Tissue engineering of corneal stroma with rabbit fibroblast precursors and gelatin hydrogels

PURPOSE: To isolate fibroblast precursors from rabbit corneal stroma using a sphere-forming assay, to engineer corneal stroma with the precursors and gelatin, and to establish the therapeutic application of precursors in a rabbit corneal stroma. METHODS: In the in vitro study, a sphere-forming assay was performed to produce precursors from rabbit corneal stroma. Corneal stroma was engineered by cultivating precursors in porous gelatin for one week. In the in vivo study, the engineered corneal stromal sheet with precursors (precursor/gelatin group) or with fibroblasts (fibroblast /gelatin group) or without cells (gelatin group) was transplanted to a pocket of rabbit corneal stroma. Gene expression and extracellular matrix production were examined immunohistochemically in each group one week and four weeks after surgery. RESULTS: In the in vitro study, cells in the spheres were BrdU-positive, and their progeny were keratocan-positive. The study also showed that the corneas transplanted with a porous gelatin sheet did not show any opacity four weeks after transplantation in any group. In the gelatin sheet of the precursor/gelatin group, a more intense expression of type I collagen was observed relative to the other two groups four weeks after the surgery. CONCLUSIONS: Our findings demonstrate that the transplantation of fibroblast precursors combined with gelatin hydrogel into the corneal stroma is a possible treatment strategy for corneal stromal regeneration.