(628c) A 3D Hydrogel Culture System to Determine Impacts of Biomaterial Stiffness and Topographical Cues on Oligodendrocyte Progenitor Cell Viability, Growth and Differentiation

Introduction: The prevalence of demyelinating disorders and injuries necessitates development of therapeutic strategies to regenerate myelin. While factors which influence the differentiation of neural stem cells (NSCs) into neurons are well-established, a knowledge gap persists in determining factors which lead to oligodendrocyte (OL) fates. Hydrogel properties such as matrix stiffness influence the proliferation and morphology of encapsulated oligodendrocyte progenitor cells (OPCs)¹. By tuning the polymer concentration during crosslinking, the crosslinking density of in vitro 3D hydrogel systems can be varied to provide encapsulated cells with matrix environments which approximate native neural tissue stiffness. Here we encapsulated OPCs within polyethylene glycol dimethacrylate (PEG-DM) and norbornene-functionalized hyaluronic acid (NorHA) hydrogels at storage moduli representative of native brain tissue (G’ = 200-2000 Pa) and evaluate the effects of hydrogel stiffness on cell fate over a seven-day period. Electrospun fibers co-encapsulated into the hydrogel system mimic the structural and mechanical properties of neuronal axons, which may be an important cue in driving OPC differentiation into oligodendrocytes².

Materials and Methods: Polyethylene glycol (PEG) was functionalized with methacrylate groups to allow for radical-mediated photo-crosslinking. Similarly, hyaluronic acid (HA) was functionalized with norbornene groups to allow radical-mediated crosslinking in the presence of a dithiol crosslinker. On stage crosslinking was used to determine the UV-initiated changes in shear rheology of both Nanoindentation was used to measure the local elastic moduli directly atop fibers encapsulated in 1.5% NorHA gels as compared to elastic moduli of the gel alone.

Results and Discussion: PEG-DM gels demonstrated storage moduli of 72 Pa (6 wt%), 779 Pa (7.5 wt%), and 5807 Pa (10 wt%), indicating that higher weight percentages of polymer correspond to greater storage moduli. Similarly, NorHA gels demonstrated storage moduli of 341 Pa (1 wt%), 1395 Pa (1.5 wt%), and 3105 Pa (2 wt%). Preliminary nanoindentation measurements suggest that elastic moduli near encapsulated electrospun fibers (1324 Pa) may be greater than elastic modulus of 1.5% NorHA alone (1207 Pa). ATP and DNA concentrations of encapsulated OPCs increased for all stiffness conditions over a seven-day period. Encapsulated OPCs remain viable and extend processes in the presence of electrospun fibers. data suggests that OPCs co-encapsulated with fibers exhibit an increased number of process extensions compared to OPCs within gels that do not contain fibers.

Conclusions and Implications: Hydrogels composed of 6-10 wt% PEG-DM and 1-2 wt% NorHA demonstrate a range of storage moduli comparable to native brain tissue. Encapsulated OPCs grow and proliferate in PEG-DM gels at all tested stiffness conditions over a seven-day time period. OPCs retain viability in the presence of electrospun MeHA fibers, and more process extension is observed in fiber-containing gels as compared to no-fiber controls.

Acknowledgements: UVA Dean’s Fellowship to RAM; NSF CMMI-1904198.

References: 1. LN Russell, KJ Lampe, ACS Biomaterials Science and Engineering, 3, 3459–3468 (2017).

2: S Lee, MK Leach, SA Redmond, SYC Chong, SH Mellon, SJ Tuck, ZQ Feng, JM Corey, JR Chan, Nature Methods, 9, 917-922 (2012).