Substrate stiffness is one key factor to influence cell behavior because cells constantly probe the mechanical properties of their environment by exerting traction forces and receive the resistance to the induced deformation.1 Therefore, besides the stiffness on the top surface, the depth of a material that cells are able to sense is important in understanding cellular mechanosensitivity and designing cell carriers and biomedical devices. Poly(ε-caprolactone) triacrylate (PCLTA) recently developed in our group using a facile method is biodegradable and can be efficiently photo-crosslinked into hydrophobic networks with controllable mechanical properties.2 In this study, we used two PCLTAs with different molecular weights to prepare two-layer substrates for investigating nerve cell behavior. PCLTA2k was spin-coated and photo-crosslinked on the stiff network of crosslinked PCLTA20k with different coating thicknesses. A mirror system was fabricated in the same way. Rat Schwann cell precursor line (SpL201) cells with the glial nature was used to demonstrate the dependence of cell spreading and proliferation on the coating thickness. Besides the better support of SpL201 cells on stiffer substrate, the effective stiffness cells can respond depended on the coating thickness. A gradual change was found in SpL201 cell spreading and proliferation when the coating thickness increased. A critical coating thickness was required to completely screen the influence from the base layer and this value was higher for the softer top layer.

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