The Effect of Micro- and Nanoscale Surface Topographies on Silk on Human Corneal Limbal Epithelial Cell Differentiation.

We previously reported that micro- and nano-scale topographic pitch created on silk films mimic features of the corneal basement membrane by providing biophysical cues to direct corneal epithelial cell adherence and migration. However, the effect of these topographical features on corneal limbal epithelial cell differentiation has not been explored. We hypothesize in the current study that various topographical pitch created on silk may affect corneal epithelial stem cell differentiation and alter the expression of genes involved in cell differentiation and self-renewal. We patterned silk films with different topographic pitch via soft lithography and observed human corneal limbal epithelial cell behavior. Colony forming assay demonstrated increased colony forming efficiency on patterned silk films. Cells cultured on nanoscale patterned silk films also expressed lower levels of putative keratocyte differentiation markers and higher levels of putative limbal stem cell markers. RNA-Seq analysis further implicated the involvement of pathways related to stem cell differentiation and self-renewal, including Notch, ERK/MAPK and Wnt/β-catenin signaling. We conclude that patterned silk film substrates can be used as scaffolds and provide biophysical cues to corneal limbal stem cells that may maintain corneal epithelial stem cells at a less differentiated state.