| Summary: | 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.
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