Addition of Microscale Topographies to Silk Fibroin Film Modulates Corneal Endothelial Cell Behavior

Biomimicry in tissue engineering has been used to improve the function of a structure by closely replicating the native architecture. One such method is the introduction of micro- and nanotopographical patterns on biomaterials that mimic the native extracellular environment to enhance cell behavior and function before and after clinical transplantation. Earlier studies from our laboratory had shown that silk fibroin films offer promising potential for corneal endothelial regeneration because of their optimum optical, mechanical, and functional properties. In this study, we hoped to improve upon the design by incorporating micropatterns that are present in the native tissue on the surface of silk films. Fibroin protein from Antheraea assamensis worms was used to prepare films with and without patterns (hexagons and microgrooves) on their surface. The mechanical and optical properties of these films were analyzed by measuring the Young's modulus and light transmittance in the visible spectrum. Cell adhesion and proliferation were determined using the MTT assay and Ki67 staining, respectively. Morphometric analysis of cell shape and size was performed using the ImageJ software, and the expression of markers was visualized and quantified using immunostaining and Western blot. Patterned films demonstrated enhanced elasticity, roughness, and hydrophilicity compared to flat films. No significant difference was observed in cell adhesion between the flat and patterned films. The percentage of proliferating cells was significantly reduced on the patterned films, especially on hexagons. The cell area and circularity on flat films were comparable to microgrooves, whereas cells on hexagons displayed larger and more variable sizes. Notably, the expression of Na-K ATPase (a critical pump protein) was significantly higher in cells grown on microgrooves than on other substrates. These findings suggest that incorporating simple micropatterns on the surface of silk fibroin films can improve the morphology and functional quality of corneal endothelial cells, providing insights into the development of biomaterial-based strategies for endothelial transplantation.