Abstract
The myelin sheath is a critical tissue component in the central nervous system (CNS) enabling the fast and efficient communication of neurons. This myelin sheath is the primary tissue component damaged in demyelinating diseases such as multiple sclerosis. Oligodendrocyte precursor cells (OPCs) give rise to myelin forming cells, oligodendrocytes, however current in vitro tissue models fail to adequately represent OPC and oligodendrocyte phenotypes, preventing the development of therapeutic strategies to reverse demyelinating diseases. Traditional tissue culture methods in 2D polystyrene dishes fail to mimic the 3D dimensionality of cells in native tissue as well as the stiffness and mechanical properties of native extracellular matrix. 3D hydrogel biomaterials are an attractive alternative to traditional 2D cell culture because they can be engineered to more closely mimic native tissue in terms of mechanics, degradability, or topography. In particular, poly(ethylene glycol)-based (PEG) hydrogels are an exciting avenue for neural tissue engineering due to their ability to recapitulate the highly hydrated and compliant native tissue. In this work, PEG-dimethacrylate (PEG-DM) hydrogels were engineered to create an in vitro tissue model that enables OPCs to proliferate and differentiate into oligodendrocytes, and investigate conditions suitable for the ultimate goal of an in vitro myelination model.
First, the effects of hydrogel mechanics were investigated to determine their impact on OPC proliferation. When OPC- like cells were encapsulated in the most compliant hydrogels with the largest mesh sizes, cells proliferated more than cells in the stiffer hydrogels, as shown through higher values of ATP and DNA and a greater propensity for EdU staining. In the least compliant materials, cells expressed more PDGFRα, platelet derived growth factor receptor-α, suggesting that cells in the least compliant materials may de-differentiate into a more proliferative cell type.
Hydrogels that degrade over the course of weeks were made by incorporating polylactic acid (PLA) into the hydrogel by mixing ratios of PEG-DM with PEG-PLA-DM. PLA was chosen as the degradable unit due to the mild antioxidant properties of lactic acid, which is released upon hydrolytic degradation. OPC-like cells encapsulated in the degradable hydrogels were able to extend processes due to the degradation, but did not respond metabolically to the release of lactic acid. These results are not surprising given the differing role of lactic acid in metabolism between cancer cells and primary OPCs. Primary OPCs were also investigated for preliminary differences between metabolism and degradable macromer content, however minimal metabolic differences were found, likely due to general poor cell viability from the OPC isolation process.
In the CNS, OPCs are known to respond to neuronal topography to differentiate and create the electrically insulating myelin sheath, thus the differentiation of OPCs in fiber containing hydrogels was also assessed. Electrospun fibers, engineered to mimic the high aspect ratio and diameter of neuronal axons, were encapsulated in the degradable PEG hydrogels. When cells and fibers were co-encapsulated, OPC-like cells remained more viable compared to cells in hydrogels without fibers. Additionally, OPC-like cells were observed extending processes towards and along fibers, similar to native OPCs or oligodendrocytes in the brain. However, gene expression results from primary rat OPCs showed minimal differences in both OPC and oligodendrocyte genes, indicating that differentiation may not occur from the introduction of topographical cues alone.
Finally, an important observation is that primary rat OPCs encapsulated in PEG hydrogels remained more viable over 7 days when compared to traditional 2D cell culture, measured through apoptosis and viability quantification assays. Together these results suggest the importance of hydrogel mechanics, degradability, and topography on OPC fate and the potential biomaterials have in developing better myelination models. Despite these strides, more work remains to fully develop an in vitro myelination model.
