The bone tissue engineering community has been striving to develop novel approaches that mimic natural bone formation. The rapid generation of mineralised bone tissue with a capacity for vascularisation and the selection of highly osteogenic cell sources are still the focus of research today. This study addresses three novel approaches in these key areas. Mineralisation in bone tissue involves stepwise cell – cell and cell – extracellular matrix (ECM) interactions. Regulation of the osteoblast culture microenvironment can manipulate osteoblast proliferation and mineralisation rates and consequently the quality and/or quantity of the final calcified tissue. Therefore, an in vitro model to investigate possible influential factors would be highly sought after. We developed a facile in vitro model through the modification of culture surfaces in which an osteoblast cell line and aggregate culture was used to mimic intramembranous ossification. Conventional monolayer culturing was used as a comparative control. The effects of multiple culture parameters, including culture duration and aggregate size, on mineralisation rates and subsequent mineral quantities and distributions have been examined by numerous well established methods alongside certain innovative techniques. Ultimately, spatial and temporal production of minerals differed depending upon aggregate size with larger aggregates mineralising faster with a distinct gene expression pattern compared to the smaller aggregates. We also demonstrated that mineralisation in the larger aggregates initiated from the periphery, whilst mineralisation in the smaller aggregates initiated from the centre. This implies that aggregate size influences mineral distribution and development over time. An in vivo study using a cell line and primary cell population was conducted to investigate how the observations noted during the short term in vitro studies would affect long term in vivo aggregate survival and bone formation. Both cell types saw similar results. The large aggregates appeared to disintegrate over the course of the experiment, whilst the small aggregates remained intact and produced an abundant volume of extracellular material. A monolayer cell sample was again used as a comparative control and generated a lower material volume over the same period. The data obtained from this element of the project produced some invaluable insights into how the specific variables of cellular aggregation might affect possible bone formation in vivo. In addition, a novel substrate, substrate X, was used to identify and investigate the possibility of mesenchymal stem cell (MSC) subpopulations within mixed MSC populations and their donor-dependent variations alongside their subsequent influences upon an individual’s osteogenic capacity. Substrate X successfully identified what are thought to be three subpopulations within individual MSC populations from multiple donors through distinct cellular attachments. Each of the subpopulations was shown to hold differing osteogenic capacities and their proportions were also shown to be donor-dependent. Subpopulation proportions were shown to correlate with specific cadherin levels and cellular aggregation potential was also shown to be donor-dependent. Furthermore, the novel aggregation technique developed by this study was pitted against a conventional aggregation technique to assess aggregate vascularisation and mineralisation simultaneously using cellular co-culturing. This study also investigated how mechanical stimulation would affect aggregate vascularisation and mineralisation. The method of aggregation developed earlier in this project was shown to create an inner-aggregate architecture that aided in specific cellular organisation and possible vascularisation more than the conventional aggregation technique. The mechanical stimulation reduced cellular migration from the aggregate body compared to a static culture equivalent but nodule mineralisation within the co-cultured aggregates was inconclusive due to the short culture period. To conclude, simple yet effective substrate chemistry modifications enabled us to evaluate a variety of parameters for refined bone tissue engineering. These included the development of an aggregate model for the study of developing mineralisation, possible MSC subpopulation identification, measurement and assessment and the evaluation of aggregate vascularisation.

Musculoskeletal conditions of the hand are frequent causes of pain and disability in older people, yet knowledge regarding the characteristics and patterns of hand pain and problems over time is lacking. The objectives of this project were to identify sub-groups of older individuals with distinct presentations (phenotypes) of hand pain and function, investigate how these phenotypes changed over a 6 year period, and explore what characteristics and factors were associated with long-term outcomes. In addition to this, an exploration of the longitudinal association between hand phenotypes and mental health was performed. The study population stemmed from the North Staffordshire Osteoarthritis Project (NorStOP); a large, general population-based, prospective cohort study of adults aged 50 years and over. Information on hand pain and problems was collected using questionnaires at baseline, 3 and 6 years. A total of 5,617 participants responded at all time points and were included in the analysis. Five phenotypes were identified using Latent Transition Analysis (‘least affected’, ‘high pain’, ‘poor gross function’, ‘high pain and poor gross function’ and ‘severely affected’) based on eight hand pain and function items. The most common transition between phenotypes was from ‘high pain’ at baseline to ‘least affected’ at 3 years. Individuals classified in the ‘least affected’ or ‘severely affected’ groups at baseline were the most stable. Individuals with nodes, chronic hand pain, sleep problems and bilateral hand pain at baseline were more likely to be in a more severe hand phenotype at 6 years. In addition to this, those in more severe hand phenotypes were more likely to have poor symptoms of mental health. The results provide clinically relevant information regarding the pattern of hand pain and problems over time, and the characteristics of those more likely to have an unfavourable outcome over a 6 year period.

The Wnt signalling pathways play crucial roles in development, tissue patterning, and stem cell fate determination. These pathways are therefore an attractive therapeutic target in the field of regenerative medicine and tissue engineering. Magnetic nanoparticles (MNP) are useful tools in bio-engineering. Previous work from our group has demonstrated the efficacy of targeting and activating cell signalling pathways using MNP functionalised with targeting proteins coupled with magnetic fields to remotely torque the MNP. Using this approach, in this work MNP were functionalised with ligands targeted to cell surface Frizzled receptors which are involved in Wnt signal transduction. The effects of remote stimulation with MNP on Wnt pathway activity were then assessed in human mesenchymal stem cells (hMSC). Results demonstrated that targeting of Frizzled receptors with MNP and remote stimulation using magnetic fields remotely activated Wnt signalling pathways. This was indicated by nuclear mobilisation of β-catenin and activation of a TCF/LEF luciferase reporter. The effect of remote Wnt pathway activation on hMSC osteogenesis was subsequently assessed. Activation was shown to augment hMSC differentiation in monolayer experiments where expression of osteogenic markers increased. This strategy also had beneficial effects on bone formation in an ex vivo foetal chick femur model as indicated by μCT and histology. The role of spatial Wnt gradients on bone formation is also important in development and was investigated using a tissue engineering platform utilising immobilised Wnt protein. In conclusion, these studies demonstrate the use of MNP to remotely activate Wnt signalling pathways and have shown potential in directing hMSC differentiation. This provides proof of concept for new injectable therapies that modulate cell signalling pathways with applications in regenerative medicine.

Low back-related leg pain (LBLP) is clinically diagnosed as referred leg pain or sciatica. The clinical task of differentiating sciatica from referred leg pain can be challenging but is important for the purpose of treatment choices. There is currently no agreement on which clinical criteria best identify sciatica in clinical or research settings and the spectrum of clinical presentation in patients with LBLP is variable. This thesis aimed to identify diagnostic criteria for sciatica and explore and describe clusters of LBLP patients using cross-sectional data from 609 primary care LBLP consulters. A systematic literature search of LBLP classification systems showed very few systems specifically addressed LBLP classification. Within the systems, there was wide variation in definitions and clinical features of sciatica, with most systems based on clinical opinion. Reliability was merely fair (kappa = 0.35) amongst clinicians diagnosing sciatica but at higher levels of confidence in diagnosis (≥80%), reliability improved (kappa =0.68). Using high confidence clinical diagnosis as a reference standard, with and without confirmatory MRI findings, diagnostic models for sciatica were developed and compared. A simple scoring tool based on the best performing model was devised showing the probability of having sciatica based on results from five clinical items (subjective sensory changes, below knee pain, leg pain worse than back pain, positive neural tension, neurological deficit). Latent class analysis identified five classes of LBLP patients. One class was clearly a referred leg pain group, the other four classes seemed to represent sciatica with varying clinical profiles. This thesis provides a diagnostic tool for sciatica with potential application in clinical and research settings. It also reveals clusters of LBLP patients which could represent more homogenous groups amenable to different treatment approaches. This thesis has provided a strong basis for future work to further explore the clinical utility of the findings.

Physical activity (PA) is recommended for all adults with osteoarthritis (OA). Adults aged 45 years and over with joint pain, are likely to have already developed OA or are at risk of OA. This thesis examines self-reported PA levels in community dwelling adults aged 45 years and over with joint pain and how PA can be best measured within this population. Self-reported PA levels of 14,212 adults (aged 45 years and over) with and without self-report joint pain demonstrated that adults aged 45 years and over with joint pain are less likely to be active compared to adults with no joint pain (OR= 0.75, 0.68-0.77 95%CI). A systematic review appraised the measurement properties of twenty self-report PA instruments previously used in adult aged 45 years and over and OA or joint pain populations. The International Physical Activity Questionnaire short form (IPAQ-SF) and the Physical Activity Scale for the Elderly (PASE) appeared to be most suitable self-reported PA instruments in joint pain and OA research. An analysis of measurement properties of the IPAQ-SF and PASE was conducted in 525 adults aged 45 years and over consulting primary care with joint pain. Reliability of the IPAQ-SF was lower (r=0.58, p= < 0.01) compared to the PASE (ICC=0.69, 95%CI= 0.61-0.76, p= < 0.001). Measurement error was large in the IPAQ-SF (-3942 to 4509 metabolic equivalents (METS)-1minute-1week 95% limit of agreement) and the PASE (-130.28 to 112.76 95% limit of agreement). In terms of construct validity, the IPAQ-SF and PASE correlated well with each other (r=0.62, p= < 0.01) and the SF-12 physical component score (PCS), (r=0.30, p= < 0.01 & r=0.39, p= < 0.01 respectively). The implications of this thesis are that adults aged 45 years and over with joint pain are at higher risk of being inactive and that both the IPAQ-SF and PASE are poor in their measurement properties for measuring of PA in this population.

Osteoarthritis (OA) is a debilitating joint disease characterised by pain and progressive destruction of elements such as articular cartilage. Autologous chondrocyte implantation is a cellular therapy developed to regenerate damaged cartilage and delay or negate the need for a total joint replacement. The use of mesenchymal stem cells (MSCs) provides an alternative to harvesting healthy cartilage in order to obtain chondrocytes for transplantation. In this thesis, the chondrogenic and immunomodulatory properties of human bone marrow (BM-MSCs), infrapatellar fat pad (FP-MSCs), subcutaneous fat (SCF-MSCs) and synovial fluid (SF-MSCs) derived mesenchymal stem/stomal cells were characterised to determine their suitability for cartilage repair. Synovial inflammation and the prevalence of macrophage subsets were determined in the synovium and infrapatellar fat pad from patients with and without OA. FP-MSCs and SF-MSCs from the same donor differed with regards to in vitro proliferation and their response to a proinflammatory stimulus. None of the MSC populations examined displayed levels of chondrogenic potency that were akin to their matched chondrocyte counterparts, although BM-MSCs and FP-MSCs did show a more enhanced ability to undergo chondrogenesis than SCF-MSCs and SF-MSCs. Additionally, the expression of certain surface markers commonly associated with chondrogenic potency in chondrocytes, such as CD44, were not indicative of the chondrogenic propensity of MSCs. Analyses of the phenotype of macrophages in human synovium and FP showed a co-existence of pro- (M1) and anti-inflammatory (M2) cells in both tissues. However, cell surface markers employed in the study did not permit a clear distinction between M1 and M2 cells. Image analyses revealed that the obesity related hypertrophy observed in adipocytes from subcutaneous fat, does not occur in adipocytes in the FP. To conclude, the results presented in this thesis adds to current knowledge of joint derived stem cells and immune cells.

No description available.

Fibrosis can be charactorised as abberent wound healing resulting from an increased presence of α-smooth muscle actin (αSMA)-rich, myofibroblasts and a continued influx of immune cell mediators. The pro-fibrotic and pro-inflammatory cytokines TGF-β1 and IL-1β, respectivley, have been implicated in fibrotic progression by activating hyaluronan (HA)/CD44-mediated pathways. CD44, the principal HA receptor, exists as multiple spliced variants which mediate multiple celluar functions through their association with HA. The aim of this Thesis was to investigate the expression and interactions of CD44 variants asociated with fibroblast activation induced by TGF-β1 or IL-1β. Multiple forms of CD44 spliced variants were identified in fibroblasts. Stimulation with TGF-β1 decreased the expression of all variants, whereas IL-1β-increased global CD44 expression. CD44s was the variant identified as essential for both TGF-β1 induction of myofibroblasts and IL-1β-induced monocyte binding to fibroblasts. CD147 is a matrix metaloproteinase (MMP) inducer that mediates receptor interactions within the plasma mebrane; and contributes to ECM re-arrangment in response to various stimuli. CD147-medaited-αSMA incorporation into F-actin stress fibres that were essential for the myofibroblast contractile phenotype. It associated with CD44s and the EDA-Fibronectin-associated integrin, α4β7, suggesting that through receptor interaction it mediated the mechanotransduction properties required for differentiation. Decreased expression of CD147 prevented intracellular activativation of ERK1/2, an essential kinase involved in mechanotransdction. These data suggest that CD44s regulates both a fibrotic and inflammatory response by fibroblasts through two separate mechanistic pathways. It also implicates CD44s in mechanotransduction, via its association with CD147. In conclusion, both CD44s and CD147 are essential mediators of fibrosis and further research into downstream mediators could lead to potential therapeutic targets to combat fibrotic progression.

Colorectal carcinoma (CRC) is the second most common cause of cancer deaths in the UK. More than half of patients are diagnosed at a late stage with around a quarter of patients having metastases at diagnosis (stage IV). Approximately 50% of diagnosed patients will progress to metastatic disease. The metastatic spread of malignant cells to distant sites in the body accounts for the majority of cancer-related death and significantly decreases patient survival. Whilst cell migration is a physiologically important process, when uncontrolled, it can be a contributing factor to the metastatic phenotype. Actin polymerisation enables the dynamic restructuring of the cytoskeleton which is fundamental to cell migration and is stimulated by the Arp (actin-related protein) 2/3 protein complex which in turn is activated by members of the WAVE (WASP Verprolin homologous protein) family. Clinico-pathological data was updated for a cohort of patients that had been involved in a previous colorectal tissue/carcinoma sampling study. The stored frozen tissue samples were analysed using histological and molecular biology techniques including conventional polymerase chain reaction, quantitative polymerase chain reaction, immunohistochemistry and in vitro gene knockdown studies. WAVE 1 and 3 expression was targeted separately in the RKO and CaCo2 cell lines utilising ribozyme transgene transfection to assess the effect knockdown on cell functions. A high WAVE 2 expression level is associated with more aggressive and higher stage primary tumours and also a shorter overall survival time and disease free survival time. WAVE 3 expression is higher in colorectal tissues compared to normal tissues but otherwise showed no significant difference. WAVE 1 did not show an increase in expression levels compared to normal colorectal tissues. The In vitro functional assays revealed a significant reduction in cell invasion and motility following WAVE 3 knockdown in CaCo2 cells. Knockdown of WAVE 1 in RKO cells resulted in a significant reduction in invasion and a moderate reduction in motility that was not significant. These results suggest WAVE1 and 3 proteins are involved in several metastatic traits and that WAVE 2 has significant correlation with higher stage disease. The data outlined here provides justification to further explore WAVE1, 2 and 3 as potential contributors of colorectal cancer progression.

CACNA1C codes for the alpha-1 subunit of Cav1.2 L-type voltage gated calcium channels (LVGCCs). Variation in CACNA1C has been reliably implicated in psychiatric illness, including schizophrenia and bipolar disorder. Analyses have indicated a convergence of genetic risk for schizophrenia on abnormalities in the synapse and in behaviours including associative learning. LVGCCs play a role in synaptic plasticity and learning, partly through regulation of gene transcription. Their role in specific aspects of associative learning that are relevant for symptoms of psychiatric illness is yet to be fully elucidated. A hippocampal-dependent fear conditioning paradigm was used to determine the role of Cacna1c and LVGCCs in specific aspects of associative learning in rats. Studies measured the activity-regulated expression of Cacna1c and the effect of inhibition of LVGCCs. A genetic Cacna1c knockdown rat model was used to investigate the effects of reduced expression on behaviour and the expression of brain derived neurotrophic factor (BDNF). This model was additionally tested on reward-based reversal-learning. Analyses were translated to humans, to assess whether disease relevant variation in CACNA1C was associated with similar deficits in reversal learning and expression. Inhibition of LVGCCs affected the consolidation, extinction and latent inhibition of contextual fear memory, whereas reduced expression of Cacan1c had a selective effect on latent inhibition. There were no effects on the acquisition of reward associations, but reversal learning was impaired. Similar deficits in reversal learning were associated with disease relevant variation in CACNA1C in humans. Reduced CACNA1C expression was found to be associated with changes in the expression of BDNF in both rats and humans. Results indicate a role for Cacna1c and LVGCCs in the appropriate formation and update of aversive and reward associations. Impairments in these processes can underlie specific symptoms of disease including emotion dysregulation and delusions. The cross-species effects on BDNF require further investigation.