In Vitro Profiling of the Extracellular Matrix and Integrins Expressed by Human Corneal Endothelial Cells Cultured on Silk Fibroin-Based Matrices.

Abstract

Developing a scaffold for culturing human corneal endothelial (HCE) cells is crucial as an alternative cell therapeutic approach to bridge the growing gap between the demand and availability of healthy donor corneas for transplantation. Silk films are promising substrates for the culture of these cells; however, their tensile strength is several-fold greater than the native basement membrane which can possibly influence the dynamics of cell-matrix interaction and the extracellular matrix (ECM) secreted by the cells in long-term culture. In our current study, we assessed the secretion of ECM and the expression of integrins by the HCE cells on Philosamia ricini (PR) and Antheraea assamensis (AA) silk films and fibronectin-collagen (FNC)-coated plastic dishes to understand the cell-ECM interaction in long-term culture. The expression of ECM proteins (collagens 1, 4, 8, and 12, laminin, and fibronectin) on silk was comparable to that on the native tissue. The thicknesses of collagen 8 and laminin at 30 days on both PR (4.78 ± 0.55 and 5.53 ± 0.51 μm, respectively) and AA (4.66 ± 0.72 and 5.71 ± 0.61 μm, respectively) were comparable with those of the native tissue (4.4 ± 0.63 and 5.28 ± 0.72 μm, respectively). The integrin expression by the cells on the silk films was also comparable to that on the native tissue, except for α3 whose fluorescence intensity was significantly higher on PR (p ≤ 0.01) and AA (p ≤ 0.001), compared to that on the native tissue. This study shows that the higher tensile strength of the silk films does not alter the ECM secretion or cell phenotype in long-term culture, confirming the suitability of using this material for engineering the HCE cells for transplantation.