| Summary: | Tissue engineering involves the use of cells, growth factors/cytokines, and scaffolds to regenerate damaged tissue. The choice of cells, scaffold and their delivery is crucial to the successful outcome of the treatment and this is particularly the case in bone and cartilage where the repair tissue has to recreate a structural hierarchy to restore long lasting function. One approach to deliver high numbers of cells to a defect site is as cell aggregates or spheroids. Experiments are described here that aim to understand how mesenchymal stem cells (MSCs) and osteoblastic cells behave in a cell aggregate and how this could be refined by the inclusion a self -assembling hydrogel to influence cellcell and cell-extracellular matrix (ECM) interactions. Forming cellular spheroids in vitro can be achieved using various methods, including hanging drop, static suspension culture, encapsulation/entrapment, and low adherence multi-well. Extensive analysis identified that the most efficient and reproducible method for the formation of spheroids using rat osteoblasts and human MSCs was through their culture at a specific concentration in polyHEMA coated plates. Both viability and ability of cells to differentiate was investigated. The MTT assay was used to assess cells viability while their ability to differentiate was assessed by measuring alkaline phosphatase activity as well as measuring gene osteogenic markers expression via qRT-PCR. Analysis of cell differentiation under these conditions revealed that alkaline phosphatase activity appeared more elevated in 2D cultures compared to 3D. However, it was noted that there were contrasting results between the two types of cells with expression of osteogenic genes higher when MSCs were grown in osteogenic media while with calvarial significant expression was also observed when grown in normal media. Because of the distinct regulatory cues given by cell-cell contact in the spheroid, analysis was performed for connexin (Cx)-43, a gap junction protein and members of the ephrin/Eph family. Cx-43 iv was immunolocalised to gap junction structures in cells after osteogenic treatment on a flat substrate but this was more difficult to assess in the 3D spheroids. Analysis of transcript patterns reflected the increased abundance of Cx-43 in cells treated with osteogenic supplements and parallel changes in expression of Ephrin B1 and Ephrin B2. Experiments were also performed including a Puramatrix hydrogel nanofibers scaffold that could encase the cells in an ECM-like environment, provide mechanical support and protect them and manipulate cell-cell interactions. The results obtained in this study concluded that calvaria cells viability and hence proliferation increased when grown embedded within 0.25% Puramtrix while mesenchymal stem cells increased when embedded in 0.5% Puramatrix. Similarly, alkaline phosphatase activity was higher in cells embedded within 0.25% Puramatrix while mesenchymal stem cells favoured 0.5%. On the other hand, osteogenic gene expression of both cells was enhanced with the use of Puramatrix scaffold.
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