A Native-like Corneal Construct Using Donor Corneal Stroma for Tissue Engineering

Abstract

Purpose: : Tissue engineering holds great promise for corneal transplantation to treat blinding diseases. This study was to explore the use of natural corneal stroma as an optimal substrate to construct a native like corneal equivalent with regenerated epithelium containing stem cells.

Methods: : Donor corneal stromal discs were prepared by FDA approved Horizon Epikeratome system. Human corneal epithelium was regenerated from donor limbal explants on the stromal discs in culture. The morphology, phenotype and regenerative capacity were evaluated by H&E and immunofluorescent staining as well as a wound healing model. The potential clinical application was evaluated by xeno-transplantation of the corneal constructs to nude mice.

Results: : An optically transparent and stratified epithelium was rapidly generated on donor corneal stromal substrate and displayed native-like morphology and structure. The cells were polygonal in the basal layer and became flattened in the superficial layers. The epithelium displayed a phenotype similar to human corneal epithelium ex vivo. The differentiation markers, keratin 3, connexin 43 and involucrin, were mainly expressed in the superficial layers, while progenitor markers, EGFR and integrin β1, were expressed more strongly by basal than superficial layers. Interestingly, certain basal cells were positively stained by antibodies against limbal stem cell-associated markers p63 and ABCG2, which are usually negative in corneal epithelial basal layer ex vivo. This suggests that this engineered corneal epithelium shared some characteristics of human limbal epithelium in vivo. This engineered epithelium was able to regenerate in 4 days following from a 4mm-diameter wound created by a filter paper soaked with 1 N NaOH. This corneal construct survived for 14 days after xeno-transplantation to the back of a nude mouse. The transplanted epithelium remained multilayer and became thicker with a phenotype similar to human corneal epithelium.

Conclusions: : Our findings demonstrate that natural corneal stroma is an optimal substrate for tissue bioengineering, and a native-like corneal construct has been created with epithelium containing limbal stem cells. This construct has great potential for clinical use in corneal reconstruction.

Keywords: cornea: epithelium • cornea: stroma and keratocytes • transplantation