INTRODUCTION: Damage to the musculoskeletal system through disease, injury, or ageing can have long-lasting, and detrimental effects on one’s overall well-being. By understanding the processes by which the different tissues of the musculoskeletal system function and communicate, we can apply it to a variety of medical interventions that will benefit the patient population. These include reducing the prevalence of injury-inducing ectopic bone formation in muscle and slowing the degeneration of muscle and bone tissue associated with aging. A major focus is the relationship between muscle and bone tissue, specifically the stem cell populations found in each tissue type. Two genes that are thought to mark stem cell populations associated with muscle and bone tissue are Pax7 and Prx1, respectively. OBJECTIVES: Establish the capability and define optimal conditions to culture primary stem cells isolated from the muscle tissue of the reporter animals that fluorescently tag the Pax7 and Prx1 cell populations. Manipulate culture medium conditions to characterize the differentiation potential for multiple lineages, osteogenic, adipogenic, and myogenic. Lastly, assess whether there is more adipogenic cell differentiation in older animal cell cultures. METHODS: The tamoxifen inducible Pax7^tm1(cre/ER2)Gaka/J and Prx1^CreER-GFP were both crossed with B6.Cg-Gt(ROSA)26sor<tm14(CAG-tdTomato)Hze>/J to create the tamoxifen inducible Pax7/Ai14 and Prx1/Ai14 reporter mice. These animals were then crossed to the B6,129S7-Rag1^tm1Mom/J , creating the Pax7/Ai14/Rag and Prx1/Ai14/Rag reporter mice strains. This transgenic mouse model made it possible to fluorescently identify the Pax7 and Prx1 population of cells isolated from the muscle tissue and characterize the differentiation potential to different cell lineages in vitro. Cells were harvested from both male and female mice that received two tamoxifen injections. Cells were then cultured in various culture media conditions. Determination of specific cell densities, culture conditions, and differentiation time points were determined by manipulating these variables, and assessing the levels of proliferation and differentiation. Multiple assays were run in order to quantify and identify the different cell lineages that were grown in culture under different culture conditions. RESULTS: Cells grown on gelatin coated dishes at densities of 2.2x10^4 to 2.2x10^5 showed optimal performance in proliferation and differentiation. Cells grown in Growth Media containing Chick Embryo Extract (GM) and without (GM-) produced myogenic and adipogenic cell types that were positive for Prx1 expression. Prx1 positive cells grown in the Osteo-Inductive Media (GMOI) produced osteogenic cell types validated through tetracycline uptake. Pax7 expression was low in all culture media conditions. Finally, adipogenic cells were present in both younger and older animals. The adipogenic cells for both populations showed positive Prx1 expression. Younger animals showed a larger relative expression of Plin1 in qRT-PCR analysis. CONCLUSIONS: Although Prx1 is thought to be associated with bone tissue, Prx1 positive cells are located within the muscle and able to be cultured. This muscle derived Prx1 cell population is able to differentiate toward the myogenic, adipogenic, and osteogenic cell lineages. By altering the specific components of culture conditions such as extracellular matrices, seeding density, and media constituents, it is possible to force a particular lineage differentiation for Pax7 and Prx1 muscle derived stem cells. Further studies are needed to elucidate the differentiation potential of Prx1 cells isolated from animals of various ages. Additional in vivo studies are needed to understand the mechanisms surrounding the Prx1 and Pax7 population of cells with their roles in healing and regeneration in response to degeneration and trauma.

Intimate partner violence is also known as domestic violence or spouse abuse. It affects the physical, psychological, social, and financial well-being of many people around the world. Many researchers from health/medical, social, and psychological fields have studied intimate partner violence in an effort to prevent it or to improve overall health and well-being among victims. Ideally, decisions are best influenced by high quality evidence. However, little attention has focused on the quality of this research. This thesis focuses on the theme of transparency relating to study quality, specifically highlighting non-publication bias, biases related to outcome and study methodologies, and overall reporting quality in previously published IPV research. These lessons learned from this research have informed, in part, an original study on intimate partner violence. Finally, this thesis concludes with insights to improve methodological quality and transparency for researchers in the intimate partner violence field. / Thesis / Doctor of Philosophy (PhD)

Ewing sarcoma, an aggressive primary bone and soft tissue tumour is characterised by the expression of the chimeric transcription factor EWS-FLI1 in 90% of patients. This alters expression of many genes including activation of the Insulin Growth Factor (IGF) pathway via IGFBP3 supression. Phase I/II trials with an IGF-1 inhibitor have demonstrated tumour regression in a modest number of Ewing sarcoma patients. The aim of this thesis was to identify mechanisms contributing to the heterogeneity of resistance in Ewing sarcoma following inhibition with OSI-906, a dual kinase inhibitor of IGF-1 (IGF-1R) and Insulin (IR) receptors. The hypothesis was that mechanisms of resistance relate to heterogeneity of responses to signalling pathway activation and inhibition. Through selection, disruption of the pathway would identify subpopulations of cells both sensitive and resistant in their response allowing for interrogation of resistance mechanisms. A genome wide approach was taken to model the resistance profile of cell lines. Through developing a method of unbiased quantification, a panel of validated Ewing sarcoma cell lines (EuroBoNet) were imaged and segmented to assess the responses of biomarkers on signalling pathway activation. Heterogeneity was confirmed between cell lines. The application to diagnostic biopsies led to the identification of prognostic classifiers and cellular subpopulations with clinical prognostic significance. The distribution of Ki67 was found to be predictive of survival and cells with lower levels of CD99 in the cytoplasm were most discriminative. Parallel sequencing strategies (RNA-seq, whole exome sequencing, and aCGH/ SNP array) for genome-wide screening was carried out for point mutations, copy number changes and rearrangements. Systematic detection was used to characterise genomic rearrangements and functional validation performed. Resistant clones, formed via ENU mutagenesis of cell lines, were sequenced in order to demonstrate the resistance profile of OSI-906. In summary heterogeneity of Ewing sarcoma at the genomic and proteomic level can influence the signalling dependency of tumours and response to inhibitors. Genomic and proteomic profiling of tumour cells may be relevant to future developments of novel therapies.

<strong>Osteoarthritis</strong>, the most prevalent joint disease in the United Kingdom, is a progressive condition that results in end-stage full-thickness cartilage loss and has important social and economic impacts on society. Since cartilage lacks regenerative capabilities, it is essential to develop approaches to initiate and enhance cartilage regeneration. In this context, tissue engineering is emerging as an attractive approach for the regeneration of cartilage tissue damaged due to disease or trauma. A scaffold-free cartilage construct, analogous to those found during embryonic precartilage condensation, has received much attention as an alternative novel modality for cartilage <strong>tissue engineering</strong>. Cartilage repair with <strong>scaffold-free</strong> tissue more closely resembles the natural situation and mimics the features of the original tissue. Moreover, scaffold-free cartilage implants can overcome the complications caused by the use of suboptimal scaffolds by avoiding the need for a foreign scaffold at all. Culturing cells into tissue patches without the requirement for a scaffold can be achieved through <strong>cell sheet engineering</strong>, which uses thermo-responsive culture dishes. However, the high costs of the tissue culture consumables, and the relatively low cellular yield, makes this process less attractive. This thesis presents a novel method for generating shape-, size- and thickness-adjustable 3-dimensional scaffold-free cell pellet sheets for use as implantable biological cell patches for cartilage tissue engineering. This new technique of bioengineering scaffold-free cell pellet sheets proves to be reproducible, easily applicable, sizable and thickness adjustable. <strong>Stem cells</strong> have added a new thrust to tissue engineering. Their distinctive self-renewal and plasticity have not only optimized many tissue engineering developments, but also rendered feasible some applications which would otherwise be unattainable with somatic cells. Human mesenchymal stem cells (HMSCs) were used to examine the optimal condition for generating cell pellet sheets with this new method. Furthermore, the resultant differentiated pellet sheets were compared directly with HMSCs, human chondrocytes and human fibroblasts alone to evaluate the feasibility of using this cell pellet sheet for clinical applications in terms of their biological and mechanical properties. The results of this thesis suggest that the engineered scaffold-free, chondrogenic, differentiated MSC pellet sheet not only exhibits desirable biologic features similar to chondrocytes, but also demonstrates good integrative and viscoelastic potential that might offer exciting possibilities for the development of novel biologically-based clinical therapies. In summary the data presented herein indicate the following points: <table><ul style="list-style-type:square"><li>The differentiation of human MSCs into chondrogenic cells was achieved.</li> <li>A novel approach of centrifugal seeding on a PDMS surface was shown to effectively generate chondrogenic-differentiated cell pellet sheets without impairing the biological functions of chondrocytes.</li> <li>Various cell types such as human MSCs, human chondrocytes and human fibroblasts were found to respond well to the novel methodology and generated viable, cohesive, less shrinkable, and readily-detachable cell pellet sheets, the size and thickness of which could be adapted as required. The results obtained were superior to those obtained using the conventional thermo-responsive culture dish method.</li></table> This new methodology developed in this thesis provides an approach to in vitro cell pellet sheet generation which is closer to the physiological process of cartilage development and which proved valuable for the study of in vitro generation of scaffold-free cell patches as an important adjunct to many traditional cartilage restorative procedures. Future research on in vivo assessment of the cell sheet and the functional role of these sheets in repairing damaged cartilage is recommended.

Rotator cuff tendinopathy accounts for the majority of cases of shoulder pain, however, the aetiology and pathophysiology of the condition is poorly understood and treatment interventions for the condition have been introduced on an empirical basis, guided only by unproven theories of biological and structural changes in and around the affected tissue. This thesis explores changes in the provision of surgical interventions for rotator cuff tendinopathy, showing a rapid, sustained increase over the last decade. Investigation into the use of ultrasound as a clinical and research tool led to the development of an independent learning method for surgeons using the modality for shoulder imaging and highlighted the technological limitations of contrast enhanced ultrasound in assessing the microvascularity of the supraspinatus tendon. Development of a novel biopsy method for sampling the supraspinatus tendon permitted collection of tissue samples from several cohorts of individuals with rotator cuff tendinopathy. These studies, for the first time, described tissue changes across the whole spectrum of pathology suggesting the possibility of an early inflammatory phase of the condition followed by tissue senescence and reduced viability as pathology progressed. Paired samples taken before and after treatment identified reduced tissue activity in response to glucocorticoid therapy and a potential healing response from the supraspinatus tendon following surgical repair. Significant deterioration in tissue activity and viability was shown as age increased in a large cohort of individuals, highlighting the major impact of ageing as a factor in the onset and progression of rotator cuff tendinopathy. The techniques introduced provide standardised, reproducible methods for further evaluation of rotator cuff tendinopathy and the development of novel therapeutic interventions.

Metal-on-metal hip resurfacing arthroplasty (MoMHRA) has undergone a recent resurgence as an alternative treatment option for young and active patients with significant hip osteoarthritis. Despite the satisfactory short-term implant survivorship, abnormal periprosthetic soft-tissue masses relating to the hip joint (pseudotumours) are being increasingly reported. These were found to be locally destructive, requiring revision surgery in a high proportion (75%) of patients, the outcome of which is poor. However, there is currently no definitive evidence of potential mechanisms involved in the occurrence of such destructive soft tissue masses. The broad aims of this thesis were: 1) to determine the extent of this emerging clinical complication (pseudotumour) with a high revision burden; and 2) to determine whether pseudotumour results from a local biological reaction to an increased wear debris burden generated by excessive MoMHRA implant wear. A clinical study investigated the prevalence of, and association between, pseudotumours and serum metal ion levels in 158 MoMHRA patients (201 hips). The overall prevalence of pseudotumour was found to be 4%, considerably higher than previously reported. The study also established that pseudotumour only occurred with elevated metal ion levels, suggesting the underlying cause is increased wear. It was postulated that pseudotumours are likely to be a biological reaction to the large amount of metal debris generated in vivo due to excessive wear. Two potential biological reactions mediating the occurrence of pseudotumours were then investigated. An in vitro clinical study demonstrated that systemic hypersensitivity type IV reaction, mediated by lymphocyte reactivity to these metals, is not the dominant biological response involved in pseudotumours. A further in vitro experiment demonstrated that metal particle-induced cytotoxicity is likely to be an important factor leading to pseudotumours. Subsequent research focused, firstly, on assessing the magnitude of bearing surface wear that occurs in pseudotumour patients, and secondly on elucidating the potential wear mechanism responsible for the increased implant wear in these patients. A retrieval study demonstrated that MoMHRA implants revised due to pseudotumour were associated with significantly greater linear wear rates. This combined with the metal ion data confirmed that pseudotumour is associated with increased wear at the MoM articulation. An edge-loading wear pattern was always found in the pseudotumour group. An in vivo study was then developed to investigate whether the edge-loading occurs during functional activities. Edge-loading in the pseudotumour group was found to occur with up to 4-fold increase in duration and up to 7-fold increase in force. This in vivo finding supported the in vitro retrieval study finding of an edge-loading wear pattern in the pseudotumour group, suggesting that edge-loading during functional activities is an important in vivo mechanism responsible for localised high wear and subsequent elevation of metal ion levels in MoMHRA patients with pseudotumours. It is concluded that pseudotumour is likely to result from a local biological reaction to increased metal debris load, generated by excessive MoMHRA implant wear due to edge-loading. In susceptible patients, the dose-dependent cytotoxicity of wear debris leads to subsequent necrosis of periprosthetic soft tissues. Clinicians need to be aware of pseudotumours and surgeons should undertake MoMHRA with great care in the knowledge that even with satisfactory component positioning, the problem can occur. In evaluating MoMHRA patients with unexplained symptoms with normal plain radiographs, further investigation with soft-tissue imaging modalities such as ultrasound or MRI is recommended.

Degenerative conditions of the spine are a major public health problem, leading to severe back pain, reduced quality of life and chronic disablement in a proportion of sufferers. For some of these patients, spinal fusion surgery is a treatment that can alleviate back pain and restore normal function. However, limitations in the availability of graft material mean that alternative grafts are needed and tissue-engineering approaches have been employed. Using a novel self-organising collagen scaffold combined with nano-hydroxyapatite and chondroitin sulphate and by employing the latest materials techniques, I have studied the osteogenic capability of a biomimetic graft for use in spinal fusion surgery. The mineralised collagen scaffold has compressive strength comparable to human cancellous bone and can support the proliferation of viable human mesenchymal stem cells. This porous scaffold can be combined with human mesenchymal stem cells to further promote bone growth, as evidenced by an upregulation in the levels of bone-forming genes and mineralisation of the scaffold. This scaffold can act as a carrier system for BMP-2, with wider application for other growth factors or drugs, providing sustained release when fabricated as a layer-by-layer scaffold. An alternative bone substitute for use in spinal surgery has been designed and characterised, with exciting potential for use in vivo.

Severe ankle injuries can result in ligament rupture or a fracture. A major problem after such injuries is limitation in mobility. Weight bearing tasks, such as walking, become a problem because of pain, deficits in joint range of motion and muscle strength. This thesis studies a key dilemma in early rehabilitation, whether to immobilise the ankle or allow joint motion to improve mobility outcomes. Studies have focused on two scenarios, severe ligament rupture, and unstable fractures managed through open reduction and internal fixation (ORIF). The analysis of gait outcomes was an important component of this thesis and a novel analytical method was developed to normalise gait velocity in the estimation of speed-dependent gait outcomes. A systematic review and meta-analysis was conducted including evidence to July 2014. The reporting and design of trials was universally poor. In the 6 weeks of recovery following ankle ORIF surgery, there was insufficient evidence that early ankle movements offered a benefit to mobility recovery compared with immobilisation in a cast. Ankle movements compared with immobilisation reduced the risk of venous thrombosis/thromboembolism. However, compared with cast immobilisation, the risk of deep and superficial surgical site infection and fixation-related complications were higher when ankle movements were permitted. To investigate the role of ankle supports in rehabilitation of walking after ORIF, two randomised cross-over studies were completed. In healthy participants with non-pathological gait, a walker boot induced gait abnormalities when compared with Tubigrip (elasticated bandage). There were no important differences in gait between a stirrup brace and Tubigrip. In people who had undergone ankle ORIF 6 weeks previously, a walker boot and to a lesser extent a stirrup brace offered improvements in gait symmetry and lower pain scores when compared with Tubigrip. Finally, a secondary analysis of the Collaborative Ankle Support Trial cohort (n=584) was conducted, which concluded that, in comparison to Tubigrip, 10 days of cast immobilisation provided greater probability of recovery of a range of mobility outcomes 4 weeks following injury. This thesis contributes evidence favouring a role for ankle immobilisation in improving mobility following severe ankle injury in adults. Clinicians should be aware of the benefits and risk of harms outlined, as well as the limitations in the current evidence base.

Objectives: Orthopaedic and dental implants are prone to frequent infections. This can lead to detrimental and often irreversible outcomes for many patients. The objective of this study was to develop a novel system using zinc oxide nanoparticles (nZnO) as a coating material that inhibits both bacterial adhesion / growth and promotes osteoblast growth. Methods and Results: Initially bacteria (S. aureus, E. coli, S. epidermidis and P. aeruginosa) were exposed to different concentrations of zinc oxide nanoparticulate suspensions (250 μg/mL, 500 μg/mL, 1000 μg/mL and 2500 μg/mL); with the higher concentrations of the suspensions demonstrating significant bactericidal effects. A novel electrohydrodynamic atomization coating technique (EHDA) was used to deposit mixtures of nZnO and nano-hydroxyapatite (nHA) onto the surface of glass samples (1 cm2). Exposure of the coated samples to phosphate buffered saline (PBS) and adult bovine serum (ABS) and measurement of bactericidal activity demonstrated superior antimicrobial activity for 100% and 75% nZnO composite coated samples. Lactate dehydrogenase (LDH) release from osteoblast-like cells (UMR-106 and MG-63) exposed to both nano-TiO2 and nano-ZnO nanoparticulate suspension supernatants indicated minimal toxicity. Nano-ZnO coated samples did not elicit LDH release with an increase in proliferation and viability of cells was observed. Scanning electron microscopy (SEM) and optical microscopy indicated that all cell types used (mesenchymal stem cells and osteoblast-like cells) were able to maintain their normal morphological state when adhered to the surface of the nano-coated material. Further studies as regards to patterned coated samples showed an exclusive adhesion selection by osteoblast-like cells to nZnO patterned regions that needs to be further investigated. Conclusion: ZnO NPs provide an antimicrobial and biocompatible coating material for medical and dental bone implants.

Until recently, the model for training new orthopaedic surgeons was referred to as “see one, do one, teach one”. Resident surgeons acquired their surgical skills by observing attending surgeons in the operating room and then attempted to replicate what they had observed on new patients, under the supervision of more experienced surgeons. Learning in the operating is an unideal environment to learn because it adds more time to surgical procedures and puts patients at an increased risk of having surgical errors occur during the procedure. Programs are slowly beginning to switch to a model that involves simulation-based training outside of the operating room. Wire navigation is one key skill in orthopaedics that has traditionally been difficult for programs to train on in a simulated environment. Our group has developed a radiation free wire navigation simulator to help train residents on this key skill. For simulation training to be fully adopted by the orthopaedic community, strong evidence that it is beneficial to a surgeon’s performance must first be established. The aim of this work is to examine how simulation training with the wire navigation simulator can be used to improve a resident’s wire navigation performance. The work also examines the metrics used to evaluate a resident’s performance in a simulated environment and in the operating room to understand which metrics best capture wire navigation performance. In the first study presented, simulation training is used to improve first year resident wire navigation performance in a mock operating room. The results of this study show that depending on how the training was implemented, residents were able to significantly reduce their tip-apex distance in comparison with a group that had received a simple didactic training. The study also showed that performance on the simulator was correlated with performance in this operating room. This study helps establish the transfer validity of the simulator, a key component in validating a simulation model. The second study presents a model for using the simulator as a platform on which a variety of wire navigation procedures could be developed. In this study, the simulator platform, originally intended for hip wire navigation, was extended and modified to train residents in placing a wire across the iliosacral joint. A pilot study was performed with six residents from the University of Iowa to show that this platform could be used for training the other applications and that it was accepted by the residents. The third study examined wire navigation performance in the operating room. In this study, a new metric of performance was developed that measures decision making errors made during a wire navigation procedure. This new metric was combined with the other metrics of wire navigation performance (tip-apex distance) into a composite score. The composite score was found to have a strong correlation (R squared = 0.79) with surgical experience. In the final study, the wire navigation simulator was taken to a national fracture course to collect data on a large sample of resident performance. Three groups were created in this study, a baseline group, a group that received training on the simulator, and a third group that observed the simulator training. The results of this study showed that the training could improve the overall score of the residents compared to the baseline group. The overall distribution from resident performance between groups also shows that a large portion of residents that did not receive training came in below what might be considered as competent performance. Further studies will evaluate how this training impacts performance in the operating room.