Supplementary MaterialsAdditional file 1: Rheological characterization of PAA gels that approximate sequential regions on the experimental gradients. Pa). Scale bar represents 200 is Poissons ratio, assumed to be 0.457 for polyacrylamide . Schwann cell culture Schwann cells isolated from adult rat sciatic nerve (generous gift from Dr. Mary Bunge, University of Miami, Coral Gables, FL) were maintained in Dulbeccos modified Eagles medium containing 10% fetal bovine serum, 4mM L-glutamine, 100 = 0.05. All reported data sets included at least one experimental group that was not normally distributed, therefore a non-parametric Kruskal-Wallis one-way ANOVA on ranks was used to statistically compare mean ranks and followed with Dunns multiple comparisons post-test. Significance was set at p 0.01. All results were collected from three independent experiments. Results PAA substrate characterization For this study, we fabricated substrates tuned to recapitulate stiffnesses found within the mechanical niche of the peripheral nervous system (PNS) [3, 19, 20]. Shear storage moduli ranged from 18 6 Pa to 190 4 Pa for the shallow gradient and 243 88 to 4325 773 Pa for the steep gradient (Fig.?1). Nominal stiffness gradient slopes were approximated by performing linear regression on the data. For comparison with other studies that report IL-22BP gradient slope as a function of change in Youngs modulus over distance, the gradient slopes in this study correspond to 0.04 kPa/mm for the shallow gradient and 0.95 kPa/mm for the steep gradient. Rheology frequency plots are included in Additional PSI-7977 distributor file?1. Open in a separate window Fig. 1 Mechanical characterization of PAA substrates. a Noted in the table are the percent concentrations of acrylamide (AAm) and bis-acrylamide (Bis) of the PAA substrates used in this study and the corresponding storage moduli G PSI-7977 distributor , measured by rheometry from the series of substrates UV polymerized with different percent grayscale masks. b The graph plots the same data, with percent grayscale masks, mapped to their corresponding sequential positions found on radial gradient substrates. Red dashed lines show the best fit linear regressions of data for the steep gradient (r2=0.940) and shallow gradient (r2=0.974). Black dotted line represents the equation y=0 for visual reference Substrate surface characterization was performed to verify that mechanically uniform and gradient substrates were similar with respect to laminin ligand density and topography, two variables which can also influence Schwann cell phenotype [21, 22] and migration . No difference in protein coating was observed either between substrates or across the length of gradient materials (Additional file?2). Similarly, SEM analysis of the cell-material interface between Schwann cells adherent to both uniform and gradient substrates revealed a homogeneous surface with no visible topographical differences between the substrate surfaces (Fig.?2). These observations indicated that Schwann cell behavior was not influenced by differences in either matrix ligand density or topography between the uniform and gradient substrates. Open in a separate window PSI-7977 distributor Fig. 2 a, b Relative to Schwann cells cultured PSI-7977 distributor on a uniform substrates (4325 Pa), Schwann cells cultured on b steep gradient (243-4325 Pa) substrates had increased expression of paxillin (red), which co-localized to actin staining (green), indicating increased focal adhesion formation. Scale bar represents 100 0.01, ** for 0.001, and *** for 0.0001, assessed by Kruskal-Wallis one-way ANOVA with Dunns post-test Schwann cells altered their morphology in the presence of a steeper mechanical gradient Qualitatively, Schwann cells cultured on the steep gradient substrate had a distinct morphologic phenotype compared to those cultured on the uniform substrates (Fig.?2). In Schwann cells adherent to the steep gradient substrate, we observed increased paxillin staining, which was co-localized to F-actin, indicating an increase in the formation of focal adhesions, which are necessary.