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Cobalt toxicity, arising from the release of metal nanoparticles and ions during wear of Co/Cr metal-on-metal (MoM) hip implants has become a recognised internal source of Co poisoning. Co ions enter the bloodstream during wear and corrosion processes of MoM implants, and then travel throughout the body causing systemic toxicity. Cardiac toxic manifestations have been shown to be among the most prevalent systemic symptoms following exposure. In this thesis we have investigated the effects of short and/or long term CoCl2 exposure on the Swiss 3T3 fibroblast cell line (3T3s) and primary adult cardiac fibroblasts (CFs) in vitro. We have measured effects on cell viability, morphology, proliferation, assessed intracellular Co uptake, and detected and quantified protein expression by western blotting. Further, we also studied the effects of chronic in vivo CoCl2 treatment (4 weeks of daily 1mg/kg intraperitoneal injections) of rats carrying out echocardiography, and determining the Co distribution into various organs. The expression of potential transporter proteins, TRPC6, TRPM7, DMT1, and of CAMKIIδ, a serine/threonine kinase with pivotal roles in cardiovascular function through regulation of Ca2+ handling and excitation-contraction coupling, were also investigated. To complete the in vitro and in vivo studies, the molecular mechanism(s) underlying the overall changes were screened by RNA-Seq analysis, and gene expression was subsequently validated by quantitative real-time-PCR (RT-qPCR) and western blotting. In terms of proliferation of 3T3 cells and CFs treated with CoCl2, the CFs were much more sensitive than 3T3 cells with IC50 values for CoCl2 in the range of ~20 μM inCFs and ~250 μM in 3T3 cells. Using phalloidin to stain the actin inside cells showed that in both types of cells actin fibres were disrupted, and the membrane formed blebs at high Co concentrations. Co uptake, evaluated at the cellular level by using inductively coupled plasma mass spectrometry (ICP-MS), revealed 3 to 4-times greater Co uptake into CFs than 3T3 cells at 48 h. In vivo studies using echocardiography showed evidence of altered cardiac function in Co treated rats after 28 days exposure. Reduction of the percentage of fractional shortening (%FS), from 60.29±0.53%, to 54.01±0.90% n=6, p < 0.05, (Co-treated compared to normal rats) occurred. This could imply early indications of cardiac dysfunction. Co accumulated in the organs of the rats and significant increases in Co ion levels (compared with control untreated animals) were detected in liver, kidney and heart (1,839.86±177.30, 1,536.01±83.95, and 307.82±35.74 μg/L respectively) by ICP-MS after 7 days exposure. Liver and kidney are the primary organs of excretion, and higher concentrations than other organs might be expected. However, these data provide strong evidence that Co accumulates in the hearts of the treated rats and this may result in cardiac dysfunction. Western blot analysis of CFs, and heart tissue lysates from animals treated with CoCl2 for 28 days, showed a significantly increased level of protein expression for CaMKIIδ, TRPC6 and TRPM7. In contrast, in 3T3 cells, the expression of TRPs was decreased at high concentrations of Co. TRP transport channel proteins are likely to act as ubiquitous metal ion flux pathways, and may play a role in uptake of Co2+ into the heart enabling it to contribute to compromised heart function. Gene expression analysis of Camkiid, Dmt1, Trpc6, Trpm7, and Trpv1 using RNA-Seq and RT-qPCR, found all had increased expression levels in heart RNA after 28 days exposure. RT-qPCR analysis showed there were increased levels in 4 out of the 5 selected genes (Camkiid, Dmt1, Trpc6, and Trpm7) in CFs after 72 h Co treatment. In contrast, Trpc6 mRNA expression was absent in 3T3 cells using RNA-Seq and RT-qPCR. These results suggest the differential uptake of Co2+ between CFs and 3T3 cells might be mediated through Trpc6. Sequestration of Co into cardiac cells in vitro and in vivo, suggests that in vivo the metal may accumulate in the hearts of MoM patients with high circulating Co blood levels. This may lead to systemic adverse effects including cardiotoxicity. Inhibition or downregulation of Co uptake mechanisms into cardiac cells may offer a therapeutic intervention to minimise adverse effects of Co in patients with MoM implants. We recommend careful monitoring of cardiac function in MoM patients, in addition to the blood metal ion level monitoring and scans already instigated by MHRA.

Functional improvement is an important outcome following total knee arthroplasty (TKA). According to recent research, three-dimensional motion analysis is the most scientific method of measuring dynamic knee function. Nevertheless, current protocols are too time consuming and complicated for routine clinical use. This study developed a clinic-appropriate motion capture system, and investigated the feasibility of its use in a clinical environment. A compact motion capture system (Dimensions: 3.5(L)x2.1(H)x1.1(W)m) and bespoke cluster-based biomechanical model were developed. Assessments for quantifying knee range of motion (ROM), knee strength, gait kinematics, and gait stability were incorporated into the software. Most results were reported in real-time. Validation studies of the assessments against clinical standard tools showed few clinically significant differences between the results, suggesting that the assessments could be used as accurate and reliable alternatives to the traditional tools. The system was then used clinically to report the functional outcome of Medacta GMK Sphere TKA patients. Patients underwent functional testing pre-, 6-weeks, and 1-year post-operatively. Average recorded assessment time was 16.8±2.4 minutes. On average, knee ROM, gait kinematics, spatio-temporal parameters of gait and gait stability improved post-operatively. Knee strength decreased over the first year however, suggesting that TKA patients require strength training post operatively in order to optimise functional outcome. The results reported in this trial were generally consistent with the current literature, implying that the system returned valid data for this patient cohort,and that the Medacta GMK Sphere TKA was successful at improving knee function, especially in frontal and transverse planes during gait. To conclude, this thesis has shown that motion capture technology can feasibly be used in the clinical environment to assess the function of TKA patients in an acceptable clinical time frame. The system developed and presented here can therefore justifiably be used clinically to better report the functional outcome of TKA.

Background: Missing data are an unavoidable feature in longitudinal studies and inad- equate handling can result in bias. The South London Stroke Register (SLSR) follows up participants at three months and annually after stroke. The majority of data collected are categorical in nature. Typically a third of survivors miss each follow-up and the impact of, and `best' methods for, dealing with these missing data are not clear. The aim of the thesis is to compare and determine the most appropriate methods for handling non-continuous missing data in the SLSR. Methods: Exploratory analyses identi ed predictors of incomplete follow-up and in- formed a simulation study in which the biases associated with prevalence rates of four indicators of poor outcome were estimated and analysis methods compared across four scenarios. Models making di ering assumptions about the missing data assessed the im- pact of missing data on associations between baseline characteristics and outcomes. Results: Missing data were strongly associated with disability and activity level after stroke and likely missing not at random (MNAR). Estimates of prevalence of poor out- comes from available case analyses were relatively unbiased apart from when a strong MNAR assumption was made and outcomes were strongly associated with dropout, with prevalence underestimated by up to 7% points. Bias was reduced after using multiple imputation (MI) with maximum bias of 5% points. There was no evidence that missing data in uenced associations between baseline characteristics and outcome. Conclusions: Some subgroups of the SLSR are at greater risk of non-participation than others but the resulting bias is likely to be minimal. When summarising population out- comes using rates MI should be used in addition to available case analysis. Future work will seek to further quantify potential biases using routinely collected data from GPs to compare responders and non-responders.

Distal femoral and proximal tibial joint-sparing bone tumour implants allow to preserve the knee, in limb salvage surgery. The aim of this thesis was to compare implant survival, functional outcomes, acceptance, proprioception and gait in patients with knee sparing implants and conventional knee sacrificing implants. Using FEA, a distal femoral implant and cadaver bone were modelled and parametrised to find the design that improves implant fixation. A survivorship study of 104 consecutive patients following knee sparing surgery (mean follow-up 36.1 ± 11.0 months) found an implant survival rate of 78% and this is comparable to the reported survival for joint sacrificing prostheses. Younger patients showed improved implant survival compared to older individuals. Plate fracture was not observed and aseptic loosening was the main reason for revision. Radiographic analysis indicated that implantation accuracy increased implant survival. Patient questionnaires showed that patients with knee sparing implants had more normal functional outcome and acceptance compared with patients with knee sacrificing implants. However, proprioception (joint position sense) was reduced in these patients. Using optoelectronic gait analysis system, hip, knee and ankle joint angle in 19 patients and 3 healthy subjects were measured. Ground reaction force and time in stance were also investigated. Joint symmetry in the joint sacrificing group was improved compared to the joint-sparing group, however overall, the joint-sparing tibial group demonstrated a more normal gait pattern. FEA results indicated that lower resection levels, reduced plate thickness and implant materials with lower modulus, decreased the stresses in the bone adjacent to the implant while loaded the bone more to reduce risk of stress shielding. To conclude, knee sparing endoprostheses provide a reliable alternative to knee sacrificing implants in limb reconstruction in selected patients. However, the current design of joint-sparing implants can be optimised to potentially improve bone remodelling and implant fixation.

It is envisaged that in order to achieve the stringent quality requirements of future human cell and tissue manufacturing processes, implementation of automated processing systems will be required. As automated systems are capable of operating at high speeds, throughput is likely to be constrained by sensitivity of cellular materials to mechanical stress. The effect of laminar capillary shear stress (pipe flow) on rat aortic smooth muscle cells was investigated. Initial studies showed that repeated exposure to high shear stress (>100 Pa) causes substantial cell damage, with surviving cells able to grow normally. A flow cytometry assay for cell membrane integrity was developed and exhibited high assay resolution, precision and speed. Further shear studies indicated minimal cell damage or loss in the shear stress range 5-25 Pa, with some evidence of cell damage at high shear stresses (35-75 Pa). Cell loss at very low shear stress ( 2 Pa) was attributed to surface adhesion. Manufacture of cell based therapeutics requires the generation and processing of concentrated cell suspensions. Volume mean cell diameter for rat aortic smooth muscle cells was measured to be 22.4 mum and enabled conversion between cell concentration and cell volume fraction in suspensions. Concentrated cell suspensions behaved as 'power law' (shear thinning) fluids and showed similarity with reported rheology of concentrated yeast and plant cell suspensions. Predictions based on literature values for oxygen uptake rate of mammalian cells indicated that oxygen depletion is likely to occur during processing of cell pellets and concentrates. Two semi-automated cell concentration protocols (centrifuge-resuspend) were developed and compared. Both protocols achieved cell volume fraction of around 0.3-0.4 (wet pellet) and the superior protocol exhibited intact cell yield of ~75%. Apparent loss of cells was mainly attributed to formation of large aggregates during concentration. A 'windows of operation' analysis was constructed to examine process feasibility in the context of experimental results. Three different processing scenarios were predicted to be feasible and it was shown that processing yield is a critical factor in determining overall process feasibility and manufacturing costs.

Amyloid is a term used to describe a set of diseases caused by protein misfolding. There are 36 proteins known to undergo amyloidosis to date. They each unfold from their unique native states and refold into a specific cross β- sheet motif to which various macromolecules can bind. These protein deposits invade tissues and organs disrupting tissue structure and function. The protein serum amyloid P (SAP) is a universal component to all amyloid deposits regardless of the protein involved. It is a disc shaped protein which is known to protect amyloid fibres against degradation. The role of SAP in amyloidosis has been a subject of discussion for many years. Some have found SAP to enhance fibre formation whereas others have found SAP to prevent it. This thesis can be divided into two parts. The first half focuses on the molecular analysis of SAP in relation to amyloid fibre growth whereas the second half focuses solely on the structural aspects of SAP. Chapters 2 and 3 demonstrate that SAP actually exhibits a dual role in relation to fibrillogenesis. It is capable of acting as both a chaperone and a fibrillogenic enhancer depending on its conformation at the time. As a monomer SAP is responsible for the enhancement and stabilisation of fibres whereas in its dimerised form it demonstrates characteristics similar to that of a molecular chaperone. Recent developments in amyloid treatment involve the removal of SAP from deposits using anti-SAP antibodies. As of yet, structural analysis has not been carried out on the SAP:antibody complex. Chapters 4 and 5 conclude this thesis by using crystallographic techniques to demonstrate that a single antibody binds to an SAP molecule during dissociation from amyloid deposits.

A method of 3D modelling of high intensity focused ultrasound (HIFU) treatment through ribcage using patient data is described. This is further tested experimentally to derive at various parameters which could be indirectly useful in defining amount of energy and alteration needed in treatment protocols for the upper abdominal pathologies covered under ribcage. In the last ten years, feasibility and safety of HIFU treatment have been tested in a growing number of clinical studies. However, there are still challenges, such as the treatment through the ribcage. HIFU propagates through the ribs could lead to insufficient energy delivery to the focal target and heat deposition at the ribs may lead to surrounding soft tissue damage due to the bones properties of absorption and reflection to the ultrasound wave. Current research has not involved the physical human anatomical structures into the experimental study of the HIFU treatment through the ribcage. Therefore, our aim was to build a patient-specific physical ribcage model that possesses human anatomical structures to investigate its effect on HIFU treatment. Ribcage model was reconstructed based on patient data. This prototyping model possessed human anatomical structures and provided useful information in the treatment planning by applying HIFU sonication on tissue mimicking phantoms. In order to determine the optimal phantoms for medical imaging and HIFU application, the properties of tissue mimicking materials (TMMs) were characterised, including acoustic, thermal and elastic properties. Acoustic pressure distribution, acoustic power delivery and focus splitting were investigated with and without the presence of ribcage model in the focused ultrasound field. Multi-channel phased array transducer was applied to investigate the changes in temperature at focus and temperature was monitored by magnetic resonance imaging (MRI) thermometry. Sonications on phantom with varied focal lengths and ribcage locations using robotic arm were performed. Thermometry revealed that heating at focus can be controlled either by changing power or duration of application in order to achieve optimal results. Focal temperature change related to the shadow area on the transducer elements caused by the ribcage model in HIFU beam path. The experimental data based on the patient-specific ribcage model may contribute to design and optimise the sonication protocol in pre-clinical planning stage. It is believed that the HIFU sonication experiments using realistic phantoms based on patient data would be a step towards translation of this technology into more widespread clinical practice.

Ultrasonic surgical instruments actuated by piezoelectric transducers are usually driven at relatively high power levels to generate high vibration amplitude at the probe, which interact with biological tissues to achieve the desired effects. In operation, the high power level introduces nonlinearities into the behaviours of the piezoelectric transducer and the biological tissues place a load onto the transducer. Both the nonlinearities and the load result in variations in resonant frequency and electric impedance, which negatively affect the performance of the ultrasonic surgical instrument. The aim of this work was to develop an adaptive driving system to address the issue of the resonant frequency shift and impedance variation and to study its effectiveness in optimising the performance of ultrasonic surgical instruments. An ultrasonic planar tool based on piezocrystal PMN-PT was designed with various configurations as an alternative to the existing ultrasonic scalpels. To address the problem of resonant frequency shift and impedance variation observed on the planar tools in characterisation stage, an adaptive driving system was developed to track the resonant frequency and stabilise the vibration velocity. The system was carefully calibrated, and its effectiveness in optimising the performance of unloaded transducers in a broad frequency range was validated. The performance variation of the planar tool under the combined influence of high power level and external tissue loads was then investigated. The capacity of the adaptive driving system to optimise the performance of the planar tool under such conditions was then accessed by performing soft tissue penetrating test. Furthermore, a needle actuating device was designed to increase the visibility of medical needles in ultrasound guided percutaneous procedures. Its modal behaviour was studied in finite element analysis and verified by experimental characterisation. The ability of the adaptive driving system to improve the performance of the needle actuating device was accessed in pre-clinical trials. The results demonstrate that the adaptive driving system developed can track the resonant frequency and stabilise the vibration velocity of ultrasonic surgical instruments in frequency up to 5 MHz and power up to 300 W. By using the adaptive driving system, optimal performance of the planar tool and needle actuating device can be achieved in both unloaded and loaded conditions. The ultrasonic planar tool with PMN-PT is potentially useful in surgical operations. However, its performance is limited by the intensive heat generated in the joint area and the low Curie point of PMN-PT. The needle actuating device can increase the visibility of standard medical needles in colour Doppler imaging.