Alkylsilane self-assembled monolayer substrates to control mesenchymal stem cell differentiation

Mesenchymal stem cells (MSCs) have been proposed as a promising cell source for musculoskeletal tissue engineering due to their multipotentiality. So far, bioactive substances have been used to induce MSC differentiation \textit{in vitro}, which can also act as a limiting factor when translating into the clinic. With these limitations in mind, efforts are now centred on controlling biomaterial surface properties as a tool to control MSC fate. In particular, modification of surface chemistry in biomaterials is an appealing option to obtain a desirable cell response for the intended function of a biomaterial. As such, chemical modifications on a surface can induce changes in focal adhesions, which in turn, could be used to promote the desired cellular response (e.g. induce chondrogenesis, osteogenesis or adipogenesis in MSCs). With a relatively high degree of reproducibility, self-assembled monolayers (SAMs) seem to be the most promising chemical modification as they are formed spontaneously on surfaces by chemical adsorption. The aim of this work was to explore and understand how different alkylsilane SAMs affected protein adsorption and how changes in adsorption could lead to induce MSC differentiation \textit{in vitro}. The project focused on five different methyl-terminated alkylsilane SAMs, two amine SAMs and two hydroxyl SAMs that presented varying carbon chain lengths (CCLs) and surface energies (i.e. wettabilities). Following fabrication, the presence of SAMs, their wettabilities and topographical profiles were assessed using XPS, FTIR, contact angle tensiometer and AFM, respectively. Viability assays confirmed that none of the SAMs caused a cytotoxic effect on MSCs or enhanced proliferation when compared to control. Cell morphology on each chemistry was also assessed using immunofluorescence. Although no major differences were found in MSC shape, cell area was clearly reduced by all chemistries compared to control. Assessment of chondrogenic, adipogenic and osteogenic markers was then undertaken qualitatively and quantitatively. While none of the assessed chemistries promoted chondrogenesis, hydroxyl and amine SAMs induced osteogenic and adipogenic differentiation simultaneously with primary MSCs. The use of a clonal MSC line demonstrated that hydroxyl SAMs promoted osteogenesis while silencing adipogenesis in the absence of external stimuli. Overall, longer CCLs enhanced lineage-specific marker expression and secretion, which highlighted the importance of this parameter with regards to MSC behaviour. It is likely that the correlation between wettability, cell proliferation and differentiation was linked to the different protein adsorption profiles observed on alkylsilane SAMs, which was also influenced by the CCL of SAMs. Data suggested that vitronectin might be more important in mediating MSC adhesions on alkylsilane SAMs than fibronectin, which was not adsorbed on any substrates. In addition to this, these findings suggested that in the presence of complex media, ITGA8 and VCAM1 seem to play a key role in determining MSC fate. Altogether, this thesis has established that surface chemistry in the form of alkylsilane SAMs can influence MSC differentiation in the absence of external stimuli, and that CCL is a parameter that MSCs react to.