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Bone loss disorders associated with an imbalance in osteoblast and adipocyte differentiation, such as osteoporosis, are fast-growing health challenges in our aging populations. Cell fate differentiation shifts towards fat-storing adipocytes at the expense of bone-forming osteoblasts resulting in bone loss and increased fat mass. There is published evidence that murine models lacking specific scavenger receptors show alterations in bone and metabolic phenotypes. Herein, mice lacking scavenger receptor class A member 5 (Scara5) exhibited significantly greater bone mass and markedly reduced bone marrow adipose tissue than control WT animals. This was consistent with in vitro differentiation of Scara5-deficient (Scara5-/-) bone marrow stromal cells (BMSCs), showing a reduction in adipocyte differentiation and an increase in osteoblast bone nodule formation. The underlying mechanism was investigated using recombinant SCARA5 and bone-related ligands, including ferritin, HMGB1, and WNT-related proteins. These in vivo and in vitro findings underscore the crucial role of Scara5 in BMSC lineage allocation and demonstrate that Scara5 is a negative regulator of bone mass and osteogenesis. Additionally, Scara5-/- animals carried leaner body mass and a smaller percentage of subcutaneous inguinal fat than the age and sex-matched controls. This project proposes Scara5 as an important modulator of bone and adipocyte formation, which could be reciprocally manipulated as a potential therapeutic target for body fat and bone formation.

It is now widely accepted that musculoskeletal imaging has a useful role to play in the early diagnosis of inflammatory arthritis, as well as providing a rapid and sensitive means of assessing treatment response, not only for individual patients in clinical practice, but also in clinical trials. However, the most frequently employed imaging modalities assess morphological changes in inflamed tissues. In contrast, Positron Emission Tomography (PET) generates images based on the detection of a desired molecular target of interest in vivo, and has been proposed to have potential as a highly sensitive and specific means of imaging inflammatory arthritis. The major goal of the work documented in this thesis was to undertake a proof of principle pilot study to assess the ability of the purportedly macrophage-specific second generation translocator protein (TSPO) targeted PET tracer [11C]PBR28, to detect and quantify synovitis in a small cohort of patients with inflammatory arthritis. Measures of tracer uptake were compared with clinical examination and ultrasound imaging, as standard means of assessment for evidence of synovitis. Additionally, the relationship between TSPO PET tracer signal and immunohistochemical staining for TSPO on synovial tissue samples was assessed. The positive correlation between TSPO tracer signal in the joint compartment and synovial tissue staining for TSPO, provided strong evidence that the tracer signal was the result of target expression, and not due to confounding factors. In order to ascertain which cell groups in inflamed synovium were likely to be significant contributors to the TSPO PET signal observed in RA, an expression profiling of the tracer target (i.e. TSPO), in cell types comprising human pannus was conducted. Using immunofluorescence studies of human RA synovial tissue, ex vivo expression of TSPO on macrophages as well as fibroblast-like synoviocytes and CD4+ T cells was noted. By measurement of mRNA expression by real-time PCR, and TSPO protein quantification, using [3H]PBR28 radioligand binding saturation assays, I have provided the first data comparing TSPO expression in major cellular components of human pannus. Collectively, data presented herein demonstrated that TSPO was not solely a macrophage specific marker in synovium. Rather, the most significant cellular contributors to TSPO PET signal were activated 'M2 phenotype' macrophages, and activated fibroblast-like synoviocytes. These findings establish the likely cellular correlates of the TSPO PET signal generated on imaging of inflamed joints in human RA. Finally, this thesis also provided preliminary data regarding the expression of TSPO in macrophages of different phenotypes, and demonstrated a consistent downregulation of TSPO in macrophages polarised to the pro-inflammatory 'M1' end of a spectrum of macrophage phenotypes, whereas TSPO expression remained unchanged in macrophages of 'M2 phenotype'. These novel findings suggest a role for TSPO in the negative regulation of inflammation, which we hypothesise is secondary to a function of TSPO in cholesterol transport within macrophages.

Osteoporosis, a metabolic disorder of the skeletal system, currently affects 200 million people worldwide. Osteoporosis is caused by an imbalance in bone resorption and formation due to either molecular mutations, oestrogen reduction at menopause, oxidative stress, as well as life style, leading to a characteristic low bone density and/or thinning that predisposes patients to skeletal fracture and breakage upon minimal physical trauma. Depending on the severity of the disease, most patients are often treated with calcium/vitamin D and anti-resorptive drugs such as bisphosphonates or calcitonin. However, there are few options for bone formation using anabolic agents - such as Teriparatide (PTH1-34), which have limited success, creating an unmet need for novel anabolic interventions. The rapidly-advancing field of osteoimmunology recognises the close and finely-tuned interaction between the immune system and musculoskeletal organisation. In an attempt to identify immune-derived cells and soluble factors that induce the differentiation of stem cells towards the osteoblast lineage, the interaction between monocytes and osteoblast precursors has proven a potentially promising approach. One of the factors identified from this screening was TSG-6 - a secreted protein with immunomodulatory and chondroprotective properties. This study aimed to generate adequate amounts of biologically active TSG-6 protein to test its role on bone formation in vivo during physiology and disease - using oestrogen-deficiency induced bone loss in mice as a model of osteoporosis. TSG-6 was not found to protect bone tissue against the effects of oestrogen-deficiency induced bone loss, as determined by global TSG-6 knockout and systemic exogenous administration studies. However, local delivery of full-length TSG-6 significantly enhanced mineral apposition rate in a dose-dependent manner, and in vitro studies found TSG-6 significantly enhanced alkaline phosphatase levels of primary bone marrow-derived human mesenchymal stem cells in vitro, in the presence of BMP-2. Contrary to its previously reported inhibitory effects on osteoblastogenesis, this thesis shows, for the first time, that TSG-6 displays context-specific anabolic properties. This finding implies a therapeutic value for the protein, with possibilities of being used in conjunction with BMP-2 to treat local bone injuries such as fractures.

While the major source of vitamin D is D3 from ultraviolet exposure, some supplements supply D2. The relative potency of vitamin D2 versus vitamin D3 remains controversial. The aims of the current study were, 1. To optimize the in vitro model, including use of cell lines, vitamin D concentrations, and outcome biomarkers. 2. To compare the potency of vitamin D2 and D3 metabolites on mouse and human bone cellular activity. 3. To explore the expression of VDR in osteoarthritic (OA) bone tissues as well as cellular responses to vitamin D2 and D3 metabolites ex-vivo. In mouse 2T3 osteoblasts, at physiological doses, both vitamin D2 and D3 metabolites increased ALP activity and mineralisation and up-regulated osteoblastic signature genes and proteins. At supra-physiological doses D3 metabolites were more potent inhibitors of 2T3 function than D2 metabolites. Although hBMS cell proliferation was inhibited by both 25(OH)D2 and D3, ALP activity was enhanced by both metabolites. However, 25(OH)D3 was a more potent stimulator of ALP and mineralisation of hBMSCs. D2 and D3 equally stimulated expression of CX43 and PHEX markers in osteocytic cell lines. Immunohistochemistry of femoral heads showed much reduced VDR expression in OA osteocytes and osteoclasts, yet both 25(OH)D2 and D3 increased OA-hBMSCs mineralisation more than non-OA-hBMSCs ex-vivo. While vitamin D2 or D3 increased mouse 2T3 osteoblastic activity at physiological doses, OA and non-OA hBMSCs differentiation was more responsive to 25(OH)D3. Key bone cells such as osteocyte and osteoclasts expressed less VDR in OA. For the first time vitamin D2 metabolites have been thoroughly examined and emerged as a potent stimulator of bone cell differentiation, at least in vitro. Vitamin D3 in contrast is confirmed as highly potent in bone cells, but with toxicity at much lower doses than D2.

Osteoarthritis, the most prevalent joint disease in the United Kingdom, is a progressive condition that results in end-stage full-thickness cartilage loss and has important social and economic impacts on society. Since cartilage lacks regenerative capabilities, it is essential to develop approaches to initiate and enhance cartilage regeneration. In this context, tissue engineering is emerging as an attractive approach for the regeneration of cartilage tissue damaged due to disease or trauma. A scaffold-free cartilage construct, analogous to those found during embryonic precartilage condensation, has received much attention as an alternative novel modality for cartilage tissue engineering. Cartilage repair with scaffold-free tissue more closely resembles the natural situation and mimics the features of the original tissue. Moreover, scaffold-free cartilage implants can overcome the complications caused by the use of suboptimal scaffolds by avoiding the need for a foreign scaffold at all. Culturing cells into tissue patches without the requirement for a scaffold can be achieved through cell sheet engineering, which uses thermo-responsive culture dishes. However, the high costs of the tissue culture consumables, and the relatively low cellular yield, makes this process less attractive. This thesis presents a novel method for generating shape-, size- and thickness-adjustable 3-dimensional scaffold-free cell pellet sheets for use as implantable biological cell patches for cartilage tissue engineering. This new technique of bioengineering scaffold-free cell pellet sheets proves to be reproducible, easily applicable, sizable and thickness adjustable. Stem cells have added a new thrust to tissue engineering. Their distinctive self-renewal and plasticity have not only optimized many tissue engineering developments, but also rendered feasible some applications which would otherwise be unattainable with somatic cells. Human mesenchymal stem cells (HMSCs) were used to examine the optimal condition for generating cell pellet sheets with this new method. Furthermore, the resultant differentiated pellet sheets were compared directly with HMSCs, human chondrocytes and human fibroblasts alone to evaluate the feasibility of using this cell pellet sheet for clinical applications in terms of their biological and mechanical properties. The results of this thesis suggest that the engineered scaffold-free, chondrogenic, differentiated MSC pellet sheet not only exhibits desirable biologic features similar to chondrocytes, but also demonstrates good integrative and viscoelastic potential that might offer exciting possibilities for the development of novel biologically-based clinical therapies. In summary the data presented herein indicate the following points:

• The differentiation of human MSCs into chondrogenic cells was achieved.
• A novel approach of centrifugal seeding on a PDMS surface was shown to effectively generate chondrogenic-differentiated cell pellet sheets without impairing the biological functions of chondrocytes.
• Various cell types such as human MSCs, human chondrocytes and human fibroblasts were found to respond well to the novel methodology and generated viable, cohesive, less shrinkable, and readily-detachable cell pellet sheets, the size and thickness of which could be adapted as required. The results obtained were superior to those obtained using the conventional thermo-responsive culture dish method.

Tendon, ligament, and joint capsular injuries represent 45% of the 32 million musculoskeletal injuries each year in the United States. Tendon injuries are especially common, requiring surgical repair for the shoulder’s rotator cuff tendons (51,000 per year), the Achilles tendon (44,000 per year), and the patellar tendon (42,000 per year). Tissue engineering provides an alternative in the treatment of tendon lesions through replacement of an injured tendon segment. The purpose of this study was to develop a tendon construct in vitro for clinical reconstructive surgery. Human tenocytes were isolated from hamstring tendons of patients who had undergone anterior cruciate ligament (ACL) surgeries. These tenocytes were cultured with culture media (α-MEM) supplemented with various concentrations of foetal bovine serum (FBS) (0%, 1%, 5% and 10%) and in the presence of different growth factors such as PDGFBB (0, 5, 10 and 50ng/ml), basic FGF (0, 5, 10 and 50ng/ml), IGF-1 (0, 10 and 50ng/ml) and TGFβ-3 (0, 1 and 10ng/ml). Fractional factorial design was utilized to select the combinations of growth factors that supported the following criteria: (1) the maximal cell proliferation with a minimum differentiation of the tenocytes in the presence of the least concentration of FBS possible and (2) maintaining cell survival and promoting tenocyte differentiation in FBS free culture media. The results have shown that: (i) The tenocyte cell number when cultured for 14 days in media supplemented with 1% FBS, 50ng/ml PDGFBB and 50ng/ml bFGF matched that of the positive control (10% FBS-treated cells). Not only was the collagen synthesis significantly reduced in these growth factor-treated cultures compared to positive control tenocytes, but also a significant inhibition of the mRNA expression of various tenocyte differentiation markers (Scleraxis, Tenomodulin, Collagen type I and Decorin) was evident. IGF-1 did not promote significant cell proliferation under low serum conditions but did induce tenocyte differentiation in vitro. Examination of the cell morphology confirmed that tenocytes were capable of less differentiation when cultured with 1% FBS, 50ng/ml PDGFBB and 50ng/ml bFGF, this culture condition was termed “the expansion phase”; (ii) The cell survival was maintained for up to 14 days in serum free culture media supplemented with 50ng/ml IGF-1 and 10ng/ml TGFβ-3 whilst cell differentiation was enhanced and evident by the increase in collagen synthesis and cell morphology. Furthermore, mRNA expression of the aforementioned cell differentiation markers were also significantly increased, this culture condition was termed “the differentiation phase”; (iii) By combining the culture condition optimized for the expansion and differentiation phase sequentially, it was possible to maintain a long term 2-D tenocyte culture in vitro for up to 28 days. In these cultures, the presence of dense collagen formation was clearly evident whereas in positive control group (10% FBS group) such observation was not noted even after prolonged culturing period of up to 45 days. These results suggested that the sequential treatment of tenocytes with growth factors identified for the expansion and differentiation phases was significantly more superior than the standard 10% FBS treatment; (iv) By combining the expansion and differentiation phases optimized for the 2-D cultures, it was possible to maintain human tenocytes in a 3-D scaffold (Bombix silk) for up to 28 days. The tendon like constructs that were formed, macroscopically and microscopically resembled the human hamstring tendon. This observation was confirmed by using H&E staining, scanning electron microscopy and by detecting collagen type I immunohistochemically; (v) It was possible to further validate these findings using in vivo animal models. This was undertaken by implanting the tenocytes cultured sequentially in the defined culture media described above, into the quadriceps of Balb/c nude male mice for up to 30 days. The nature and specificity of the tendon like structure that was formed after this implantation was investigated by H&E staining and immunohistochemistry. It was revealed that the culture conditions that were optimized during the expansion and differentiation phases were suitable for generating a human tendon reconstruct; a finding which is of significance due to its potential for tendon reconstructive surgery.