Date Published: November 15, 2012
Publisher: Public Library of Science
Author(s): Jing Lin, Kyung-Chul Yoon, Lili Zhang, Zhitao Su, Rong Lu, Ping Ma, Cintia S. De Paiva, Stephen C. Pflugfelder, De-Quan Li, Che John Connon. http://doi.org/10.1371/journal.pone.0049571
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.
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 ). Transplantation of a corneal limbal graft that contains corneal epithelial stem cells (also referred to as limbal stem cells) can help to restore vision. Although corneal transplantation has achieved clinical success, there is an increasing shortage of corneal donors worldwide. There are over 10 million global blinding patients caused by corneal disease due to the lack of cornea donors for corneal transplantation. Corneal tissue engineering is becoming an important discipline that holds great promise for corneal transplantation to treat the blinding corneal diseases –.
Limbal stem cells and optical substrates are known to be the most important factors for corneal tissue bioengineering in regenerative medicine . In the present study, human corneal stroma was used as a native optical substrate to reconstruct human corneas with stratified multilayer epithelium. The natural corneal stroma is a good substrate with two advantages: 1) providing appropriate scaffolds where the stem cells can proliferate and recapitulate native corneal structure and functions ; and 2) containing highly organized collagen lamellae that provides mechanical support and light diffraction properties appropriate for transparency , . 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.