(386e) Graduate Student Award Session: Incorporating Electrospun Fiber Topography in a 3D PEG Hydrogel Promotes Oligodendrocyte Maturation

The central nervous system is composed of highly ordered tissue that forms in response to complex topographical and directional cues. During neurogenesis, neurons migrate and extend axons along the radial glia to create the directional phenotype inherent to the CNS. In response to the chemical and topographical cues provided by neurons, oligodendrocyte precursor cells (OPCs) then extend processes towards neurons to generate the electrically insulating myelin sheath. Cell loaded biomaterials are not only a potential course for regeneration following insult or injury to the central nervous system, but can also create in vitro models of health or diseased tissue. Despite their potential, amorphous biomaterial hydrogel systems lack the directional and topographical cues provided during development of native central nervous system tissue. Here, we have developed methods of incorporating topographical cues in 3D poly(ethylene glycol) (PEG) based hydrogels through the inclusion of electrospun fibers and further investigated their impact on neural cell types.

Hydrolytically degradable PEG hydrogels with an initial stiffness of 800 Pa were made with a 50:50 mixture of PEG-dimethacrylate (PEG-DM) (8000 g/mol PEG) and PEG-polylactic acid-DM (PEG-PLA-DM). 2 μm diameter fibers were electrospun from 15% (wt/v) polystyrene (280,000 MW) in dichloromethane:dimethylformamide: tetrahydrofluran (7:2:1) using a Spraybase electrospinning system with 22 gauge emitter and 0.17 mL/hour flow rate. Fiber-loaded, degradable PEG hydrogels containing 10⁶ cells/mL were made from a 7.5% (wt/v) macromer solution upon exposure to UV light for 10 minutes with the photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate. Fibers were incorporated in the PEG hydrogels in one of two methods. In the first method, fibers were spun directly into silicone molds, coated with polyornithine, and mechanically separated before cell/macromer solution was added over the fibers and placed under UV light to gel. In the second, fiber mats were electrospun, and cut using a Leica Cryostat. Cryosectioned fibers were collected, coated with polyornithine, and suspended in phosphate buffered saline. Suspended fibers were then mixed with cell/macromer solution and exposed to UV to encapsulate fibers in the 3D PEG hydrogel. The first method yielded long fibers encapsulated in the gels throughout a z-height of up to 200 μm. The second method encapsulated fibers across the entire z-height of the gel, however yielded much shorter fibers as determined by the cryostat section height. Both fiber methods enabled cells to be encapsulated in the 3D hydrogels with cell viabilities around 90%, similar to fiber-free hydrogels. When OPCs were encapsulated with fibers in the hydrogels, OPCs were found to extend processes towards fibers and along fibers. This observed morphology of cells in gels with fibers were more similar to OPCs in native neural tissue than those of cells in gels absent of fibers. Together these findings suggest that incorporating topographical cues into amorphous hydrogels is necessary for OPC maturation into the functional oligodendrocytes that generate the electrically insulating myelin sheath.