These data show for the first time that EAATs are functional in human osteoblasts and that inhibition of their activity can modulate the osteoblast bone-forming phenotype. Manipulation of these mechanically regulated glutamate transporters may represent a new therapeutic approach for the treatment of fractures and disorders such as osteoporosis.

In the investigation of the correlation between VON and clinical severity, the mean VON was definitely greater than that in healthy Achilles tendon. Neovascularization was noted in 97.5% (n = 39) symptomatic Achilles tendons in 30 patients. The VAS showed a positive correlation with VON with a Spearman correlation coefficient of 0.326 (<italic>p</italic> = 0.04, power = 0.89), while no significant correlation was found between VISA-A score and VON.

The main aggrecan catabolite, found in samples of synovial fluid from patients with arthritis, and released from cartilage explant cultures exposed to IL-1, both have the amino-terminal amino acid sequence 374ARGSV ... (human sequence enumeration) corresponding to cleavage at the 'aggrecanase site' within the IGD of aggrecan (Sandy et al., 1992, and Lohmander et al., 1993). Loss of aggrecan is a primary event in the destruction of cartilage in arthritic disease. The Glu373- Ala374 bond ('aggrecanase site') within the IGD of the aggrecan core protein is cleaved by members of the ADAMTS family, including ADAMTS-4 and -5 (Tortorella et al., 1999, Abbaszade et al., 1999, and Sandy et al., 2000). In this investigation the model system of chondrocyte-agarose cultures, pioneered by Aydelotte and Kuettner 1988, was used to study the degradation of aggrecan by ADAMTS-4 and -5 in cartilaginous extracellular matrices. Low molecular weight co-migrating 37kD ADAMTS-4 and -5 isoforms were detected in apparently increased amounts in IL-1 a treated cultures compared to controls. These isoforms were bound by heparin and required de novo protein synthesis in the presence of IL-1 a for their generation. As previously reported, de novo protein synthesis in the presence of IL-1 a was also required for 'IGD aggrecanase activity' (Arner et al., 1998). Heparin bound media fractions from IL- 1a treated cultures possessed 'IGD aggrecanase activity' against exogenous aggrecan, which was inhibited by the amino-terminal region of TIMP-3 and was shown to be due to a 37kD isoform of ADAMTS-4. This implicated low molecular weight isoforms of ADAMTS-4 in the aggrecanolysis detected in the presence of IL-1 a. Carboxy-terminal truncation of furin cleaved ADAMTS-4 has previously been proposed as both an activation mechanism for the enzyme (Pratta et al., 2003, Kashiwagi et al., 2004, Gao et al., 2002, Gao et al., 2004 and Flannery et al., 2002) and a means of deregulation of the enzyme's catalytic activity.( Gao et al., 2002, Gao et al., 2004, and Kashiwagi et al., 2004). Therefore high molecular weight Furin cleaved ADAMTS-4 isoforms may be required to play normal physiological roles, whereas the low molecular weight forms are likely to be the enzyme isoforms involved in the destruction of aggrecan and other proteoglycans in articular cartilage during arthritis.

Introduction: Articular cartilage comprises a dense extracellular matrix (ECM) of primarily collagen, proteoglycans and water interspersed with the cartilage cell- the chondrocyte. Osteoarthritis (OA) is a disease characterised by articular cartilage degradation and a change in chondrocyte phenotype. Increased or abnormal joint loading is a risk factor for OA and can regulate chondrocyte phenotype. The chondrocyte cytoskeleton comprises actin microfilaments, tubulin microtubules and vimentin intermediate filaments and has been implicated in the propagation of physical signals to the chondrocyte nucleus, termed 'mechanotransduction'. In addition, the organisation of chondrocyte cytoskeletal networks has been observed to differ in both human OA and in a rat model of OA when compared with normal chondrocytes. We hypothesise that dysregulation of cytoskeletal networks prevents normal ECM-chondrocyte signalling and promotes a catabolic phenotype as in OA. Results: When compared with normal human chondrocytes, OA chondrocytes exhibited differences in the gene expression of components of the cytoskeleton and in the spatial organisation and architecture of the cytoskeleton, both in situ and in vitro. In normal and OA human chondrocytes cultured in agarose hydrogels, disruption of each of the three main cytoskeletal elements resulted in gene expression changes in both normal and OA cells. A number of gene responses to cytoskeletal disruption were similar in normal and OA cells, such as SOX9, MMP14, TGFB1, CASP3 and PTGS2 (COX-2). Other genes responded differently to the same treatment in normal versus OA cells, including ADAMTS5, COMP, FGFR3 and NOS2A (iNOS). Cyclic compression (15% strain, 0.5 Hz) for up to 40 minutes induced cytoskeletal reorganisation in normal and OA human chondrocytes and up-regulation of p-tubulin and destrin mRNA expression. Recovery in free swelling conditions for five hours post-load showed the chondrocyte phenotype was enhanced in OA chondrocytes. Cyclic compression in the presence of cytoskeletal disruption altered the transcriptional response of the actin depolymerising proteins cofilin and destrin in normal and OA chondrocytes, and the transcriptional response of SOX9 and the actin sequestering protein thymosin p4 in OA chondrocytes. Conclusions: Changes in the cytoskeleton of OA chondrocytes are not simply a result of the altered mechanical environment in OA articular cartilage. Changes in the cytoskeleton can affect chondrocyte phenotype and the response of chondrocytes to cyclic compression, therefore the observed differences in organisation and expression could result in the altered phenotype of OA chondrocytes. Differences between the effect of cyclic compression on normal and OA human chondrocytes support the existence of different or divergent mechanotransduction pathways that are mediated in part by elements of the chondrocyte cytoskeleton.

Introduction: Total knee replacement (TKR) is a common orthopaedic procedure, with 20-70% of patients needing 1-3 units of blood, although allogeneic transfusion is not risk free. Tranexamic acid (TXA) is a synthetic antifibrinolytic agent used intravenously to stop bleeding in TKR and other surgical procedures. Objectives: To determine whether intra-articular TXA is effective, safe and cost-effective in reducing blood loss and subsequent blood transfusion after TKR. Design: This thesis describes three research projects to address the objectives: 1. A systematic review and meta-analysis of the use of intravenous (IV) TXA in TKR; 2. A randomised controlled trial of the topical (intra-articular) use TXA in TKR; 3. A biomechanical study of the effect of local TXA on TKR materials. Outcome measures: The primary aim of intra-articular TXA was to reduce the blood transfusion rate. Secondary outcomes included reduced blood loss, length of stay, complications and cost and improved functional outcome measures. To explore whether TXA degrades TKR materials, tensile properties, wear rate and surface topographic profile were biomechanically tested. Results: The systematic review found that IV TXA reduced blood loss and transfusion significantly but there was significant heterogeneity between trials. A first trial of topical (intra-articular) TXA in TKR found TXA to be effective and safe in reducing blood loss and transfusion. Thirteen patients (16.7%) were transfused in the placebo group versus 1 (1.3%) patient in the TXA group (c2; P=0.001). Blood loss was reduced from 465 ml in the placebo group to 297 ml in the TXA group (t-test; P=0.00025). TXA use resulted in a net cost saving of £333 per patient (P=0.044). There was no adverse effect of TXA on the biomechanical properties of the joint materials. Conclusion: Topically Applied TXA in TKR is effective, safe and cost-effective in reducing blood loss and transfusion in TKR, and avoiding the potential complications of intravenous administration.

No description available.

Hallux valgus is a complex progressive foot deformity of uncertain aetiology. The disorder is characterised by a lateral deviation of the hallux at the first metatarsophalangeal joint; an angle ≥ 15° is considered as clinical hallux valgus. A model that predicts first metatarsophalangeal joint angle and thus, hallux valgus is potentially very useful; enabling the clinician to identify individuals at risk of developing the disorder and to predict prognosis. The aim of this study is to develop such a model. The literature relating to hallux valgus identifies eight potential aetiological factors of hallux valgus. The scientific evidence presented in support of these suspected aetiological factors, and the theories of pathology of hallux valgus in association with these factors were critically evaluated by a review of the literature. Methods to evaluate the significance of these factors in hallux valgus were identified and appraised. These methods were applied to a large sample of genetically related individuals. The genetic and environmental influences affecting first metatarsophalangeal joint angle, pes planus, metatarsal formula, digital formula and first ray neutral position were explored through the statistical analysis of the data obtained from the sample. The results of analyses suggest that all of these variables are gender influenced, multifactorial traits. Further analysis of a subset of data generated a statistical model that relates the degree of hallux deviation at the fast metatarsophalangeal joint (and thus, the degree of hallux valgus) to clinically measurable predictor variables. A further subset of data was applied to test the model. The model was found to accurately predict first metatarsophalangeal joint angle in 92% of cases. Application of the model allows the clinician to evaluate an individual's risk of developing hallux valgus enabling accurate prognosis. Recommendations for achieving improved prognosis and the implications for future research are proposed.

Joint degeneration requiring arthroplasty surgery and the consequences of osteoporosis are the two fundamental pathologies in orthopaedics. There are around 44,000 Medline-indexed journals about osteoporosis, and around 30,000 concerned with arthroplasty. However despite both typically occurring in a similar elderly population, only 350 (less than 0.5%) are cross-indexed. Aseptic loosening is the commonest cause of hip arthroplasty failure, with revision surgery being the only current treatment. Recent work has increased the understanding of the aetiology of aseptic loosening and studies suggest that this process may be inhibited by the use of drugs that are normally used to treat osteoporosis, such as the bisphosphonates. It has also been shown that the occult incidence of metabolic bone disease may be as high as 40% in patients undergoing primary hip arthroplasty. This study is a progression of similar work on the aetiology and control of aseptic loosening done in the same department over the proceeding few years. In the first instance a cellular model of aseptic loosening was investigated by Ong and Taylor [published in 2003]. This laboratory based project used mouse bone, and exposing it to interface membrane tissue sampled at the time of revision arthroplasty surgery. This model was described by Reynolds and Dingle in 1970, and shown to activate osteoclasts. Ong and Taylor demonstrated that osteoclast activation could be inhibited with doxycycline, suggesting that matrix metalloproteinases may be important in the pathophysiology of aseptic loosening, and that the process is potentially preventable. The work was progressed further by Ibrahim and Taylor [2004] who developed a live model of particle induced osteolysis. They measured radio-labelled calcium uptake in mouse femora following implantation of ceramic particles, sham surgery and in controls. This was shown to be a useful model of quantifying osteolysis, although they did not find a difference between the controls and those exposed to ceramic particles. The original aim of this work was to follow on from the previous work and demonstrate that osteolysis could be inhibited or reversed using pharmacological agents. Ideally this would be done in a human clinical model, and a number of drugs were considered, including doxycycline, bisphosphonates and statins. Such a project would have involved recruiting patients to a clinical trial, followed by either randomisation to treatment or control groups before commencing treatment on participants. The ideal end-point would be revision for aseptic loosening (although radiological development of loosening would be an alternative). Because hip arthroplasty is such a successful operation these end-points are both rare and often not seen for many years. Even if we assume a rather optimistic reduction in loosening of 50% using our agent, we would have to recruit several hundred participants and wait at least 10 years to get meaningful results. We therefore have had to sacrifice some of the principles of strong research in favour of a project that could be completed with a limited time-frame and a limited budget. We studied patients that had already had an arthroplasty in situ for a number of years, and in view of the multi-factorial nature of loosening (as discussed below), limited this to one type of arthroplasty. The hypothesis of this study is that patients who have an underlying disorder of bone metabolism (such as osteoporosis or vitamin D deficiency) are more likely to develop aseptic loosening. In addition we hypothesise that there are measurable clinical, radiographic and biochemical markers that help predict those likely to develop loosening. This hypothesis was investigated in 127 patients (78 patients with a loose cemented total hip replacement matched by age, gender, race, prosthesis and time from surgery with 49 patients with a well-fixed stable hip replacement)/ We then conducted four connected studies involving, clinical, radiological, DEXA and biochemical assessment for markers of loosening. The aims are detailed below, but were principally to see whether patients with loosening are more likely to markers of osteoporosis or poor bone health. Unfortunately, this study takes us no further forward with regard to whether aseptic loosening can be inhibited by specific therapeutic agents, but hopefully it helps us to better understand the pathophysiological processes involved with arthroplasty failure. These can be used in future research to help improve arthroplasty function and longevity.

No description available.

Cellular changes occur during osteoarthritis (OA), which lead to an alteration in phenotype of the resident cartilage cell, the chondrocyte, and subsequent destruction of the tissue. Many of these changes are unknown. I hypothesise these changes may be due in part to microRNAs (miRNAs), small non-coding RNAs that regulate the expression of a discrete repertoire of genes through base-specific interactions within the target genes 3’untranslated region. A previous screen in our laboratory has identified a number of miRNAs differentially expressed in OA cartilage. The objectives of this study were; (1) to investigate the genetic association of the most extensively studied cartilage miRNA (miR-140), and its targets, with OA, and (2) to assess the function of other, differentially expressed, and less well studied miRNAs, in development signalling pathways, namely miR-125b and miR-324-5p, but focusing on miR-324-5p. Here I identified; (1) SNPs within the miR-140 locus and a predicted, OA-associated, miR-140 target, that may affect function; and (2) miR-125b and miR-324-5p as regulators of Hedgehog (Hh) signalling, likely to play a role in skeletal development. miR-324-5p regulates Hh signalling in human and mouse, yet the mechanism appears unconserved. In humans, miR-324-5p targets SMO and GLI1. Using Stable Isotope Labelling with Amino acids in Cell culture (SILAC) mass spectrometry and whole-genome microarrays, I identified novel miR-324-5p targets, and validated Glypican1 (Gpc1) as a direct target of miR-324-5p and a regulator of Hh signalling in mouse. In addition to regulation of Hh, miR-324-5p regulates Wnt signalling, in which it forms a negative feedback loop. Together, this body of work demonstrates how miRNAs, their targets and their function can be linked in their expression and association with OA.