurately present the bone of interest in 3D space. Secondly, a computer-aided post-processing pipeline is presented to automatically track the bone of interest in 3D space. A globally optimal registration method is employed to align the 3D surfaces of the target bony structure reconstructed from the manual segmentation in US images at different positions to locate the target bony landmark from one position to another. The globally optimal registration overcomes the issue of getting trapped into local minima for the conventional iterative closest point registration. The accuracy of the globally optimal registration is validated with a proximal femur phantom. The average rotation error is found to be 0.38° and the average translation error is found to be 0.33 mm, both of which are within the clinical tolerance of computer-aided orthopaedics surgery ±3° and ± 1 mm). The 3D globally optimal registration guarantees an accurate track of the same target bony structure at different positions instead of estimating the similar bony structure at each position by eyeball with previous MAUS system. However, in practice, it takes around 20 minutes to manually segment the bone structure for a single surface reconstruction, which is extremely laborious and time consuming. In order to automate the CAT & MAUS system, a novel automatic segmentation of the bone structure in a 2D US image is then developed as a precursor for an accurate globally optimal registration. The automatic bone segmentation introduces the local phase features and acoustic characteristics to enhance the bone probability for detection. The result of the automatic segmentation is validated with a pilot phantom study before applying to the in-vivo experiment and achieves an accuracy of 0.13 mm. After fully developing the CAT & MAUS system, hip joint kinematics of healthy subjects are quantified in six degrees of freedom using the CAT & MAUS system and compared to the results from the optoelectronic motion analysis system. It is shown that CAT & MAUS results describe a greater rotation range than MA results by up to 4.03° in the sagittal plane because the optoelectronic motion analysis system alone suffers from severe soft tissue artefact as the skin markers shift away from the underlying bony structures during movement. The deformation of body segments captured by MA during movements caused by soft tissue artefact is explained using Procrustes analysis to indicate the accuracy and repeatability of the CAT & MAUS system. Finally, possible future directions are proposed based on preliminary investigations using the developed CAT & MAUS system with particular reference to both the technical and clinical perspectives.

Anti-citrullinated protein antibodies (ACPA) are the main autoantibodies in rheumatoid arthritis (RA). Citrullination is the conversion of arginine to citrulline by peptidylarginine deiminase (PAD) enzymes. Periodontitis (PD) is a risk factor for RA, and there is evidence this may be due to citrullination by the unique prokaryotic PAD enzyme expressed by PD pathogen Porphyromonas gingivalis (PPAD). The aim of this work was to characterise PPAD, and investigate its potential role in the aetiology of RA. The 3D crystal structure of PPAD was solved, and functional residues identified. The catalytic domain comprises five ββαβ motif repeats, which create a canonical binding groove into the active site, and the immunoglobulin-like domain is an anti-parallel β-sandwich. PPAD lacks significant homology to human PADs, and forms distinct citrullinated peptides from those formed by human PAD isoforms PAD2 and PAD4 due to unique C-terminal substrate specificity. In vivo, immunisation with P. gingivalis proteins: PPAD, arginine gingipain (Rgp), and enolase (PgEnolase), induced ACPA responses in mice. In human cohorts, antibodies to PPAD, Rgp, and PgEnolase were increased in RA patients compared to osteoarthritis or healthy controls, and in RA patients with PD compared to those without. Six months of PD treatment saw a significant reduction in antibodies to P. gingivalis in RA patients with PD, but no change in ACPA. These results demonstrate P. gingivalis induces ACPA responses comparable to those observed in RA, potentially through formation of novel citrullinated peptides by PPAD. Structural and mechanistic information obtained here can be used in future studies targeting PPAD as a potential therapeutic strategy in RA.

Sjögren’s syndrome (SS) and systemic lupus erythematosus (SLE) are distinct autoimmune rheumatic diseases (ARD) characterised by shared clinical features and immune abnormalities. A proportion of 15-20% of patients with SLE also have features of SS, manifesting as an overlap ARD called secondary SS associated with SLE (SS/SLE). Both SLE and SS are B cell driven diseases, with a recognised female predominance. SS is characterised by distinctive chronic inflammatory process leading to destruction of the exocrine glands orchestrated by T and B lymphocytes, dendritic cells (DCs), macrophages and other mononuclear cells. These cells have been demonstrated to also play a role in SLE and SS pathogenesis, while the immune phenotype of SS/SLE patients has not been investigated before. The results of my research demonstrated that the clinical, serological and histological parameters used by clinicians in routine practice have not been able to distinguish between patients with different diagnoses when stratified using unsupervised hierarchical clustering. However, in-depth immune phenotype have found that these patients also had both unique and shared defects in their CD19+ B cells, CD4+ and CD8+ T cells peripheral blood profiles, as well as defects in lipid raft expressions in these cell subsets, as assessed by flow-cytometry and ImageStream analyses. Overall, in-depth immune phenotyping was able to stratify pSS, SLE and SS/SLE with common underlying B and T cell abnormalities based on their immune signatures rather than diagnosis, which can have significant therapeutic implications in the selection of treatment strategies.

Systemic sclerosis (SSc) is an autoimmune, connective tissue disease characterised by endothelial dysfunction, collagen deposition and fibrosis. The leading causes of mortality, contributing to over 55% of deaths, are pulmonary hypertension (PH) and pulmonary fibrosis (PF). This thesis explored the contribution of endothelial cells in the development of pulmonary vascular complications in SSc. Blood-outgrowth endothelial progenitor cells (EPCs) are considered to support vascular repair, however their ability to do so in SSc patients is unclear. This thesis developed a robust method to culture EPCs from SSc and healthy donor (HC) blood, and explored their phenotype compared to pulmonary artery endothelial cells (PAECs). EPCs exhibited a number of endothelial characteristics including morphology, the ability to form barriers, and respond to permeability-inducing factors such as TNFα. EPCs exhibited greater Rac-1 activity and formed stronger cellular barriers which was Rac dependent. HC-EPCs were also more resistant to apoptosis compared to SSc-EPCs and PAECs. These observations suggest that HC-EPCs may aid vascular repair by providing apoptotic resistance and reducing endothelial permeability. In contrast to HC-EPCs and PAECs, SSc-EPCs supported greater levels of leukocyte trafficking. Thus SSc-EPCs may fail to ‘heal’ the endothelium and exacerbate endothelial dysfunction in SSc patients. Further, the differences between SSc-EPCs and HC-EPCs support their use as a surrogate for exploring endothelial dysfunction in SSc. The contribution of endothelial to mesenchymal transition (EndoMT) in SSc was also explored. Transitioning EndoMT cells were present in pulmonary arteries of SSc-PAH patients and pre-clinical models. The functional impact of EndoMT on endothelial function was explored by establishing a cytokine induced-EndoMT (I-EndoMT) model in vitro. I-EndoMT cells ‘lost’ endothelial and ‘gained’ mesenchymal cellular markers. EndoMT cells failed to form effective cellular barriers and secreted elevated levels of pro-inflammatory cytokines. Collectively this suggests that EndoMT may contribute to endothelial dysfunction and pathological remodelling in SSc-PAH patients.

Fibrosis is a major pathological feature of many chronic diseases characterised by activation of fibroblasts, accumulation of extracellular matrix (ECM) and persistent inflammation which can lead to impaired organ function and ultimately, to organ failure. To date, there is no effective treatment to delay, halt or reverse fibrosis. Therefore, there is an urgent unmet need for a better understanding of the mechanisms leading to pathological fibrosis to identify potential new targets and biomarkers. The aims of this project are to identify genes altered in fibrosis in 3 major organs (lung, skin and kidney); to shortlist relevant genes using key criteria; and, to explore in vitro and in vivo the function of selected genes under normal and fibrotic conditions. An extensive in silico analysis using published literature and microarray datasets from 1988 to 2015 was performed. Hundreds of genes were identified with a wide variety of functions. Among them, 91 were common for pulmonary, dermal and renal fibrosis while 180 were specific genes altered in lung (60 genes), skin (60 genes) and kidney fibrosis (60 genes). A subset of 12 genes was selected using key criteria including potential drugability and the availability of reagents for further study. Expression of the 12 short-listed genes was explored in human fibrotic fibroblasts and tissues and their function was examined in vitro and in vivo. Data showed that 3 of the 12 selected genes were significantly associated with fibrotic fibroblasts and tissues: Tetraspanin 13 (TSPAN13), Hyaluronan synthase 2 (HAS2) and Cell migration-inducing protein, hyaluronan binding (CEMIP). TSPAN13 has not previously been explored in fibrotic diseases. Here, TSPAN13 was found up-regulated in fibrosis and appears to be a potent pro-fibrogenic molecule with critical functional activities relevant to fibrosis. HAS2 and CEMIP, both genes involved in the hyaluronic acid (HA) pathway, were up- and down-regulated in fibrotic settings respectively. These data showed that TSPAN13, HAS2 and CEMIP are significantly regulated in fibrotic settings suggesting an important role in fibrosis. Therapeutic antibodies are being developed against the two up-regulated targets, TSPAN13 and HAS2, as potential anti-fibrotic therapies.

Aggrecan is a large- aggregating proteoglycan that is essential for the function of articular cartilage. A loss of aggrecan is one of the major event in osteoarthritis, a debilitating and degenerative joint disease. To understand how a gene is lost and what fails to prevent transcription in disease state, an understanding of the bases of transcription must first be sought. Transcription is the first step in gene regulation and the chromatin plays an important role in blocking or allowing transcription to occur. Enhancers are non-coding DNA sequences that allow the binding of proteins, such as transcription factors that drive transcription irrespective of distance or orientation. Enhancers are marked by histone modifications that distinguish them from promoters and divide them into poised or active and are generally highly evolutionary conserved. Using publically available data on histone modifications found on ENCODE and transgenic mice this study has added to the understanding of the transcriptional regulation of aggrecan along with the known enhancer at -10kb. 3 intergenic regions, -35kb, -65kb and -87kb and one intronic (+26kb) from the transcription start site of aggrecan expresses primarily in the chondrocytes at E15.5. The -87kb enhancer marks chondrocytes that commit to an articular cartilage fate, and is strongly active in adult mice. The -65kb appears developmentally active, marking hypertrophic chondrocytes. The -35kb and +26kb are expressed in all chondrocytes at E15.5 mice and the -35kb is active in adult tissue. These enhancers bind to Sox9 and a loss of Sox9 in the -35kb enhancer shifts the expression from chondrocytes to fibroblast or perichondrium cells. These enhancers may play a role in response to diseases such as OA, as when these enhancers are transfected into differentiated murine ADTC5 cells and treated with Il-1β or hOSM there is a reduced level of expression from the -87kb element and an increase expression of the -65kb in response to Il-1β. A highly conserved intronic sequences (+55kb) was identified that does not express in chondrocytes, rather in kidney and lungs and may play a role outside of aggrecan gene regulation. Using the Acan promoter and the -35kb enhancer a cartilage-specific Cre recombinase line was generated providing a tool to explore the pathogenesis of cartilage genes in disease such as OA or in lineage tracing.