Abstract
Demyelination occurs when the protective myelin sheath surrounding mature neurons is degraded. Neural stem cells (NSC) and oligodendrocyte progenitor cells (OPC) are responsible for replacing damaged myelin by migrating to the injured area, differentiating into mature oligodendrocytes (OLs), and extending processes to re-wrap exposed neurons. Unfortunately, oligodendrocyte differentiation and remyelination are understudied. Most research in this area has been conducted using in vivo models, which are complicated by the presence of extraneous cell types and biological processes. Existing in vitro studies have been conducted in 2D, which does not appropriately reflect the 3D environment of native tissue. 3D hydrogels are a promising alternative approach to in vivo or 2D in vitro culture due to their ability to replicate natural tissue characteristics without the presence of confounding variables. Norbornene-functionalized hyaluronic acid (NorHA) is an attractive option since HA is a key component of the extracellular matrix, and NorHA gel stiffness can be easily tuned within the range of native brain tissue. We can also replicate structural cues by incorporating electrospun HA fibers with diameters similar to native axons. Here, we construct a 3D tissue mimetic model system by co-encapsulating cells and electrospun topographical cues within NorHA hydrogels.
In this thesis, we explore the effect of electrospun topography on NSC and OPC growth and differentiation. Chapter 2 discusses the need for an in vitro model system to elucidate the molecular-level effects of demyelinating disease and injury cascades. In Chapter 3, our model system investigates the effects of electrospun topographical cues on OPCs. Confocal imaging revealed that the presence of fibers affected OPC morphology, resulting in the extension of numerous cellular processes associated with OPC differentiation. Chapter 4 explores the effects of electrospun topography on NSCs. Unlike the OPCs used, NSCs were not genetically modified and so are more representative of native cells. Additionally, the use of NSCs represents a step back along the differentiation pathway, allowing us to determine whether cells unbiased towards glial fates would still differentiate into OL in the presence of fiber cues. Once again, NSCs in fiber-containing gels displayed more mature morphologies. Lastly, Chapter 5 focuses on practical applications of our model in determining OPC response to traumatic brain injury (TBI). A shockwave generator system was used to simulate the effects of mild TBI on OPCs encapsulated in NorHA gels. Blast overpressure did not have a significant effect on hydrogel material properties, but inclusion of electrospun fibers did result in an increase in bulk storage modulus. Cell viability and proliferation were not significantly affected by overpressure exposure in either the presence or absence of fibers. As before, inclusion of fiber cues influenced OPC differentiation; however, blast injury on the scale of mild TBI was demonstrated to have minimal impact on cell differentiation.
