Abstract
Event Abstract Back to Event Oligodendrocyte survival, proliferation, and maturation is dependent on 3D hydrogel mechanics Lauren N. Russell1 and Kyle Lampe1 1 University of Virginia, Chemical Engineering, United States Introduction: Millions of people suffer from damage or disease to the central nervous system (CNS), such as through a spinal cord injury or multiple sclerosis, resulting in a loss of myelin. In the CNS, oligodendrocytes extend processes which wrap around neuronal axons to generate myelin, an electrically insulating layer conducive to rapid, controlled neuronal signaling. Diminished myelin distorts and slows neuronal communication and causes neuronal degeneration and death. Cell loaded biomaterials are a potential method of repair, but have rarely been investigated for oligodendroglial specification[1]. Here, we investigate how mechanical properties of a polyethylene glycol based hydrogel affect proliferation and differentiation of two oligodendrocyte precursor cell (OPC) lines. Materials and Methods: Hydrogels with controllable storage moduli were formed by photoinitiation of methacrylated polyethylene glycol (PEG) with molecular weights and concentrations ranging from 4600 to 8000 g/mol and 7.5% to 20% (wt/v), respectively. Gelation was accomplished by photoinitiation of LAP at 365 nm and 4 mW/cm2 with OPCs[2],[3] encapsulated at 1x107 cells/ml. Both GFP+ MADM and N20.1 OPC cell lines were studied. ATP and DNA were quantitatively analyzed at discrete time points using high throughput luminescence and fluorescence assays to determine the viability and proliferation. LIVE/DEAD confocal microscopy verified ATP and DNA results. Results and Discussion: PEG molecular weights of 4600, 6000, and 8000 g/mol at 7.5% (wt/v) yielded hydrogels with storage moduli of 1,420 Pa, 795 Pa, and 418 Pa, respectively, similar to different regions of native brain tissue. LIVE/DEAD staining at 1 through 7 days indicated highest viability in the 4600 g/mol condition with high viability for all conditions at 7 days (Figure 1). DNA, cell number, and presence of colonies revealed that proliferation occurred in all conditions with the largest colonies present in less stiff hydrogels. When laminin was incorporated at 25 μg/ml, OPCs spread and extended processes, indicating maturation. Viability and proliferation was also impacted by hydrogel mechanics as the most compliant materials (lowest % wt/v) led to greatest ATP and DNA values with a significant increase over time (Figure 2). Conclusions: The material properties of PEG hydrogels influence oligodendrocyte-like cell proliferation, indicating their potential use in controlled growth of oligodendrocytes for CNS regeneration. In particular, these cell lines favor the more compliant matrices found with either the higher molecular weight conditions (Figure 1) or the lower weight percents of macromer during polymerization (Figure 2). Cellular morphology and process extension is also impacted by these features (Figure 1) and is being investigated toward maturation and oligodendrocyte regeneration. This is the first research to date showing a specific effect of hydrogel modulus on OPC proliferation and maturation. Current research is focused on incorporating topographical cues and degradable subunits into the hydrogel to promote a favorable intracellular redox state. UVA Biotechnology Training Program for fellowship to LNR; Zong and Gaultier labs at UVA for OPC cell lines
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