(335f) A 3D Hyaluronic Acid Hydrogel Model for Studying Myelination

Deniz B. Unal¹, Emily Zezas¹, Steven R. Caliari^1,2, & Kyle J. Lampe¹

¹University of Virginia, Charlottesville, VA Department of Chemical Engineering, ²Department of Biomedical Engineering

Multiple Sclerosis (MS) affects approximately 1 million people in the US over the age of 18. The hallmarks of the disease are immune system attack on the oligodendrocytes that produce the insulating myelin sheath as well as attack on the myelin sheath itself, which leads to impaired motor skills and even paralysis. Current disease therapies are limited to immunomodulatory and immunosuppressive methods, which do not relieve symptoms in the progressive stage of the disease where axonal loss occurs. Better models are needed to understand myelination and demyelination and ultimately develop therapeutic strategies to promote regeneration of oligodendrocytes and the myelin sheath. Demyelination and remyelination events in live tissues are difficult to study because of the overlapping impact of both biochemical cues and mechanical cues from the neuronal axons. As an alternative in vitro approach, oligodendrocyte progenitor cell (OPC) adhesion, proliferation, differentiation, and migration can be stimulated in 2D using artificial fibers to act as axon mimics. Axon mimic diameter is a tunable biophysical property that can encourage myelination (or remyelination after injury or disease). This work introduces 3D in vitro models composed of crosslinked hydrogels with electrospun fibers as artificial axons that builds on previous work by mimicking native tissue dimensionality while removing the complications of biochemical signals from live axons.

In this work, we encapsulate MADM oligodendrocyte progenitor cells (OPCs) in 3D hyaluronic acid hydrogels with co-encapsulated hyaluronic acid electrospun fibers to act as artificial axons. The storage moduli of the hydrogels are tuned to that of brain tissue (70 – 400 Pa) by crosslinking norbornene-functionalized hyaluronic acid with dithiothreitol (DTT) crosslinker groups under UV light. Methacrylated hyaluronic acid is electrospun into fibers of 0.32 ± 0.04 µm diameter and fibers are crosslinked. Hydrogels support 55% viability in the stiffest gels (~ 400 ± 50 Pa storage modulus), 75% viability in the medium stiffness gels (~ 120 ± 20 Pa storage modulus), and 85% viability for the most compliant gels (~ 70 ± 10 Pa storage modulus), indicating a trend of increasing viability with decreasing stiffness according to live/dead assay 16 hours post-encapsulation. Cells proliferate at least two-fold in all gels after 8 days of culture with the most compliant gels showing the highest statistically significant proliferation in comparison to the stiffer gels. ATP production (normalized to DNA content) reaches a maximum at 84 hrs for all hydrogel groups with no statistical difference between gel stiffnesses. Cells coexist with electrospun fibers incorporated in hydrogels, however they do not extend processes toward the fibers. Future work will incorporate RGD peptide (1 mM), providing integrin-binding adhesion cues for the cells to link to the hyaluronic acid matrix. Additionally, MMP-degradable peptide crosslinker incorporation will provide cleavable crosslinks for cells to remodel their extracellular environment to promote differentiation and migration.