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Mesenchymal stem/stromal cells (MSCs) release Extracellular vesicles (EVs) that are believed to play a major role in nerve regeneration after stroke. However, a major complication when trying to transition MSC-EVs from a pre-clinical to clinical setting is the convenient long-term storage of MSC-EVs. Therefore, we developed a strategy to freeze dry MSC-EVs to store them for more practical clinical applications. We first determined the optimal trehalose concentration for freeze drying the MSC-EVs, and we subsequently investigated the optimal freezing conditions. It was determined that 100 mM of trehalose and freezing temperature at -20°C were the optimal conditions to freeze dry the EVs. The therapeutic capabilities of the freeze-dried MSC-EVs was tested via tube formation assay and co-culturing them with neural stem/progenitor cells (NSPCs). It was found that human vein umbilical endothelial cells (HUVECs) treated with rehydrated MSCEVs promoted tube formation suggesting the trophic factors in the MSC-EVs survived the freeze-drying process. As for the NSPC co-culture, all treatments involving rehydrated MSC-EVs protected by trehalose during the freeze-drying process promoted proliferation and did not affect their ability to differentiate into oligodendrocytes, astrocytes, or neurons. Determining the optimum freezing-drying conditions allows us to stockpile a large amount of MSC-EVs at room temperature for on-demand applications.

Due to the similar nature 3D synthetics share with in vivo cell conditions, peptide-based hydrogels pose an attractive strategy for the culturing of stem cells. One aspect of this unique cell culturing technique is the tunability of the hydrogel’s stiffness, a quality linked to stem cell differentiation. Due to this linkage, a methodology in which specific cell lineages are achieved within IIFK hydrogel cultures is proposed. This work provides an analysis for the peptide scaffold IIFK; it characterizes the effect between different peptide and PBS concentrations over the resulting hydrogel stiffness and develops a mathematical model to further elucidate this interaction. Nine different hydrogel formulations were made (with a minimum of eleven replicates each) and each of its replicate’s stiffness (storage modulus, Pa) was measured through rheological experiments. Then, two different methods of replicate selection were conducted and various models were derived, each using either of the two replicate selection methods and incorporating a specific number of replicates in their creation. Regardless of sample selection and replicate number, the generated models show extremely high significances between IIFK hydrogel stiffness and PBS concentrations over the resulting hydrogel stiffness. Data analysis shows that for IIFK, the hydrogel stiffness bears a strong behavior that can be modeled by a full quadratic equation. However, the data also shows that the dependency of the model is strongly correlated with the datasets chosen to produce it, with number of replicates and replicate values both resulting in differences in each model’s predictive reliability (e.g., 82% vs 91%). Therefore, while this thesis demonstrates the ability to model IIFK hydrogel behaviour with high predictability ratings, it also establishes the necessity of both producing more replicates as well as selecting the best values for IIFK-based hydrogel modelling.

The use of nanowires, nano and micro needles in biomedical applications has markedly increased in the past years, mainly due to attractive properties such as biocompatibility and simple fabrication. Specifically, these structures have shown promise in applications including cell separation, tumor cell capture, intracellular delivery, cell therapy, cancer treatment and as cell growth scaffolds. The work proposed here aims to study two platforms for different applications: a vertical magnetic nanowire array for mesenchymal stem cell differentiation and a micro needle platform for intracellular delivery. First, a thorough evaluation of the cytotoxicity of nanowires was done in order to understand how a biological system interacts with high aspect ratio structures. Nanowires were fabricated through pulsed electrodeposition and characterized by electron microscopy, vibrating sample magnetometry and energy dispersive X-ray spectroscopy. Studies of biocompatibility, cell death, cell membrane integrity, nanowire internalization and intracellular dissolution were all performed in order to characterize the cell response. Results showed a variable biocompatibility depending on nanowire concentration and incubation time, with cell death resulting from an apoptotic pathway arising after internalization. A vertical array of nanowires was then used as a scaffold for the differentiation of human mesenchymal stem cells. Using fluorescence and electron microscopy, the interactions between the dense array of nanowires and the cells were analyzed, as well as the biocompatibility of the array and its effects on cell differentiation. A magnetic field was additionally applied on the substrate to observe a possible differentiation. Stem cells grown on this scaffold showed a cytoskeleton and focal adhesion reorganization, and later expressed the osteogenic marker osteopontin. The application of a magnetic field counteracted this outcome. Lastly, a micro needle platform was fabricated through lithography and electrodeposition, characterized using the previously mentioned techniques and then evaluated as a vector for intracellular delivery. Fluorescence and electron microscopy imaging were first performed to assess the biocompatibility, cell spreading and the interface of the cells and the needles. Intracellular delivery of a fluorescent dye was achieved via inductive heating of the needles, with the results showing a dependency of delivery and cell survivability on the exposure time.

This research examines the role of actin cytoskeleton filaments in chondroinduction by cyclic hydrostatic pressurization. A chondroinductive hydrostatic pressurization system was developed and characterized. A pressure of 5 MPa at 1 Hz frequency, applied for 7200 cycles (4 hours intermittent) per day, induced chondrogenic differentiation in C3H10T1/2 cells while 1800 cycles (1 hour intermittent) did not induce chondrogenesis. Quantitative analysis of chondrogenesis was determined as sulfated glycosaminoglycan synthesis and rate of collagen synthesis while qualitative analysis was obtained as Alcian Blue staining and collagen type II immunostaining. Actin disruption using 2 uM Cytochalasin D inhibited the enhanced sGAG synthesis in the chondroinductive hydrostatic pressurization environment and significantly inhibited rate of collagen synthesis to the mean level lower than that of the non-pressurized group. These results suggest an involvement of actin cytoskeleton filaments in mechanotransduction of cyclic hydrostatic pressure.

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