Osteoarthritis (OA) is a chronic and highly prevalent degenerative disease of the synovial joint leading to cartilage destruction and bone remodelling. The current management of end-stage OA is joint replacement, however, this procedure is not suitable for a subset of patients hence there is a growing need for alternative treatments and technologies to address this limitation. One such approach to this problem is the application of cell-based therapies that regenerate areas of damaged cartilage. Recently discovered articular cartilage progenitor cells (CPC) have been hallmarked as a potential cell source for repair and/or regeneration of damaged articular cartilage. Initial focus was on the characterisation of human CPC isolated from healthy donors and compared with OA derived CPC and patient matched OA Bone Marrow Mesenchymal Stem Cells (BM-MSCs). Comparison of all cell types showed similar morphology and proliferative capacity. In addition, all cell types isolated showed positive expression of the putative mesenchymal stem cell makers; CD-90, CD-105 and CD-166 while lacking expression of CD-34. All cell types investigated showed successful osteogenic, chondrogenic and adipogenic differentiation, hence providing evidence of the mesenchymal stem cell properties of isolated CPC. A gene profiler array was used to identify the expression of Wnt pathway genes from RNA isolated from CPC cell lines originating from healthy and OA cartilage. Interestingly, the expression of Dkk-1 was observed to have the highest up-regulation in OA-derived CPC. The role of Dkk-1 was further studied in a number of CPC and chondrocyte cell lines from healthy and OA cartilage. It was found that normal CPC cell lines showed homogenously low expression and secretion of Dkk-1, however, OA-derived CPC cell lines exhibited a heterogeneous expression and secretion of Dkk-1. In a pellet culture model of chondrogenic differentiation, CPC cell lines secreting high levels of Dkk-1 failed to undergo chondrogenic differentiation, measured by diminished expression of chondrogenic differentiation markers, Type II collagen, ACAN and Sox-9 at both molecular and protein levels. Immunolocalisation of Dkk-1 in OA osteochondral plugs showed peri-cellular expression in chondrocytes located in all zones and around migratory endothelial cells invading articular cartilage where there was a quantifiable increase of blood vessel invasion. This later observation was further studied through a series of experiments to investigate the role of Dkk-1 in relation to endothelial cell migration and angiogenesis using an in vitro model of angiogenesis and migration/invasion assays. A novel finding emerged from these studies, which provides evidence for a pro-angiogenic and pro-migratory role of Dkk-1 and to a lesser extent Dkk-2 in human endothelial cell lines. A novel in vitro Transwell co-culture model was developed to study the interaction between chondrocytes and endothelial cells mimicking the osteochondral interface. A novel finding from these studies included the observation that normal or OA-derived chondrocytes appeared to induce an endothelial to mesenchymal transformation (EndMT) of the co-culture endothelial cells. This was assessed by a loss of the endothelial cobble stone morphology and a down-regulation of key factors implicated in endothelial cell phenotype, including VE-cadherin, Tie-2, e-NOS, PDGF-AA and PECAM-1. As endothelial cells lost their phenotype they adopted a spindle morphology and expressed mesenchymal cell markers including: Lumican, Snail, α-SMA, Vimentin and MMPs. Interestingly, this was also associated with an increase in Dkk-1 expression. To confirm a role for Dkk-1 in this process endothelial cells were cultured in the presence of Dkk-1 and were found to undergo EndMT when compared to the control. In summary, this thesis has uncovered several interesting differences in CPC phenotype. In addition, my results suggest that Dkk-1 has potential as a biomarker of OA pathology. This thesis highlights further the complex role of the Wnt Pathway and in particular Dkk-1 may play a role in the pathogenesis of osteoarthritis.

With the current crisis of organ shortage and an increasing number of dialysis patients,studies directed at ameliorating such a substantial organ discrepancy are of considerable importance to the transplant community. The use of extended criteria donation has helped to compensate the disparity of organs however, we are still a long way from achieving satisfactory targets. Still there are many organs from older donors that are discarded primarily on the basis of chronological age. It is here that biological age may display a crucial role in allowing the transplant team to characterize donor organs with greater accuracy. Indeed both biological and chronological age are very closely related and ECD criteria are based very much on the latter, albeit with other clinical variables. However the biomarker of ageing CDKN2A which is suitably represented by Baker and Sprott’s criteria, displays closer variabilities with post-operative transplant function, at least up to one year. Telomere length known as the “Gold standard” biomarker of ageing does not display as robust a role in predicting organ function as CDKN2A. The classification of organs represented in this text from category I-IV serves merely as a guide to future studies and is yet to be validated in larger clinical trials. It is however a simple and rapid assessment tool (Gingell-Littlejohn et al PLOS One 2013). Relatively advanced cellular senescence was displayed in the mutant AS/AGU rat kidney when compared to the parent AS strain. This was exploited in a unique animal model to study the effects of ischaemia reperfusion injury on the mutant kidney, hence mimicking to a certain degree the transplant related injuries in ECD kidneys. Although ischaemic times in the model were moderate in nature, there was nonetheless a difference in the tolerance to IR injury between parent and mutant strain as evidenced by increased p16 and p21 staining in AS/AGU rats. Such a model therefore is exclusive in that interventions to improve ECD renal function post-transplantation can accurately and conveniently be represented and studied at a pre-clinical level. Anti-ischaemic compounds have been the subject of much debate over the years, however a single “Holy Grail” compound able to completely abolish the injurious effects of IR injury has never been elicited. mTOR inhibitors however, display several cellular effects and act as potent immunosuppressants. They (AZ-6) have also been shown to partially mediate the detrimental effects of IR injury on the native kidneys of AS rats in a specifically designed animal model as shown. Further studies encompassing transplanted kidneys from mutant AS/AGU rats exposed to such a promising agent would be of undoubted importance to the clinical field of transplantation, potentially leading to immeasurable economic and patient benefits.

Feline Osteoarthritis (OA) is a pathological change of a diarthrodial articulation which primarily occurs in older cats. The aims of this study were:- 1) to define the radiographic features of OA in the cat for each individual appendicular joint; 2) to relate the radiographic features to the gross pathologic and histopathologic features; 3) to explore underlying causes of OA in cats, 4) to identify the presence of Protease Activated Receptor-2 (PAR-2) and matriptase in feline articular cartilage and synovial membrane and to determine their role in OA pathogenesis. The present study has defined five radiographic features of OA for each appendicular joint:- presence of osteophytes, enthesiophytes, areas of abnormal mineralisation,synovial effusion and joint remodelling. The study furthermore suggested that increases in radio-opacity beneath the semilunar notch, along the femoral trochlea, beneath the tibial plateau and on the femoral head/neck are also important radiographic features. The radiographic prevalence was highest in the elbow (23.9%, 93/389) and stifle (23.9%,93/389) joints, followed by the hip (21.1%, 82/389), tarsal (17.7%, 69/389), shoulder(6.7%, 27/389) and carpal (6.4%, 25/389) joints. The results from this study demonstrate that the presence of a radiographically apparent supinator sesamoid bone(SSB), meniscal mineralisation (MM) and two fabellae are related to cartilage pathology and can be indicators of OA. Prevalence rates for gross pathology changes were highest in the elbow (20.2%,102/506) joint, followed by the stifle (19.6%, 99/506), hip (18.4%, 93/506), shoulder (17.8%, 90/506), tarsal (15.0%, 76/506), and carpal (9.1%, 46/506) joints. Eight key gross pathologic features were identified- cartilage discolouration, cartilage fibrillation,cartilage ulceration, cartilage erosion, osteophytes, thickening of joint capsule, synovium discolouration and joint remodelling. The radiographic and gross pathologic total scores were positively correlated in each appendicular joint and the joint most likely to have cartilage damage without radiographic evidence of OA is the shoulder (71.1%, 64/90) followed by the elbow (39.1%, 9/23), hip (32.4%, 11/34), stifle (26.1%,6/23), carpal (23.1%, 21/91) and tarsal (14.9%, 7/47) joints. Four possible underlying conditions that lead to secondary OA were identified:- radioulnar incongruity, hip dysplasia (HD), cranial cruciate ligament (CCL) disease and primary meniscal mineralisation. The identification of PAR-2 and matriptase proteins and gene expression in feline articular tissues is a novel and important finding supporting the hypothesis that serine proteases are involved in the articular cartilage degradation seen in feline OA.