[Reconstruction tissue-engineered corneal epithelium using xenogeneic acellular corneal stroma as scaffold].

OBJECTIVE

To compare two methods for preparing acellular corneal stroma and evaluate the possibility of culturing corneal epithelium on xenogeneic acellular corneal stroma.

METHODS

Experimental study, applying completely randomized design method. Dispase followed by Triton-X-100 detergent and sodium chloride SDS detergent followed by trypsinase were applied respectively to treat the rabbit cornea. The characteristics of corneal stroma and acellular status after treatment were examined with slit lamp, optical microscope and transmission electron microscope. The rabbit limbal cells were then cultured on the acellular porcine Bowman's membrane/stroma. Rabbit corneal epithelium lamella reconstructed in vitro and evaluated in morphology, histopathology and immunohisto-chemistry.

RESULTS

Acellular corneal stroma prepared by two different methods is quite similar in morphology, being gray and opaque with visible edema and soft texture. Collagen fibers of the stroma were regularly in histopathology and ultrastructure. But the one prepared by the NaCl-SDS-Trypsinase method retained a amounts of cell debris, while there was none in the other did by Dispase-Triton-X-100 method. The limbal cells began to shift out at 24 hours after being inoculated on xenogeneic acellular corneal stroma, then attached on it, formed a confluent monolayer containing normal-appearing in 7 days. The tissue engineering corneal epithelium was cryosectioned and characterized immunohistochemically at 14 days after inoculation. Meanwhile, epithelium associated antigen CK3 in endochylema was stained.

CONCLUSIONS

Dispase-Triton-X-100 was proved better in obtaining acellular corneal stroma. It is possible to reconstruction tissue-engineered rabbit's corneal epithelium on acellular porcine corneal Bowman's membrane/stroma.