Biomedical Engineering - Theses
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ItemNo Preview AvailableTuning the Bioactivity of Cell-derived Extracellular Matrices through Control of Substrate PropertiesYang, Michael ( 2021)Mesenchymal stromal cells (MSCs) are the subject of thousands of clinical trials for treatment of innumerable human pathologies. However, their widespread clinical use is still hampered by difficulties in retaining their stem cell-like properties during prolonged ex vivo expansion. The use of cell-derived extracellular matrix (ECM) has recently garnered much interest as a culture substrate because ECM significantly improves cell viability in ex vivo culture. However, these biomaterials are themselves produced in an environment which is not physiologically accurate, and therefore, in this study the aim was to optimise bioactivity of cell-derived ECM by controlling substrate properties to more accurately mimic the in vivo niche. MSCs are innately sensitive to substrate stiffness and their surrounding mechanical environment. The first results chapter (Chapter 2) describes investigation into whether manipulation of substrate stiffness increases bioactivity of cell-derived ECM. Polyacrylamide hydrogels with tunable stiffnesses were fabricated, and MSCs induced to deposit ECM on these surfaces. Subsequent culture and behavior of primary MSCs on the ECM was then observed. Primary cells cultured on ECM deposited on soft substrates demonstrated the highest levels of proliferation. On the other hand, there were significantly higher levels of osteogenesis when culturing MSCs on ECM deposited on stiff substrates. These results show that the bioactivity of ECM can be taken even further by controlling mechanical properties of substrates that mimic the cellular microenvironment. In addition to substrate stiffness, surface chemistry was controlled to determine whether this affected ECM bioactivity in a similar fashion, described in Chapter 3. The surface chemistry of glass coverslips was modified with various silanes, introducing amine, carboxylic acid, propyl, and octyl functional groups onto the surfaces, and studied how introduction of these moieties affected the ECM. ECM on these surfaces improved cellular proliferation and adipogenesis. However, the bioactivity of the ECM did not depend significantly on the surface chemistry for the range of chemistries and culture timescales tested. These results allow for greater flexibility in fabricating tissue engineering materials with a variety of surface chemistries for use as ECM scaffolds. Lastly, Chapter 4 describes an investigation to determine whether the beneficial effects observed by tuning substrate stiffness could be scaled up and applied to 3D culture systems. Porous polyacrylamide cryogels were fabricated for the culture of MSCs and subsequent ECM deposition. MSCs cultured in this manner did not demonstrate improved cell behavior. However, these 3D scaffolds were a suitable material for the deposition of cell-derived ECM, providing a future avenue of research for the scaleup and clinical translation of these matrix biomaterials. This dissertation presents findings which demonstrate that the effects of varying substrate stiffness can be combined with use of ECM to increase MSC proliferation and osteogenesis. Altering surface chemistry is also shown to not have a significant effect on dECM bioactivity for the range of functional groups tested. Fabrication of tunable porous cryogels with ~65 um pores also revealed that secretion of ECM by MSCs is not affected sufficiently to result in changes to its bioactivity. These findings contribute to the field by showing that parameters such as substrate stiffness can be manipulated and used with ECM in cell culture to result in improved cell behavior compared to using ECM alone, and also illustrates that strict control of other variables such as surface chemistry and three-dimensionality are not necessary in order to maintain the bioactivity of cell-derived ECM.