Background: Electromyography (EMG) is a measure of neural activation to muscles and as such can give a window into neuromuscular dysfunction in patients. Although it was the primary focus of early clinical gait analysis (CGA), it has become progressively less common since the widespread adoption of optoelectronic measuring systems capturing three dimensional kinematics and kinetics. This is surprising since EMG has considerable potential to explain gait deviations observed in the kinematic and kinetic data. Apart from the extra time required for collecting data there are a number of barriers to the use of EMG in modern CGA. The most obvious is that EMG data has traditionally been collected, analysed and, most importantly, presented using quite different techniques which prevents a streamlined integration of EMG with the kinematic and kinetic data. Secondly, although the general characteristics of normative EMG patterns in the larger muscles are reasonably well understood, there is considerably less consensus on those which are smaller but still clinically important. Finally several of the most clinically important muscles, such as the tibialis posterior (TP), can only be accessed using fine-wire techniques and there is no consensus on how such data should be presented clinically. Objectives: This research aims to define rigorous data capture, analysis and presentation protocols for incorporation of both fine-wire and surface EMG measurements into CGA. The secondary aim is to provide definitive normative EMG profiles in the selected lower limb muscles across the gait cycle in healthy adults as reference for CGA purposes. Finally, a case series aim to explore whether the methods of collection, analysis and data presentation established in this work could be used to detect patterns of muscle dysfunction underlying kinematic impairments in the gait of stroke participants. Methods/results/discussion: A systematic review was conducted and the synthesised EMG profiles with and without between-subject variability from all included papers showed a wide range of variability in lower limb EMG profiles, a lack of studies in deep muscles which potentially play important roles in gait such as TP, no standardisation of fine-wire EMG acquisition and processing (compared to the surface EMG) and various methods of EMG normalisation. These variety of collection and analysis techniques resulted in large variability, in the current literature base, of EMG profiles between different studies. The majority of EMG studies currently available in the literature focus on larger superficial muscles. Studies on TP were scarce in spite of its important role in foot posture and gait. One reason for the lack of information on deep lower limb muscles may be that these can only be assessed using fine-wire sensors, for which there are no guidelines for standardised collection procedures amenable for use in CGA. A series of experiments aimed at addressing these limitations of fine-wire EMG in the current literature base (identified in the systematic review) and ultimately using improved collection and analyses techniques to allow direct comparison between fine-wire and surface EMG and provide a normative database for clinical application were carried out on TP for which little normative reference data exists, tibialis anterior (TA), and medial gastrocnemius (MG). The normalisation study mean normalisation appears to be the best method to reduce variability and this is true across muscles, sensors and different measures of variability: standard deviation can be reduced by 18%-62% of the mean signal and standard errors of measurement can be reduced up to 42% of the mean signal. A peak normalisation is equally effective with small difference (<5%). The second study revealed six gait cycles were necessary to collect fine-wire EMG which showed similar patterns ( r >0.9) at the same standard error of an ensemble average of surface EMG for TA and MG. The grand ensemble average of fine-wire EMG showed slightly greater between-session variability than surface EMG (9%-10% for fine-wire and 4%-7% for surface). Normative EMG data was then collected using normalisation with respect to the peak over six gait cycles from TP, TA and MG alongside kinematics and kinetics at five different speeds from eight young participants. Finally a case series of EMG collections with participants with stroke were used to explore the proof-of-concept of how standardised EMG methods could be implemented in clinical gait analysis and the potential benefits of using EMG to support identification of reasons for gait deviations in CGA. A normative database collected using these established methods was effective to identify pathological features and changes of muscle activity in three participants with post-stroke when using ankle-foot orthosis (AFO). However, the sensitivity of the database to detect changes under AFO condition depended on the severity of the impairment. Conclusion: As there was no previous standardised guidelines for the use of fine-wire EMG in CGA, this PhD defined a protocol for EMG measurement of TP, TA and MG using fine-wire and surface sensors in combination with kinetics and kinematics for CGA. The results of a series of systematic examinations of different normalisation techniques as well as between subject and between-session variability indicate that six gait cycles of data is sufficient for the collection of fine-wire EMG in CGA and that normalisation relative to the mean or peak during the gait cycle is the most appropriate if EMG data is to be used to aide CGA. A case series of stroke participants demonstrated data collected in this way could be used to detect impaired muscle activation underlying impaired kinematics of walking when compared to a normative database, and that the EMG data could add useful information to understanding typical CGA outputs.

Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory disease. Stiffness is a major symptom of RA which is commonly reported by patients, affects patients’ daily life, and is relevant to patients in relation to fluctuating aspects of RA such as flare and low disease activity. Morning stiffness is also frequently used as an outcome measure both clinically and in research. Despite the relevance and uses of stiffness, it remains poorly understood and was omitted from the RA core set because of poor measurement properties. A pragmatic, mixed methods approach was used to better understand the patient experience of stiffness in people with RA and to develop and test a new RA stiffness patient reported outcome measure (PROM). It involved a systematic literature review, semi-structured interviews, focus groups, cognitive interviews, the development of appropriate candidate items to characterise stiffness and multivariate analysis of a survey using these items. The systematic literature review found that current stiffness assessment is based on items that capture the duration or severity of morning stiffness. However, items were often poorly defined, highly variable in wording and format, had limited measurement property evidence and had not been developed according to current standards including collaboration with patients. Overall, there was no evidence regarding the most appropriate way to assess stiffness in RA, indicating the need for a new measure developed according to best practice PROM guidelines. Semi-structured interviews with RA patients provided an improved understanding of their experience of stiffness, demonstrated its relevance to patients and enabled the development of a conceptual model. These data also highlighted inconsistencies between current stiffness assessment and the patient perspective of this symptom. Focus groups with RA patients reinforced the stiffness conceptual model in a new sample, using a different method of data collection. They also provided information specifically addressing stiffness assessment from the patient perspective, including a number of concepts for measurement instrument development. These patient-driven concepts and qualitative data were tempered with measurement theory to develop a conceptually sound yet practically appropriate preliminary set of items for a new RA stiffness PROM. Preliminary items were reviewed and modified by RA patients in cognitive interviews. Following refinement, 45 candidate items (39 new items and 6 traditional stiffness items) were taken forward to a postal survey to develop and test the structure of a new RA stiffness PROM. Analysis of the survey responses involved rigorous statistical testing including a series of iterative principal component analyses (undertaken initially with two different approaches), balancing Cronbach’s alpha for internal consistency, bootstrapping for stability, and expert judgement for clinical appropriateness. The emergent structure was the Rheumatoid Arthritis Stiffness (RAST) questionnaire with 21 items in 3-components capturing ‘stiffness severity’, ‘physical impact’ and ‘psychosocial impact’. The initial qualitative work enhanced its content validity and statistical testing for appropriate relationships with other measures of disease demonstrated good construct validity. These results provide support for RAST as an appropriate tool for use in future stiffness assessment. The development of the RAST is important in recognising stiffness as a relevant patient symptom and is a significant step towards standardised stiffness assessment. Further testing in a fresh population will generate additional evidence of reliability and sensitivity to change to support its use. The RAST provides a measure for use in new investigations of disease mechanisms and response to therapy.

Articular cartilage is a highly specialised tissue composed of a mechanically competent matrix and a single cell population - the chondrocytes. The maintaining of a specialised phenotype requires the integration of intracellular signalling, that in response to appropriate extracellular stimuli, results in expression of cell-specific genes. Previous work in our laboratory has identified hypoxia as one such key stimulus, which through HIF-2α, enhances expression of cartilage master regulator SOX9 and its matrix-encoding target genes (COL2A1, AGC and COL9A1). MEF2C transcription factor is known to be involved in muscle and cardiovascular development; however, recently it has been shown to play a key role in chondrocyte hypertrophy co-ordinately with SOX9. In a previous microarray analysis, we found that MEF2C was upregulated during hypoxia-induced re-dedifferentiation of human articular chondrocytes (HACs); interestingly where its suggested genetic target - COL10A1 - was barely detectable. In this research we therefore investigated a possible new and unknown function of MEF2C transcription factor as a potential genetic regulator of the permanent articular chondrocyte phenotype. In this study, MEF2C protein was detected with a nuclear localisation in chondrocytes in situ in intact healthy human articular cartilage. Experiments in isolated HACs revealed that, at both gene and protein levels, hypoxia enhances MEF2C expression in a HIF-2α and SOX9 dependent fashion. Subsequently, depletion experiments of MEF2C indicated that it is required for SOX9 gene expression both in normoxia and hypoxia. Our results, therefore suggest a mutual positive regulation between MEF2C and SOX9 transcription factors in articular cartilage. Thus, based on our studies a new and critical function for transcription factor MEF2C in HACs has been identified, where it helps promote expression of the differentiated chondrocyte phenotype through mutual regulation with SOX9. These findings give important new insights into our understanding of the transcription factor networks that regulate expression of the articular chondrocyte phenotype.

Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. The direct and indirect cost of managing back pain posts heavy socioeconomic burden to the society. Improved technologies and techniques together with well documented experiments will benefit the research field by saving effort in doing the optimization in every laboratory and avoiding experiment failure due to incomplete understanding of the procedures. These can accelerate scientific discovery, reduce the sacrifice of animals and enable a more effective use of funding. Nucleus pulposus (NP) is the central part of IVD. Differences in matrix compositions in human NP clinical samples demand different cell isolation protocols for optimal results but there is no clear guide about this to date. Sub-optimal protocols may result in low cell yield, limited reliability of results or even failure of experiments. We experimented different isolation protocols to study different parameters involved and suggested some rules for cell isolation in three main applications: RNA extraction for phenotyping, cell isolation for cell culture, and characterization by flow cytometry. In addition, instead of extracting RNA from isolated cells, extraction of RNA from tissues directly may avoid the change of RNA levels during the cell isolation process. However, extraction of RNA directly from human and large animal IVD tissue is technically challenging due to its tough nature, low cell-to-matrix ratio and high proteoglycan content. Thus we developed a method for RNA extraction from bovine disc tissues by integrating the use of cryosectioning, additional phase separation and high salt precipitation into conventional guanidinium thiocyanate based method. With this method, RNA could be extracted from the NP tissue directly but the concentration was low. A shift toward 270 nm was observed in its UV spectrum which was due to phenol contamination. This caused an overestimation of RNA concentration. Hence we developed a computational method based on UV spectra for correcting the overestimated concentrations of RNA contaminated with phenol. The accuracy of concentration increased substantially with the use of the correction formula. Mesenchymal stem cells (MSCs) have great potential in IVD engineering and hence we studied the isolation and culturing of MSCs from different sources. Effect of cell shape was reported for MSCs but not for NP cells. Micropatterning can be used to pattern cells into different shapes and arrangements. Therefore, we optimized the preparation of bovine NP cell micropatterns and also investigated the preparation of cell micropatterns for confocal microscopy. Besides, we developed methods to study the gene expression of cells on patterns by RT-qPCR with and without prior selection of cells of interest by laser capture microdissection (LCM). In short, different methods related to IVD research were developed and optimized. With improved methods together with a better understanding of the underlying rationale, researchers can save time and cost in their experiments and reduce experiment failure rate. This will help to accelerate researches. New methods also enable studies which were not feasible in the past.

Approximately 8.5 million people in the UK are affected by osteoarthritis (OA), a multifactorial, polygenic disease characterised by articular cartilage loss. In 2012, the arcOGEN Consortium reported on the largest OA genome-wide association scan (GWAS) to date, in which five regions of the genome were significantly associated with the disease in Europeans. I aimed to characterise two of these regions: rs10492367 is intergenic between PTHLH and KLHL42, while rs9350591 is intergenic between FILIP1 and SENP6. MYO6, TMEM30A, COX7A2 and COL12A1 also surround rs9350591. There are no non-synonymous polymorphisms within either association region that could account for the signals. I first confirmed that the genes were expressed throughout in vitro chondrogenesis and osteoblastogenesis. Using quantitative real-time polymerase chain reaction, I identified the differential expression of PTHLH, KLHL42, SENP6, MYO6, COX7A2 and COL12A1 in articular cartilage stratified by disease state, joint and/or sex. There were no differences between the genotypic groups of either signal, corroborated by pyrosequencing which quantified allelic outputs. Expression quantitative trait loci, irrespective of rs9350591 genotype, acted upon MYO6 and COL12A1. I used data from an Illumina BeadChip array to identify the hypermethylation of cg26466508 in rs9350591 risk allele carriers relative to non-risk allele homozygotes. In luciferase reporter assays, the alleles of polymorphisms in high linkage disequilibrium with rs10492367 displayed differential enhancer activity. Electrophoretic mobility shift assays were used to investigate protein binding to four of these polymorphisms, with RELA, SUB1 and TCF3 binding to rs10492367: chromatin immunoprecipitation confirmed these findings. Finally, I used silencing RNAs to knockdown the transcription factors in human articular chondrocytes, with SUB1 depletion resulting in a downregulation of PTHLH. Overall, I have highlighted the complexity of characterising GWAS signals. The data suggest functional roles for the regions, perhaps by mediating OA susceptibility during joint development rather than in end-stage diseased cartilage.

Porous structures are used in orthopaedics to promote biological fixation between metal implant and host bone. In order to achieve rapid and high volumes of bone ingrowth, the structures must be manufactured from a biocompatible material and possess the required porosities, pore sizes and mechanicfll strength. The research presented within this document describes the development of a unit cell modelling technique for the manufacture of highly porous wireframe metal structures using the Selective Laser Melting (SLM) rapid manufacturing system. SLM uses an ytterbium fibre laser to selective melt 75J.lm layers of metal powder. As each layer is melted it is bonded to the previously manufactured layer, and so parts are effectively 'grown' from the bottom up. Using unit cell modelling techniques and SLM, titanium constructs are manufactured that exhibit fully interconnected porosities, pore sizes in the range of 100-700J.lm, compression strengths in excess of 50MPa and which are 65% porous by volume. A post-manufacture high vacuum sinter process is defined which optimises the mechanical strength and surface topographies of the structures. The structures are shown to promote volumetric bone ingrowth rates of between 20 and 30% in in-vivo rabbit models. SLM is proven capable of manufacturing complex hierarchical structures, which is demonstrated by the direct manufacture of orthopaedic components with surfaces optimised for bone ingrowth and polymer bearing attachment. Finally this thesis describes the progress that has been achieved in developing commercial SLM manufacturing system. The innovative concepts described in this thesis have been submitted as a patent application.

Antineutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) is a multi-systemic disease with autoantibody generation to myeloperoxidase (MPO) and proteinase 3 (PR3), components of neutrophils and monocytes. It is widely believed that ANCA are pathogenic. The most convincing evidence for this comes from the ability to induce crescentic glomerulonephritis in mice with passive transfer of MPO-ANCA. Studies have also shown with limited numbers of ANCA, the autoantibodies can activate neutrophils in vitro. The role of monocytes in pathogenesis though is scarcely explored. The alternative pathway of complement has also been implicated as vital to the pathogenesis of AAV, but the activating factors of complement are unknown. Contrary to previous studies we failed to consistently activate neutrophils with a large panel of randomly selected ANCA. We investigated the effect of these ANCA on peripheral blood monocytes. Interestingly, we found that MPO-ANCA derived from AAV patients cause a reduction in IL6 and IL10 production from lipopolysaccharide treated monocytes, which is Fc dependent and MPO enzyme dependent. Using gene expression microarrays we show that MPO-ANCA cause a widespread reduction in toll-like receptor (TLR) 4 signalling. We show using mass spectrometry analysis that MPO-ANCA leads to the production of a subset of oxidised phospholipids that can inhibit TLR4. We also demonstrate that MPO-ANCA lead to an increase in survival of macrophages differentiated in vitro from monocytes, which is dependent on colony stimulating factor-1. The differentiated macrophages have an M2-like phenotype that induce IL10 and TGFβ production from CD4 T cells, which would have a potential role in fibrosis. Secondly, we address potential triggers for complement activation during disease and show that there is a redundancy between the classical and alternative pathway. C3 deficient mice are protected from disease, but factor B deficient and C4 deficient mice are not. We also demonstrate cross talk between the coagulation and complement cascade. Using bone marrow chimeras, we demonstrate a role for circulating and not bone marrow-derived C5 in disease pathogenesis, which may help refine complement-targeted therapy.

Avascular necrosis (AVN) of the femoral head is a debilitating disease of the bone that may result from a variety of aetiologies, and progresses to the death of the bone and collapse of the femoral head, and ultimately to deformation of the hip joint. In vitro models of AVN have been previously developed in live animals; however none of these models produce the mechanical failures that are observed in AVN femoral heads. The aim of this study was to produce a mechanical simulation model of AVN in vitro. To achieve this, a series of preliminary studies were carried out. First, femoral heads from AVN patients undergoing total hip arthroplasty were studied to understand the effects of the disease on the mechanical and structural properties of the bone. The findings were compared with mechanical and structural properties of bone from nonpathological control femoral heads, and the results demonstrated mean reductions in the mechanical properties of bone with AVN. Methods of reducing mechanical properties of bone were analysed to develop a mechanical simulation model of AVN in vitro. For this, chemical methods were analysed which either demineralised or dissolved the collagen matrix in the bone. The effects of treatment time were analysed on mechanical properties of bone, and it was found that demineralising bone plugs in hydrochloric acid was the most effective method of reducing the mechanical properties of bone. In order to develop a model of AVN, porcine bone sections were treated with hydrochloric acid and returned to the femoral heads to simulate lesions in AVN. The resultant change in the structural mechanical properties of the femoral heads were analysed to determine the most suitable method of simulating AVN in porcine bone, and to provide recommendations for achieving a mechanical bone model representing the structural and mechanical properties of AVN femoral heads.

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