Tissue-engineered human corneal endothelial cell sheet transplantation in a rabbit model using functional biomaterials

Background. This study was performed to investigate whether transplantation of bioengineered human corneal endothelial cell (HCEC) sheet grafts into corneas denuded of endothelium could restore corneal function and clarity in a rabbit model. Methods. After being labeled with PKH26 fluorescent dye, the adult HCECs derived from eye bank corneas were cultivated on the thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) -grafted surfaces for 3 weeks at 37 degrees C, and were harvested as transplantable cell sheets after incubation for 45 min at 20 degrees C. Attached by gelatin hydrogel discs, the bioengineered cell monolayers were transplanted to rabbit corneas denuded of endothelium (HCEC sheet group). Traumatized rabbit corneas were served as controls. Postsurgical corneas under-went clinical observations and histological examinations for 6 months. Results. By transmission electron microscopy and Western blot analysis of zonula occludens- 1 and Na+,K+-adenosine triphosphatase proteins, the structure and function of HCEC sheets resembled those of native corneal endothelium. After endothelial cells were removed, corneas of each group turned severe edematous and opaque. In the HCEC sheet groups, corneal clarity was gradually restored and corneal thickness was significantly less than that in the control groups (P<0.05). The attached PKH26-positive HCECs spread on rabbit Descemet's membrane after receiving cell sheet grafts. lntraocular delivery of HCEC sheets by means of a minimally invasive technique (i.e., small-incision surgery using biodegradable hydrogels) demonstrated long-term graft integration with damaged corneas. Conclusions. These results indicate that using cultured HCECs and functional biomaterials, PNIPAAm and gelatin, an effective cell sheet-based therapy can be developed for the treatment of corneal endothelium deficiency.