Abstract
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. Human corneal epithelium was cultivated from donor limbal explants on corneal stromal discs prepared by FDA approved Horizon Epikeratome system. The morphology, phenotype, regenerative capacity and transplantation potential were evaluated by hematoxylin eosin and immunofluorescent staining, a wound healing model, and the xeno-transplantation of the corneal constructs to nude mice. 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 superficial layers. The epithelium displayed a phenotype similar to human corneal epithelium in vivo. The differentiation markers, keratin 3, involucrin and connexin 43, were expressed in full or superficial layers. Interestingly, certain basal cells were immunopositive to antibodies against limbal stem/progenitor cell markers ABCG2 and p63, which are usually negative in corneal epithelium in vivo. It suggests that this bioengineered 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 well 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. 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 may have great potential for clinical use in corneal reconstruction.
Highlights
Ocular surface diseases with corneal epithelial stem cell deficiency, such as Stevens-Johnson syndrome, chemical, thermal and radiation injuries, extensive microbial infection, and inherited disorders such as aniridia, are sight threatening and often cause blindness
When stem cell-containing limbal explants cultured on corneal stromal discs, the human limbal epithelial cells (HLECs) grew earlier, reached confluent more rapidly, and in all cases formed stratified multi-layer epithelia after air-lifting for 5–7 days, in comparison with the limbal explants grown on culture inserts alone as a control
Our study suggests that the donor corneas that do not meet the criteria for direct clinical transplantation are a potential treasure for corneal tissue regeneration by using their natural stroma as a high quality optical substrate
Summary
Ocular surface diseases with corneal epithelial stem cell deficiency, such as Stevens-Johnson syndrome, chemical, thermal and radiation injuries, extensive microbial infection, and inherited disorders such as aniridia, are sight threatening and often cause blindness (see review [1]). The first clinical success of the cultivated corneal epithelial transplantation was noted in a report of two patients published by Pellegrini and colleagues in 1997 [5]. After this initial report, more investigators reported transplantation of in vitro cultivated corneal epithelium on the substrate carriers using amniotic membrane [6,7,8], fibrin [9,10], and collagen hydrogel [11]. Ideal substrates should be optically transparent, should support corneal epithelial progenitor cell survival, should promote cell differentiation to form a native-like stratified epithelium, and should serve as a carrier for easy transplantation to patients
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