(19a) Matrix Stiffness and Ligand Guide Schwann Cell Phenotype Specification
AIChE Annual Meeting
2019 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Cell Biomechanics and Adhesion
Sunday, November 10, 2019 - 3:30pm to 3:48pm
Materials and Methods: Polydimethylsiloxane (PDMS) was prepared with varying precursor ratios to yield a range of Youngâs modulus (E) from 1119.01 to 3.85 kPa in order to examine the impact of matrix stiffness on Schwann cell phenotype. PDMS was coated on a glass coverslip by spincoating the precursor solution at 2,500 rpm for 30 seconds and left overnight to solidify. Schwann cells were then cultured on PDMS substrates coated with 10 Âµg/ml fibronectin, collagen I, laminin, or left uncoated in order to assess impact of ligand in combination with substrate stiffness. Cells were analyzed for morphology, proliferation, and c-Jun expression. c-Jun expression was quantified via pixel intensity from immunofluorescence staining and western blot analysis. Micropatterning of cell adhesive areas was accomplished on PDMS substrates using microcontact printing of laminin to further explore the relationship between cell shape, cell area, and c-Jun expression. Data was analyzed and significance determined via One-way ANOVA and Tukeyâs post hoc test where P < 0.05 was considered statistically significant.
Results and Discussion: Our results showed that an intermediate stiffness (E=8.67kPa) promoted the highest level of c-Jun expression across all ligand types. It was also determined that both relatively stiff (E=1119.01kPa) or soft (E=3.85kPa) substrates inhibit c-Jun expression. In all experiments, having a ligand present on the surface enhanced cell spreading, proliferation, and c-Jun expression in comparison to uncoated PDMS. Fibronectin had the highest cell spreading and proliferation in comparison to collagen I and laminin, however c-jun expression was greatest on laminin coated surfaces. Cells were analyzed for both cell and nuclear morphology in relation to c-jun expression, with higher levels of c-jun expression corresponding to a higher level of cellular elongation across each ligand and substrate stiffness. Therefore, microcontact printed proteins were used to constrict cells to specific spreading area and degree of elongation, and it was found that elongated cells displayed higher levels of c-Jun expression compared to rounded cells. It was also observed that less spread, elongated cells showed higher c-Jun expression in comparison to well-spread, elongated cells.
Conclusion: To summarize, we have demonstrated c-Jun expression level can be regulated by both substrate stiffness and ligand types and have quantified the combinatorial effect of substrate stiffness and ligand type on c-Jun expression. These studies also confirmed higher levels of cell elongation in Schwann cells leads to increased expression of the regenerative Schwann cell marker c-jun. Taken together, these results highlight the ability of matrix bound factors in the microenvironment to play key roles in the phenotype specification of Schwann cells. Thus, utilizing engineered microenvironments to investigate Schwann cells may hold great potential in producing regenerative Schwann cells, which can lead to enhanced therapeutic technologies for nerve injuries. Looking forward, these results can provide inspirations for the future design of nerve guidance conduits as a potential replacement for autografts, in addition to potential cell therapies with Schwann cells that may offer improvements to functional repair and outcomes in PNS injuries.