Under normal circumstances bones are kept strong enough to withstand mechanical loads without fracture because bone cells sense the deformation that occurs when they are loaded and adjust bone structure accordingly. This process of functional adaptation allows us to lead active lives without a high incidence of fracture. The situation changes in later life when age-related bone loss occurs, leading to osteoporosis. Current treatments are unable to recover the bone lost with age, nor can they selectively target bone formation to regions of the skeleton under the greatest strain. Clinical trials are currently investigating the potential of blocking sclerostin, an endogenous inhibitor of bone formation which is down-regulated by loading, to cause bone formation. However, the mechanisms by which sclerostin expression is controlled by loading are not known. The experiments in this thesis were designed to establish how osteocyte-like cells translate the mechanical strain signals they encounter during loading into appropriate down-regulation of Sostlsclerostin. These studies delineate novel mechanisms whereby prostaglandin and oestrogen receptor signalling, previously known to influence bone's adaptation to loading, cooperate to promote Sost down-regulation following strain. We demonstrate that sclerostin inhibits the proliferation of osteoblast-like cells following strain, providing a mechanism whereby blocking sclerostin may lead to new bone formation. In the absence of sclerostin, osteoblastic cells' proliferation following strain requires the oestrogen receptor a and protein kinase Ca. Strain also orients the division of these cells through Planar Cell Polarity signalling, thereby potentially influencing bone architecture. These findings identify new mechanisms whereby loading stimulates bone cells to produce a structure strong enough to resist fracture. Deciphering the cellular processes by which sclerostin is naturally down-regulated following loading may lead to the development of improved treatment regimens for bone loss diseases based on the combination of sclerostin blocking therapies with other bone-strengthening strategies such as exercise.

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The aim of this thesis was a preliminary investigation of nutritional and biochemical factors which may be implicated in ethnic differences in fracture risk. Investigation of polymorphic loci of the vitamin D receptor gene revealed that the frequency of B and t alleles was considerably greater in the British subjects than in their Gambian or Chinese counterparts (p<0.001). The frequency of the "s" allele of the polymorphic SP1 binding site in the collagen 1α1 gene was also considerably greater in the British subjects than in the Gambian (p<0.02) or Chinese (p<0.001) subjects. The vitamin K status of bone, indicated by the γ-carboxylation of plasma osteocalcin, was determined in eleven premenopausal women of each group and in postmenopausal Gambian (n=50), British (n=31) and Chinese (n=23) subjects. In all groups, undercarboxylation was significantly higher post- than premenopause, but this was related to the higher total osteocalcin concentration present postmenopause. After consideration of differences in total osteocalcin, undercarboxylation was highest in the British, intermediate in the Gambian and lowest in the Chinese subjects irrespective of menopausal status. Possible determinants of osteocalcin γ-carboxylation were investigated. There was a direct relationship between plasma vitamin K concentration and osteocalcin γ-carboxylation in the British subjects, and a direct relationship between plasma triglyceride concentration and osteocalcin carboxylation in the Gambian and Chinese subjects. Apolipoprotein E2 allele was associated with increased γ-carboxylation in the British and Chinese subjects, but there was little indication of a an influence of plasma vitamin D concentration. Differences in plasma vitamin K concentration were observed between the ethnic groups, being significantly higher in the postmenopausal Chinese compared with the British (117% p≤0.0001) and Gambian subjects (103% p≤0.0001). These data suggest genetic differences may exist in calcium homeostasis and the collagen structure of bone, along with differences in vitamin K status and the γ-carboxylation system of osteoblasts, which may be implicated in ethnic variation in osteoporotic fracture risk.

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The consequences of irradiation on the molecular structure, crystal morphology and mechanical properties of UHMWPE, have recently been implicated as a potential major factor influencing the wear, prompting several manufacturers to consider alternative sterilisation methods. The current work has contributed to this area through investigation of the effects of irradiation dose (representative of current orthopaedic practice) and post-sterilisation ageing on subtle changes in the microstructure of UHMWPE using a wide range of experimental techniques, including X-ray diffraction, scanning electron microscopy and differential scanning calorimetry to probe the microstructure. In particular, an analysis of retrieved human knee tibial implants of both earlier designs and a recently introduced total knee system design, has been conducted using the above techniques in order to understand the fundamental wear mechanisms leading to premature failure of these components. This has been complemented with laboratory simulation tests to investigate the effects of the above physical and chemical changes on the abrasive and fatigue wear resistance of sterilised, aged and unaged UHMWPE. A new micro-scale abrasion test and a new rolling contact fatigue test have been used to achieve this. The <I>in vivo</I> wear mechanisms observed under these test conditions have been compared with those seen in the retrieved tibial implant material to emphasise the clinical relevance of this study. This work has shown that gamma sterilisation in air, followed by long-term shelf and/or <I>in vivo</I> ageing, significantly reduces the fatigue and abrasive wear resistance of UHMWPE, associated with notable changes in its microstructure. Alternative sterilisation methods which eliminate long term free radical activity have been recommended.

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Particles of a variety of orthopaedic materials have been implicated with bone resorption both <I>in vivo </I>and <I>in vitro</I>. The polymer investigated in this study is poly(L-lactide), a bioresorbable material which has been used in orthopaedics to manufacture screws and pins for fracture fixation. Poly(L-lactide) implants degrade and release particulate debris prior to their resorption. So far, the particles generated during the degradation of poly(L-lactide) implants have not been implicated with bone resorption in human clinical trials however, osteolysis in the vicinity of degrading resorbable implants made of a similar material known as poly(glycolide) has been observed radiographically. The biocompatibility of poly(L-lactide) particles was investigated using both <I>in vitro</I> and <I>in vivo</I> techniques in this project and the findings can be summarised as follows: 1. Exposure of fibroblasts to poly(L-lactide) particulates caused an increase in prostaglandin E<SUB>2</SUB> (PGE<SUB>2</SUB>) release from intact viable cells whereas exposure of macrophages to these particles caused PGE<SUB>2</SUB> release due to cell death. 2. The presence of lactate monomer [the ultimate degradation product of poly(L-lactide)], caused alterations in cell mortality, percent lactate dehydrogenase (LDH) release and increases in PGE<SUB>2</SUB> release which were not attributable to osmolarity changes alone. 3. An <I>in vitro</I> transformation assay revealed that poly(L-lactide) particles have the capacity to transform C3H/10T1/2 cells, although there is no evidence to suggest that these particles would cause tumours in humans or other animals. 4. Finally, a "rat pin" model did not show up any evidence of bone resorption in the vicinity of a loaded metallic pin implant, caused by high molecular weight poly(L-lactide) particles although fluorescence microscopy indicated that mineralisation may have been reduced.

To enhance our understanding of the pathophysiology of bone disease associated with liver transplantation and of the mechanisms underlying bone loss in the three month period following transplantation, this prospective study was undertaken as follows: (1) bone pathophysiology was evaluated pre- and three months post-transplantation in transiliac biopsies using tetracycline-assisted histomorphometry; (2) cellular activities of bone formation and resorption pre- and post- transplantation were studied using quantitative enzyme cytochemistry in combination with histomorphometric methods; (3) cellular activities for markers of bone energy metabolism and biosynthesis and/or cell proliferation were investigated using quantitative enzyme cytochemistry; (4) plasma markers for bone metabolism were investigated at regular intervals in collaboration with other laboratories. It was concluded from this study that rapid bone loss early after transplantation is due both to increased bone turnover and a negative remodelling balance at the individual bone remodelling site. These changes were at least partially mediated by increased PTH levels secondary to a negative balance in plasma calcium. Cyclosporin A is known to increase intracellular calcium levels and inhibit calcium release from mitochondria. It also reduces glomerular filtration rate which could be sufficient to depress extracellular calcium levels and thereby cause the observed rise in PTH levels. The consequences of this for post transplant bone remodelling is a markedly enhanced risk of osteoporosis in these patients. Ensuring replete calcium and vitamin D levels pre-transplantation and supplementation of cyclosporin A treatment with vitamin D metabolites and calcium post-transplantation followed by careful monitoring of plasma calcium concentrations might offer a better overall outcome for preventing transplantation-associated osteoporosis at this early stage post transplantation.

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