Optimization of Corneal Epithelial Progenitor Cell Growth on Bombyx mori Silk Fibroin Membranes.

Thomas A Hogerheyde,Jennifer Walshe,Damien G Harkin,Sally A Stephenson,Shuko Suzuki,Neil A Richardson,Laura J Bray

doi:10.1155/2016/8310127

Thomas A Hogerheyde, Jennifer Walshe + Show 5 more

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https://doi.org/10.1155/2016/8310127

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Abstract

Scaffolds prepared from silk fibroin derived from cocoons of the domesticated silkworm moth Bombyx mori have demonstrated potential to support the attachment and growth of human limbal epithelial (HLE) cells in vitro. In this study, we attempted to further optimize protocols to promote the expansion of HLE cells on B. mori silk fibroin- (BMSF-) based scaffolds. BMSF films were initially coated with different extracellular matrix proteins and then analysed for their impact on corneal epithelial cell adhesion, cell morphology, and culture confluency. Results showed that collagen I, collagen III, and collagen IV consistently improved HCE-T cell adherence, promoted an elongated cell morphology, and increased culture confluency. By contrast, ECM coating had no significant effect on the performance of primary HLE cells cultured on BMSF films. In the second part of this study, primary HLE cells were grown on BMSF films in the presence of medium (SHEM) supplemented with keratinocyte growth factor (KGF) and the Rho kinase inhibitor, Y-27632. The results demonstrated that SHEM medium supplemented with KGF and Y-27632 dramatically increased expression of corneal differentiation markers, keratin 3 and keratin 12, whereas expression of the progenitor marker, p63, did not appear to be significantly influenced by the choice of culture medium.

Highlights

Corneal epithelial progenitor cells are essential for maintenance of a smooth and transparent ocular surface
In contrast to results obtained using human corneal epithelial cell line (HCE-T) cells, extracellular matrix (ECM) coating had no significant effect on the adhesion of primary human limbal epithelial (HLE) cells to BMSF films (Figure 1(b))
Based on the evidence above and the response of HCE-T cells reported in the present study, we propose that coating B. mori silk fibroin- (BMSF-)based scaffolds with ECM proteins may increase the potential of this biomaterial to promote increased epithelial cell adhesion and growth

Summary

Introduction

Corneal epithelial progenitor cells are essential for maintenance of a smooth and transparent ocular surface. These so-called “corneal stem cells” are concentrated within the peripheral or limbal margin of the cornea [1] and express molecular markers typical of epithelial progenitor cells including the transcription factor p63 [2,3,4]. Strategies available for treating LSCD are based upon implantation of corneal epithelial progenitor cells derived from either an autologous or donor tissue source [11, 12]. The most popular choice of scaffold for cultivated corneal epithelial implants is denuded amniotic membrane (AM). Limitations associated with the use of AM, including poor transparency and risk of disease transmission [13], have encouraged the development of alternative scaffolds such as membranes derived from the silk structural protein, fibroin [14]

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