Thickness sensing of nerve cells on double-layered hydrophobic polymer networks with distinct mechanical properties

摘要

Rat Schwann cell precursor line (SpL201) cells with the glial nature were used to demonstrate the dependence of cell spreading and proliferation on the coating thickness of two-layer substrates. Besides the better support of SpL201 cells on stiffer substrate, the effective stiffness cells can respond depended on the coating thickness. 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. Two poly(E-caprolactone) triacrylates (PCLTAs), PCLTA2k and PCLTA20k, were named after their number-average molecular weight of 2030 and 20020 g mol~(-1), respectively. Each substrate consisted of a top thin film layer and a base layer using either of these two PCLTAs. Different thicknesses for the top thin film layer have been achieved on the base layer. PCLTA2k and 20k were used in this study because of their distinct mechanical properties at 37°C after crosslinking. Crosslinked PCLTA2k was amorphous and soft with a tensile modulus of 4.8 ± 0.5 MPa, while crosslinked PCLTA20k was semi-crystalline and stiff with a much higher tensile modulus of 193 ± 16 MPa. SpL201 cell images on these two-layer polymer substrates at day 7 post-seeding are shown in Figure 1. Distinct cell proliferation was found for the substrates with different mechanical properties and coating thicknesses. Stiff crosslinked PCLTA20k was found to support SpL201 cell proliferation much better than soft crosslinked PCLTA2k. Much more cells were well spread on crosslinked PCLTA20k as the base material. As the thickness of soft PCLTA2k top layer increased, the cell number decreased gradually. When the PCLTA2k film was sufficiently thick, cells showed a similar density to that on pure PCLTA2k network. An opposite trend for SpL201 cell proliferation was demonstrated in the mirror system with soft PCLTA2k network as the base material. Cell spreading was shown in Figure 2. Two trends were dearly demonstrated on the two-layer substrates of distinct crosslinked PCLTAs. SpL201 cells could spread more with larger cell areas on the stiffer substrates of crosslinked PCLTA20k than on crosslinked PCLTA2k. Cell area did not differ significantly from the base material when the coating thickness was 870 nm for crosslinked PCLTA2k and 170 nm for crosslinked PCLTA20k. When the top layer became thicker, cell area gradually increased or decreased asymptotically to the value on the bulk form of the coating material. The critical coating thickness that could block the influence of the base material was ～2 μm for soft crosslinked PCUA2k and μ5 μm for stiff crosslinked PCUA20k. It indicated that the mechanical stiffness of the top layer could influence the critical coating thickness for cells to be able to sense. Figure 1. Fluorescence cell images for actin filaments (stained with RP, red) and nuclei (stained with DAPI, blue) at day 7 on the top layer of crosslinked PCLTA2k and 20k with different thicknesses covering the base layer of crosslinked PCLTA20k and 2k, respectively. Scale bar of 200 μm is applicable to all. Figure 2. Cell area of SpL201 cells at day 1 on the thin films of PCLTA2k and 20k with different thickness on a base layer of crosslinked PCLTA20k and 2k, respectively. Conclusions: We have used two PCLTAs with distinct mechanical properties to fabricate a series of substrates consisting of a top thin layer with controlled thickness from one PCLTA and a base layer of bulk networks from the other. The mirror systems demonstrated the roles of both thickness and stiffness of the top layer in mechanosensing of SpL201 cells. The stiffer substrate of crosslinked PCLTA20k could support both cell spreading and proliferation better than crosslinked PCLTA2k. A critical coating thickness for screening the effect of the base layer was higher for the softer network of crosslinked PCLTA2k, compared with crosslinked PCLTA20k.