In vitro culture of human corneal endothelium on non-mulberry silk fibroin films for tissue regeneration

Abstract

Purpose: The purpose of this study was to determine if non-mulberry varieties of silk are suitable for the culture of corneal endothelium (CE). Methods: Aqueous silk fibroin derived from Philosamia ricini (PR), Antheraea assamen-sis (AA), and Bombyx mori (BM) were cast as approximately 15 μm films with and without pores on which human CE cells were cultured. Tensile strength, elasticity, transmittance in visible range, and degradation properties of the films were characterised. Adhesion of CE to the silk films was quantified using MTT assay in addition to quantifying the number and area of focal adhesions using paxillin. Expression of CE markers was determined at the gene and protein levels using PCR and immunostaining, respectively. Barrier integrity of the cultured cells was measured as permeability to FITC dextran (10 kDa) in the presence or absence of thrombin. Results: The films exhibited robust tensile strength, >95% transmittance and a refractive index comparable to the native cornea. BM degraded significantly faster when compared to PR and AA. A comparison between the three varieties of silk showed that significantly more cells were adhered to PR and AA than to BM. This was also reflected in the expression of stable focal adhesions on PR and AA, thus enabling the formation of intact monolayers of cells on these varieties unlike on BM. Treatment with thrombin significantly increased cellular permeability to dextran. Conclusions: Our data shows that PR and AA varieties sufficiently support the growth and function of CE cells. This could be attributed to the presence of natural cell binding motifs (RGD) in these varieties. Translational Relevance: Development of a suitable carrier for engineering the CE to address a major clinical requirement of healthy donor tissues for transplantation.