Membranes Prepared from Recombinant RGD-Silk Fibroin as Substrates for Human Corneal Cells.

Elham Nili,Damien G Harkin,Rebecca A Dawson,Neil A Richardson,Traian V Chirila,Shuko Suzuki

doi:10.3390/molecules26226810

Abstract

A recombinant formulation of silk fibroin containing the arginine–glycine–aspartic acid (RGD) cell-binding motif (RGD-fibroin) offers potential advantages for the cultivation of corneal cells. Thus, we investigated the growth of corneal stromal cells and epithelial cells on surfaces created from RGD-fibroin, in comparison to the naturally occurring Bombyx mori silk fibroin. The attachment of cells was compared in the presence or absence of serum over a 90 min period and analyzed by quantification of dsDNA content. Stratification of epithelial cells on freestanding membranes was examined by confocal fluorescence microscopy and optimized through use of low molecular weight poly(ethylene glycol) (PEG; 300 Da) as a porogen, the enzyme horseradish peroxidase (HRP) as a crosslinking agent, and stromal cells grown on the opposing membrane surface. The RGD-fibroin reduced the tendency of stromal cell cultures to form clumps and encouraged the stratification of epithelial cells. PEG used in conjunction with HRP supported the fabrication of more permeable freestanding RGD-fibroin membranes, that provide an effective scaffold for stromal–epithelial co-cultures. Our studies encourage the use of RGD-fibroin for corneal cell culture. Further studies are required to confirm if the benefits of this formulation are due to changes in the expression of integrins, components of the extracellular matrix, or other events at the transcriptional level.

Highlights

Fibroin is the protein responsible for the mechanical properties of silk fibers and can be readily isolated from the cocoons of different Lepidopteran species, mainly from those produced by the domesticated silkworm Bombyx mori
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The recombinant RGD-fibroin developed by Kambe et al [20,21] displays advantages for the cultivation of corneal cells when compared to the non-modified fibroin (BMSF)

Summary

Introduction

Fibroin is the protein responsible for the mechanical properties of silk fibers and can be readily isolated from the cocoons of different Lepidopteran species, mainly from those produced by the domesticated silkworm Bombyx mori. This fibrous protein (BMSF) is a biocompatible and biodegradable material which, owing to its remarkable physicochemical properties, has been employed as a biomaterial in a variety of tissue engineering and biomedical applications [1,2,3,4,5,6,7]. BMSF is a composite of naturally designed block copolymers. The BMSF templates have been explored as a material for repairing the ocular surface, based on their high transparency and greater stability than the membranes prepared from other biomaterials [2,5,11,12,13,14]

Methods

Results

Discussion

Conclusion