| This work investigated the use of biomedically relevant, polymer substrates for in vitro human mesenchymal stem cell (hMSC)-substrate surface interaction. Two materials were identified: (i) Poly(glycerol-sebacate) (PGS), a novel biocompatible and biodegradable thermosetting rubber-like elastomer, and (ii) injection molded polystyrene (PS). PGS was selected because it has tunable mechanical properties within the range of biological tissue, and thus provides a useful model to determine the types of substrate mechanical cues that would elicit specific hMSC lineage specification and possible differentiation outcomes. PS is a relevant material for in vitro cell-substrate surface interaction analysis since it is typically the base material of cell culture dishes. Both these materials have also shown micro to nanoscale molding capabilities. Hence these materials would also serve as a model in determining topographical properties (and related mechanical properties) at the dimension-scale of the extracellular environment that modulates hMSC state and fate. The work characterized, designed, and manufactured substrates made of these materials, for in vitro hMSC culture. Micro/nanoscale PGS and PS surface features were manufactured using silicon (Si) based tooling technology. The response of hMSCs to PGS substrates of various Young.s moduli was examined. hMSC response to a nanoscale array of PS pegs was also investigated.;PGS was observed to be a semi-crystalline thermosetting elastomer that is fully amorphous above 35°C. The material acquired increasing stiffness and density of photoresist-coated with increasing levels of curing temperature and duration of cure. hMSCs were observed to respond differently on PGS with elastic modulii of 0.11, 1.11, and 2.30 MPa. The cells spread and proliferate more, and develop a stretched cytoskeleton on the stiffer substrates. On the softest substrate (0.11 MPa) the cells developed a branched and filopodia-rich morphology with a diffused actin cytoskeleton. PGS and a variety of other typical polymeric substrates such as poly(urethane) PU, poly(L-lactide-co-epsilon-caprolactone) PLCL, and poly(lactic-co-glycolic acid) PLGA, were found to produce its own fluorescence emission during fluorescence based imaging, which interfered in immunocytochemical (ICC) imaging and analysis of fluorescently labeled biomolecule structures of cells contacting these materials. The study successfully quenched this light interference by using Sudan Black, dye B (SB). Both PGS and PS sub-micron features and nanoscale peg arrays were successfully manufactured using Si based tooling technology. Cultures of hMSC on arrays of a nanopegged PS surface (400 nm diameter, 800 nm center-center, ∼ 200 nm high) revealed several interesting phenomena. The cells were observed to adhere to, migrate over, undergo mitosis, and interact over the nanopegged PS surface. The cells exhibited unique morphology in comparison to those cultured on commercial PS Petri dishes, and on flat injection molded PS templates. hMSCs on the nanopegs appear rounded, less spread out, and more motile with a filopodia-rich morphology. |
