Metal-on-metal (MoM) hip joint replacements have been increasingly used for younger and more active patients in recent years due to their improved wear performance compared to conventional metal-on-polymer bearings. MoM bearings operate at body temperature within a corrosive joint environment and therefore are inevitably being subjected to wear and corrosion as well as the combined action of tribo-corrosion. Issues such as metal sensitivity/metallosis associated with high levels of metal ion release triggered by the wear and corrosion products remain critical concerns. During the past few decades, significant research has been conducted into understanding the wear/lubrication mechanisms within the MoM hip joints in order to improve their performance and thereby prolonging their life. However, not much attention has been given to the combined effect of wear and corrosion of such devices in the hip joint environment, in addition, the role of third body particles and the effects of proteins have not been well understood. In this work, a systemic approach is presented for the first time for the mapping of abrasion and tribo-corrosion performance of a cast CoCrMo (F75) in simulated hip joint environments. The effects of third body particles have been studied in the MoM context using 4 μm SiC, 1 μm and 300 nm Al2O3, as well as sub-micron BaSO4. Modified tribo-testers (micro-abrasion, nanoindenter/scratching) incorporating a novel electrochemical cell have been used to monitor the abrasion-corrosion behaviour of the alloy in situ. The effects of solution chemistry, abrasives size / concentration and presence of proteins on the wear / corrosion level, wear-corrosion mechanisms, and the depassivation/repassivation kinetics of the CoCrMo have been explored. A variety of surface and sub-surface characterization techniques have been employed to identify the microstructual wear mechanism interactions. Results show that the change of protein concentration (0, 25% and 50% bovine serum) and pH (pH 7.4 and pH 4.0) of the test solutions can significantly influence the protein adsorption behaviour, which subsequently influence the wear rates (synergy), wear mechanisms as well as the wear-induced corrosion currents of the CoCrMo. For abrasion-corrosion tests, reducing abrasive size from 4 μm to 300 nm and/or abrasive volume concentration from 0.238 vol% to 0.006 vol% results in different abrasion-corrosion wear mechanisms (rolling or grooving abrasion) and the average wear-induced corrosion currents show a linear correlation with wear rates for 4 μm and 1 μm abrasives. For low volume concentration (< 0.03 vol%) slurries containing bovine serum, organo-metallic conglomerates have been found within the wear scars. These conglomerates help separate the surfaces, impose less damage to the surface passive film and polish the wear scars through a chemical mechanical polishing mechanism. In addition, tribo-corrosion tests at micro-/nano- scales reveal the effects of single abrasive particle on the surface/sub-surface microstructual change. This investigation has revealed the nanoscale wear mechanisms that generate nanoscale wear debris, the mechanical mixing of the surface nanostructure with adsorbed denatured protein and also the slip/dislocation systems that are present near and on abraded surfaces that are likely to disrupt the surface passive films. The findings give a better understanding of the evolution of the sub-surface nanocrystalline structures and tribo-layers formation seen for the retrieved implants. This near surface nanostructure layer and phase transformation might offer better wear resistance through these inherent self-protecting mechanisms (i.e. increased hardness); conversely, it may become the precursors to debris ejection and enhanced ion-release into the CoCrMo joints. This work established an experimental technique that gives greater understanding of the tribocorrosion behaviour of cast CoCrMo in simulated hip joint environments. In particular, the roles of third body abrasive particles and proteins have been addressed, which are relevant to clinical applications. The material multi-scale wear mechanisms as well as the evolution of the surface / subsurface microstructures and tribo-layers have been elucidated, which provide new insights into the in vivo wear mechanisms of CoCrMo. The findings of this study may provide some important indications for improved MoM joint materials, design, manufacture and evaluation.

There is an increasing demand for total knee replacements (TKR). Young patients are placing increasing functional demands on modern TKR. Clinical experience has also shown the need for high flexion in patients after TKR. In this study, assessment of TKR performance subjected to deep knee bend was investigated. Patellar resurfacing in TKR is assumed to release pain and restore knee function. Despite the recent advance and success in TKR operation, patellar resurfacing has been associated with an increase in complications at the patello-femoral joint, and hence revisions following TKR. Complications include poor tracking, instability, wear, loosening and fractures. These complications have been attributed in part to the component design features (e.g. sagittal radius, depth, and orientation of the trochlear groove of the femur and the geometry of the patellar component surface) and surgical technique (e.g. component alignment and ligament balance). However, the influence of these factors on the overall performance of TKR has not been investigated extensively. The objective of the study was to determine the variation of patellar kinematics (tracking motion) and contact mechanics (contact force, area, pressure and stress) induced by component design and surgical technique. A three-dimensional finite element (FE) model of a PFC-Sigma TKR, including the tibio-femoral and patello-femoral joints was developed. Explicit FE analysis was used to simulate TKR under a deep knee flexion. The models predicted substantial increase in patellar pressure and stress with nonconforming patello-femoral articulating surfaces. Femoral groove orientation affected patellar tracking and contact mechanics. Extending femoral groove distally reduced patello-femoral contact stress at high flexion angles. Also, externally rotating the femoral component and adjusting the line of action of quadriceps pull would be beneficial by reducing patellar lateral force. The FE model used in the current study provided insight into the effect of component design parameters and surgical technique on patellofemoral kinematics and contact mechanics.

Human organs are the most valuable gifts of life. Until today, through organ transplantation, thousands of lives have been saved and many more blessed with hope and happiness through a better quality of living. However, rapid developments in transplant technology will be meaningless if supply of the needed organs remains scarce and organ transplantation procedures cannot take place accordingly. This global problem of organ shortage is also faced by Malaysia. Despite campaigns and initiatives introduced by the Malaysian authorities, the problem remains unresolved and the situation is worsening. Malaysia is reported to have less than one donor for every one thousand of the population (Lela Yasmin Mansor, 2007). However, statistics from the National Transplant Registry Malaysia confirm a steady increase in the number of registered potential donors each year. This suggests that certain factors must be preventing potential donors from becoming actual donors. Therefore, this study will not only discuss the current scenario of the organ shortage problem in Malaysia, highlighting its underlying factors, but will also scrutinise legal and social factors causing actual donations to remain relatively small, despite the promising number of potential donors registering each year. The study will suggest practical solutions to help solve organ shortages in Malaysia, particularly by utilising brain-dead patients from serious road traffic accidents as a potential source of cadaveric organs. Clarification on the Islamic perspective concerning organ donation is also included, as Islam is the main religion professed in Malaysia.

Acrylic bone cement has been used for fixation of load-bearing orthopaedic implants for over five decades, and continues to be the 'gold standard' for elderly patients and those with systemic disease. Aseptic loosening remains a major indication for revision of cemented hip implants, and has been associated with mechanical degradation of the cement mantle via the initiation and coalescence of fatigue micro-cracks. Microstructural defects such as voids and agglomerates of radiopacifier particles have been implicated in this damage accumulation process. Improved understanding of the relative effects of these features on the mechanisms of fatigue crack initiation and failure within the cement is required in order to inform the development of more robust cement formulations and thus increase the longevity of the cement mantle in vivo. The present study utilised micro-computed tomography (μ-CT) and scanning electron microscopy (SEM), in conjunction with mechanical testing, to provide a systematic, quantitative assessment of the effect of cement formulation and microstructure (including voids and radiopacifiers) on the in vitro fatigue failure of four commercial, vacuum-mixed cement formulations. Results were compared with μ-CT data and fractographic analysis of an ex vivo cement specimen. This novel 'data rich' methodology enabled non-destructive, three-dimensional analysis of defect populations in terms of the size, morphology and spatial density of individual microstructural features, and the identification and characterisation of crack-initiating defects. The inclusion of barium sulphate as a radiopacifier was found to have a negative effect on the fatigue life of cement; radiopacifier particles showed a tendency to form numerous large agglomerates, which readily initiated fatigue cracks; furthermore, fatigue life scaled consistently with initiating defect size. In contrast, cement containing zirconium dioxide as a radiopacifier demonstrated superior fatigue performance, and failure in these cement formulations was dominated by crack initiation from voids. In all four cement formulations, void populations were found to be bi-modal, and the largest voids (> 0.5 mm equivalent spherical diameter) were surrounded by secondary satellite voids in both in vitro and ex vivo cement specimens. Extensive void formation was also noted in both moulded specimens and cement mixing gun stubs, in addition to ex vivo cement. Optimisation of cement formulations and vacuum-mixing techniques may therefore be advantageous in order to reduce the formation of barium sulphate agglomerates and large voids, and thus minimise their potential crack initiation effects in vivo.

This thesis documents the work of Andrew I. M. Greer undertaken for the fulfilment of the requirements for the Degree of Doctor of Philosophy. The project, funded by the EPSRC and MRC, is to develop a nanofabrication processing strategy compatible with titanium based orthopaedic implants. Such a development will facilitate the translation from current and historical in vitro analysis of cell-stimulating nanotopographical cues to in vivo studies upon an implant relevant material. The work presented opens by summarising the social motives and consequences before contextualising the project aims with reference to existing approaches in the field. The thesis progresses through a series of different nanofabrication approaches until an effective strategy satisfying the goals of the project is devised. Thereafter the strategy is explored with its results characterised from a material level through to a biological level. Ultimately the primary goal of the project is realised through the development of novel sol-gel chemistry capable of retaining a nanopattern and transforming into titania, the natural composition at the surface of a titanium based implant. Furthermore, nanofeatures previously too stringent to fabricate for a comprehensive biological study are readily achievable using the documented strategy and fundamental studies have been carried out which indicate that the features concerned are highly effective at up-regulating early indicators of bone formation.

Prostatic diseases are common; benign prostate hyperplasia (BPH) is almost ubiquitous in elderly men and 899,000 men were diagnosed with prostate cancer worldwide in 2008. The incidence of both is increasing and expected to continue to rise. Therefore, prostatic diseases represent a considerable economic burden, but there are currently no reliable markers available to accurately differentiate indolent from aggressive disease nor to predict who will benefit from treatment for either BPH or prostate cancer. This results in over and under-treatment of both diseases with consequent patient related morbidity and mortality. The molecular mechanisms underlying the natural history of prostatic diseases remain elusive. It is accepted that prostate cell growth and survival are exquisitely dependent upon activation of the androgen receptor (AR) by androgens. Following ligand binding, AR undergoes further phosphorylation at serine residues, which inhibit proteolytic degradation, stabilise AR and influence AR transactivation. It is therefore plausible that alterations in AR phosphorylation may drive prostatic disease progression. However, few studies have explored the significance of AR phosphorylation, or the kinases driving AR serine phosphorylation in the clinical setting. The over-riding objective of this study was to establish the clinical relevance of AR serine phosphorylation status in prostate tissue in both BPH and prostate cancer. The specific aims of the current study were: • To firstly establish and validate a panel of AR phosphospecific antibodies. • To evaluate site specific AR serine phosphorylation expression levels in prostate cancer and BPH patient cohorts, with full clinical data and follow-up. • To investigate the expression of candidate kinases mediating such phosphorylation. This involved establishing tissue banks with linked comprehensive clinical databases, and utilising this tissue to establish AR phosphorylation expression profiles for each patient. Six AR phosphospecific antibodies (pARS81, pARS94, pARS213, pARS515, pARS578, pARS650) were verified using peptide competition assays and western blotting. Cdk1, ERK1/2, Akt and PKC were identified as putative kinases mediating AR phosphorylation using the online kinase search tool Scansite 2.0. Immunohistochemistry was performed on hormone naïve diagnostic prostate cancer tissue relating to 90 patients. High expression levels of AR phosphorylation at serine sites 81, 515 and 578 were each associated with a poorer clinical outcome. Following cox regression analysis, cytoplasmic pARS515 expression (p=0.038, HR 4.5 (95% CI 1.1–20.6)) and pARS81 nuclear expression (p=0.030, HR 0.033 95% CI 0.002-0.721) were independently associated with shorter time to biochemical relapse and shorter disease specific survival respectively. Cdk1 and/or pCdk1161 were significantly associated with pARS81 and pARS515 as predicted by Scansite 2.0. Similarly, nuclear PKC expression was significantly associated with pARS578 expression both in the cytoplasm and the nucleus. In patients with PSA at diagnosis ≤20ng/ml, high cytoplasmic pARS515 expression was associated with significantly shorter time to biochemical relapse (p=0.019). This translated into significantly shorter disease-specific survival (p<0.001, 10y survival 38.1% vs 100%). Prostate cancer patients with a low serum PSA level at diagnosis may be suitable for delayed radical treatment via active surveillance. An investigation was therefore undertaken in 51 prostate cancer patients treated by active surveillance. Active surveillance is a deferred radical treatment approach which provides a potential solution to the problem of over treatment as a result of over-diagnosis. However some patients harbour occult aggressive disease and delay in treatment may result in disease progression and failure of radical therapy. Although none of the individual AR serine phosphorylation sites were associated with clinical outcome measures on univariate analysis, high expression of total AR in the cytoplasm (p=0.021, HR 4.6 (95% CI 1.3-16.8)) and presence of perineural invasion in the tumour specimen (p=0.003, HR 8.6 (95% CI 2.1-35.7)) were deemed independent with regards to shorter time to treatment intervention in a cox regression analysis. Validation of the results seen in the first active surveillance prostate cancer cohort was undertaken in a second prospectively collected cohort consisting of 84 active surveillance patients. The results in the first cohort were not replicated in the second. Although cytoplasmic pARS81 was associated with time to intervention (p=0.032) and pARS515 expression trended towards an association (p=0.072), an increase in patient numbers in both cohorts may have provided more reliable results. However even with the numbers available in contrast to the first active surveillance cohort, but in line with the pilot prostate cancer cohort, Cdk1 was associated with pARS515 expression, and pCdk1161 trended towards an association. BPH is also an androgen driven disease dependent upon the AR. Previous research into predictive and prognostic markers in BPH is scant. Therefore a comprehensive analysis of clinical and novel pathological factors, including markers of inflammation, was performed in 336 BPH patients. Following this a complete panel of AR serine phosphorylation sites, and associated kinases, was analysed with reference to clinical outcome measures in the BPH cohort. Low expression levels of total AR and AR phosphorylated at Ser-81, 515 and 650 were associated with poorer clinical outcomes. Low expression of smooth muscle pARS515 (p=0.029, HR 0.31 (95% CI 0.10-0.94)) and older age (p=0.004, HR 5.13 (95% CI 1.43-18.41)) were deemed independent on cox regression analysis with regards to shorter time to postoperative acute urinary retention (AUR). Furthermore, low expression of pARS515 in the smooth muscle was associated with increased incidence of postoperative AUR in patients over 70 years old (25.1% vs 2.8% at 10 years following transurethral resection of prostate (TUR)), (p=0.002, HR 0.20 (95% CI 0.06-0.62)). This may have important clinical implications in postoperative counselling of these patients. In addition it may influence the decision to commence early postoperative medical treatment (with 5-alpha-reductase inhibitors and/or alpha blockers) on a prophylactic basis in these patients. Cytoplasmic pARS650 expression (p=0.010, HR 0.50 (95% CI 0.29-0.86)) and PSA at diagnosis (p=0.018, HR 1.89 (95% CI 1.11-3.16)) were independently associated with time to failure of surgical intervention. Furthermore, low expression of pARS650 in the cytoplasm was associated with increased failure of surgical intervention in patients with PSA ≥4ng/ml at diagnosis (45.5% vs 13% at 5 years post TUR), (p=0.026, HR 0.52 (95% CI 0.29-0.93)). This comprehensive study on immunohistochemical expression of site specific AR serine phosphorylation and associated kinases fills a gap in the current literature. It has demonstrated the clinical significance of AR serine phosphorylation in prostate cancer and BPH and uncovered potentially exciting new avenues for future investigation. Site specific serine phosphorylation of the AR may serve as a prognostic and predictive biomarker in prostatic disease and has potential as a future target for therapeutic intervention.

Orthopaedic infections can be polymicrobial existing as a microbiome. Infections often incorporate staphylococcal species, including Staphylococcus aureus. Such infections can lead to life threatening illness and implant failure. Furthermore, biofilm formation on the implant surface can occur, increasing pathogenicity, exacerbating antibiotic resistance and altering antimicrobial mechanism of action. Bacteria change dramatically during the transition to a biofilm growth state: phenotypically; transcriptionally; and metabolically, highlighting the need for research into molecular mechanisms involved in biofilm formation. Metabolomics can provide a tool to analyse metabolic changes which are directly related to the expressed phenotype. Here, we aimed to provide greater understanding of orthopaedic infection caused by S. aureus and biofilm formation on the implant surface. Through metagenome analysis by employing: implant material extraction; DNA extraction; microbial enrichment; and whole genome sequencing, we present a microbiome study of the infected prosthesis to resolve the causative species of orthopaedic hip infection. Results highlight the presence of S. aureus as a primary cause of orthopaedic infection along with Enterococcus faecium and the presence of secondary pathogen Clostridium difficile. Although results were hindered by the presence of host contaminating DNA even after microbial enrichment, conclusions could be made over the potential increased pathogenicity caused by the presence of a secondary pathogen and highlight method and sample preparation considerations when undertaking such a study. Following this finding, studies were focused on an orthopaedic clinical isolate of S. aureus and a metabolome extraction method for staphylococcal biofilms was developed using cell lysis through bead beating and solvent metabolome extraction. The method was found to be reproducible when coupled with liquid chromatography-mass spectrometry (LC-MS) and bioinformatics, allowing for the detection of significant changes in metabolism between planktonic and biofilm cultures to be identified and drug mechanism of actions (MOA) to be studied. Metabolomics results highlight significant changes in a number of metabolic pathways including arginine biosynthesis and purine metabolism between the two cell populations, evidence of S. aureus responding to their changing environment, including oxygen availability and a decrease in pH. Focused investigations on purine metabolism looking for biofilm modulation effects were carried out. Modulation of the S. aureus biofilm phenotype was observed through the addition of exogenous metabolites. Inosine increased biofilm biomass while formycin B, an inosine analogue, showed a dispersal effect and a potential synergistic effect in biofilm dispersal when coupled with gentamycin. Changes in metabolism between planktonic cells and biofilms highlight the requirement for antimicrobial testing to be carried out against planktonic cells and biofilms. Untargeted metabolomics was used to study the MOA of triclosan in S. aureus. The triclosan target and MOA in bacteria has already been characterised, however, questions remain over its effects in bacteria. Although the use of triclosan has come under increasing speculation, its full effects are still largely unknown. Results show that triclosan can induce a cascade of detrimental events in the cell metabolism including significant changes in amino acid metabolism, affecting planktonic cells and biofilms. Results and conclusions provide greater understanding of orthopaedic infections and specifically focus on the S. aureus biofilm, confirming S. aureus as a primary cause of orthopaedic infection and using metabolomic analysis to look at the changing state of metabolism between the different growth states. Metabolomics is a valuable tool for biofilm and drug MOA studies, helping understand orthopaedic infection and implant failure, providing crucial insight into the biochemistry of bacteria for the potential for inferences to be gained, such as the MOA of antimicrobials and the identification of novel metabolic drug targets.

Soft tissue sarcomas (STS) comprise a heterogenenous group of greater than 50 malignancies of putative mesenchymal cell origin and as such they may arise in diverse tissue types in various anatomical locations throughout the whole body. Collectively they account for approximately 1% of all human malignancies yet have a spectrum of aggressive behaviours amongst their subtypes. They thus pose a particular challenge to manage and remain an under investigated group of cancers with no generally applicable new therapies in the past 40 years and an overall 5-year survival rate that remains stagnant at around 50%. From September 2000 to July 2006 I undertook a full time post-doctoral level research fellowship at the MD Anderson Cancer Center, Houston, Texas, USA in the department of Surgical Oncology to investigate the biology of soft tissue sarcoma and test novel anti- sarcoma adenovirus-based therapy in the preclinical nude rat model of isolated limb perfusion against human sarcoma xenografts. This work, in collaboration with colleagues as indicated herein, led to a number of publications in the scientific literature furthering our understanding of the malignant phenotype of sarcoma and reported preclinical studies with wild-type p53, in a replication deficient adenovirus vector, and oncolytic adenoviruses administered by isolated limb perfusion. Additional collaborative and pioneering preclinical studies reported the molecular imaging of sarcoma response to systemically delivered therapeutic phage RGD-4c AAVP. Doxorubicin chemotherapy is the single most active broadly applicable anti-sarcoma chemotherapeutic yet only has an approximate 30% overall response rate with additional breakthrough tumour progression and recurrence after initial chemo-responsiveness further problematic features in STS management. Doxorubicin is a substrate for the multi- drug resistance (mdr) gene product p-glycoprotein drug efflux pump and exerts its main mode of action by induction of DNA double-strand breaks during the S-phase of the cell cycle. Two papers in my thesis characterise different aspects of chemoresistance in sarcoma. The first shows that wild-type p53 suppresses Protein Kinase Calpha (PKCα) phosphorylation (and activation) of p-glycoprotein by transcriptional repression of PKCα through a Sp-1 transcription factor binding site in its -244/-234 promoter region. The second paper demonstrates that Rad51 (a central mediator of homologous recombination repair of double strand breaks) has elevated levels in sarcoma and particularly in the S- G2 phase of the cell cycle. Suppression of Rad51 with small interfering RNA in sarcoma cell culture led to doxorubicin chemosensitisation. Reintroduction of wild-type p53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression via transcriptional repression of the Rad51 promoter through increased AP-2 binding. In light of poor response rates to chemotherapy, escape from local control portends a poor prognosis for patients with sarcoma. Two papers in my thesis characterise aspects of sarcoma angiogenesis, invasion and metastasis. Human sarcoma samples were found to have high levels of matrix metalloproteinase-9 (MMP-9) with expression levels that correlated with p53 mutational status. MMP-9 is known to degrade extracellular collagen, contribute to the control of the angiogenic switch necessary in primary tumour progression and facilitate invasion and metastasis. Reconstitution of wild-type p53 function led to decreased levels of MMP-9 protein and mRNA as well as zymography-assessed MMP-9 proteolytic activity and decreased tumour cell invasiveness. Reintroduction of wild-type p53 into human sarcoma xenografts in-vivo decreased tumour growth and MMP-9 protein expression. Wild-type p53 was found to suppress mmp-9 transcription via decreased binding of NF-κB to its -607/-595 mmp-9 promoter element. Studies on the role of the VEGF165 in sarcoma found that sarcoma cells stably transfected with VEGF165 formed more aggressive xenografted tumours with increased vascularity, growth rate, metastasis, and resistance to chemotherapy. Use of the anti-VEGFR2 antibody DC101 enhanced doxorubicin sensitivity at sub-conventional dosing, inhibited tumour growth, decreased development of metastases, and reduced tumour micro-vessel density while increasing the vessel maturation index. These effects were explained primarily through effects on endothelial cells (e.c.s), rather than the tumour cells per se, where DC101 induced e.c. sensitivity to doxorubicin and suppressed e.c. production of MMPs. The p53 tumour suppressor pathway is the most frequently mutated pathway in sarcoma. Recapitulation of wild-type p53 function in sarcoma exerts a number of anti-cancer outcomes such as growth arrest, resensitisation to chemotherapy, suppression of invasion, and attenuation of angiogenesis. Using a modified nude rat-human sarcoma xenograft model for isolated limb perfusion (ILP) delivery of wild-type p53 in a replication deficient adenovirus vector I showed that functionally competent wild-type p53 could be delivered to and detected in human leiomyosarcoma xenografts confirming preclinical feasibility - although not efficacious due to low transgene expression. Viral fibre modification to express the RGD tripeptide motif led to greater viral uptake by sarcoma cells in vitro (transductional targeting) and changing the transgene promoter to a response element active in cells with active telomerase expression restricted the transgene expression to the tumour intracellular environment (transcriptional targeting). Delivery of the fibre-modified, selectively replication proficient oncolytic adenovirus Ad.hTC.GFP/ E1a.RGD by ILP demonstrated a more robust, and tumour-restricted, transgene expression with evidence of anti-sarcoma effect confirmed microscopically. Collaborative studies using the fibre modified phage RGD-4C AAVP confirmed that systemic delivery specifically, efficiently, and repeatedly targets human sarcoma xenografts, binds to αv integrins in tumours, and demonstrates a durable, though heterogeneous, transgene expression of 1-4 weeks. Incorporation of the Herpes Simplex Virus thymidine kinase (HSVtk) transgene into RGD-4C AAVP permitted CT-PET spatial and temporal molecular imaging in vivo of transgene expression and allowed quantification of tumour metabolic activity both before and after interval administration of a systemic cytotoxic with predictable and measurable response to treatment before becoming apparent clinically. These papers further the medical and scientific community’s understanding of the biology of soft tissue sarcoma and report preclinical studies with novel and promising anti- sarcoma therapeutics.

Demand for Total Knee Replacement (TKR) surgery is high and rising; not just in numbers of procedures, but in the diversity of patient demographics and increase of expectations. Accordingly, greater efforts are being invested into the pre-clinical analysis of TKR designs, to improve their performance in-vivo. A wide range of experimental and computational methods are used to analyse TKR performance pre-clinically. However, direct validation of these methods and models is invariably limited by the restrictions and challenges of clinical assessment, and confounded by the high variability of results seen in-vivo. Consequently, the need exists to achieve greater synergy between different pre-clinical analysis methods. By demonstrating robust corroboration between in-silico and in-vitro testing, and both identifying & quantifying the key sources of uncertainty, greater confidence can be placed in these assessment tools. This thesis charts the development of a new generation of fast computational models for TKR test platforms, with closer collaboration with in-vitro test experts (and consequently more rigorous corroboration with experimental methods) than previously. Beginning with basic tibiofemoral simulations, the complexity of the models was progressively increased, to include in-silico wear prediction, patellofemoral & full lower limb models, rig controller-emulation, and accurate system dynamics. At each stage, the models were compared extensively with data from the literature and experimental tests results generated specifically for corroboration purposes. It is demonstrated that when used in conjunction with, and complementary to, the corresponding experimental work, these higher-integrity in-silico platforms can greatly enrich the range and quality of pre-clinical data available for decision-making in the design process, as well as understanding of the experimental platform dynamics. Further, these models are employed within a probabilistic framework to provide a statistically-quantified assessment of the input factors most influential to variability in the mechanical outcomes of TKR testing. This gives designers a much richer holistic visibility of the true system behaviour than extant 'deterministic' simulation approaches (both computational and experimental). By demonstrating the value of better corroboration and the benefit of stochastic approaches, the methods used here lay the groundwork for future advances in pre-clinical assessment of TKR. These fast, inexpensive models can complement existing approaches, and augment the information available for making better design decisions prior to clinical trials, accelerating the design process, and ultimately leading to improved TKR delivery in-vivo to meet future demands.

Aims: This thesis has three main aims: • To investigate the potential role of human bone marrow stromal cells (HBMSC) in Impaction Bone Grafting (IBG). • To investigate the potential role of a synthetic graft, Poly (DL-lactic acid), (PDLLA) as a tissue engineering scaffold and a graft extender in IBG. • To investigate methods to improve graft compaction and reduce fracture risk in IBG. Methods: Part I: The biocompatibility and mechanical properties of HBMSC seeded onto allograft or PDLLA were compared to allograft or PDLLA alone in vitro. Part II: Evidence of biocompatibility, neovascularisation and new bone formation in impacted allograft and PDLLA scaffolds seeded with HBMSC, in vivo was assessed and compared to allograft and PDLLA alone. Part III: The laboratory work was translated into the clinical setting with implantation of impacted allograft seeded with HBMSC for the treatment of bone defects in two case studies. Part IV: The role of vibration in IBG technique to reduce fracture risk and improve graft compaction and prosthetic stability was assessed in an in vitro femoral IBG model. Results: Part I: HBMSC seeded onto morsellised allograft or PDLLA, and cultured under osteogenic conditions in vitro were able to withstand the forces equivalent to a standard femoral impaction and were able to differentiate and proliferate along the osteogenic lineage. The living composite formed provided a biomechanical advantage, with increased interparticulate cohesion and shear strength when compared to allograft alone. Part II: HBMSC seeded onto morsellised allograft or PDLLA, impacted and implanted subcutaneously in nude mice demonstrated cell viability and histological evidence of new bone formation and neovascularisation after 28 days. Part III: In two case studies impacted allograft augmented with marrow-derived autogenous cells was used to treat bone voids in the proximal femur. Both patients made an uncomplicated clinical recovery. Imaging confirmed filling of the defects with very encouraging initial graft incorporation. Histochemical staining of graft samples demonstrated that a live composite graft with osteogenic activity had been introduced into the defects. Alkaline phosphatase and immunohistochemical staining techniques confirmed the bone phenotype of the autotransplanted cells. Part IV: Vibration assisted compaction of morsellised allograft reduced the peak loads and hoop strains transmitted to the femoral cortex during graft compaction, improved graft compaction in the proximal and middle femoral regions, which in turn conferred improved mechanical stability of the prosthesis under cyclical loading, demonstrated by a reduction in stem subsidence. Conclusions: • HBMSC when combined with either allograft or synthetic graft (PDLLA) can survive the forces of a standard IBG and under osteogenic conditions, differentiate and proliferate along the osteogenic lineage. HBMSC and allograft / PDLLA composites confer an additional biomechanical advantage over allograft / PDLLA alone. • Increased new bone formation and neovascularisation has been demonstrated in vivo in allograft and PDLLA / HBMSC composites compared to allograft or PDLLA alone. • Tissue engineering principles combining morsellised allograft and HBMSC composites have been utilised to fill bony voids in two clinical cases, with good clinical outcome. • By reducing peak loads, hoop strains and femoral fracture risk, and improving graft compaction and prosthetic stability the use of vibration and a perforated tamp is a potential new safer more flexible IBG technique. • Utilising tissue engineering techniques and improved graft impaction methods provides avenues to augment the biological and mechanical properties of morsellised allograft, and potentially increase the longevity of revision hip arthroplasty performed using the IBG technique.