Oral Mucosal Epithelial Cells Grown on Porous Silicon Membrane for Transfer to the Rat Eye

Yazad D Irani,Keryn A Williams,Marek Jasieniak,Steven J P Mcinnes,Nicolas H Voelcker,Sonja Klebe

doi:10.1038/s41598-017-10793-1

Abstract

Dysfunction of limbal stem cells or their niche can result in painful, potentially sight-threatening ocular surface disease. We examined the utility of surface-modified porous-silicon (pSi) membranes as a scaffold for the transfer of oral mucosal cells to the eye. Male-origin rat oral mucosal epithelial cells were grown on pSi coated with collagen-IV and vitronectin, and characterised by immunocytochemistry. Scaffolds bearing cells were implanted into normal female rats, close to the limbus, for 8 weeks. Histology, immunohistochemistry and a multiplex nested PCR for sry were performed to detect transplanted cells. Oral mucosal epithelial cells expanded on pSi scaffolds expressed the corneal epithelial cell marker CK3/12. A large percentage of cells were p63+, indicative of proliferative potential, and a small proportion expressed ABCG2+, a putative stem cell marker. Cell-bearing scaffolds transferred to the eyes of live rats, were well tolerated, as assessed by endpoint histology. Immunohistochemistry for pan-cytokeratins demonstrated that transplanted epithelial cells were retained on the pSi membranes at 8 weeks post-implant, but were not detectable on the central cornea using PCR for sry. The pSi scaffolds supported and retained transplanted rat oral mucosal epithelial cells in vitro and in vivo and recapitulate some aspects of an artificial stem cell niche.

Highlights

The mammalian cornea is covered by specialised, non-keratinised epithelial cells that regenerate throughout life from adult stem cells located primarily at the limbus
We have previously shown that human corneal epithelial cells can be grown on pSi membranes coated with rat tail collagen, and that aminosilanised pSi membranes implanted under the rat conjunctiva do not erode the surrounding tissue[41]
Of the oxidised pSi membranes revealed a thickness of 276 μm (Fig. 1a) and an average pore size of 21.2 ± 6.7 nm (Fig. 1b)

Summary

Introduction

The mammalian cornea is covered by specialised, non-keratinised epithelial cells that regenerate throughout life from adult stem cells located primarily at the limbus. Bilateral disease necessitates allogeneic limbal stem cell transplantation, in which either tissue or ex vivo-expanded cells sourced from cadaveric human donors is used[9, 10]. Oral mucosa (OM) is an attractive alternative source of autologous cells for corneal surface regeneration as it is non-keratinised[13], harbours adult epithelial stem cells[14, 15], and can be relatively harvested[16]. Oral mucosal epithelial cells obtained from biopsies and expanded on various scaffolds have since been transplanted as “bandages” to treat human ocular surface disease[21, 22]. The use of autologous oral mucosal epithelial cells to repopulate a stem cell niche has far received little attention Reconstruction of such a niche requires a source of cells, and an implantable scaffold to support the transplanted cells. We have previously shown that human corneal epithelial cells can be grown on pSi membranes coated with rat tail collagen, and that aminosilanised pSi membranes implanted under the rat conjunctiva do not erode the surrounding tissue[41]

Methods

Results

Conclusion