Preliminary Study On Treatment Of Rabbit Cornea Injury By Human Bone Marrow Mesenchymal Stem Cells Carrying On Porcine Corneal Matrix

Objective To culture human bone marrow mesenchymal stem cells (MSCs) in vitro and study the possibility of porcine corneal matrix seeded with MSCs for the reparation of injury rabbit corneas.Methods MSCs were isolated from the removed human bone during the sacrectomy and purified by whole bone marrow adherence method and passaged in DMEM containing fetal bovine serum in vitro. The immunophenotype of cultured MSCs was evaluated by flow cytometry. The differentiated adipocytes and osteoblasts from the MSCs by directional induce was identified using AgNO3 staining and Oil red O stain respectively. The generations 3 of MSCs were cultured on porcine corneal matrix without corneal epitheliaum for 4 days and then transplanted onto 14 left eyes of 14 New Zealand white rabbits, and porcine corneal matrix without corneal epithelium was transplanted onto the left eyes of other 14 New Zealand white rabbits as control group. Rabbits cornea transparency and neovascularization in experimental group and control group were observed after 2,4,8 weeks of transplantation. The growth of MSCs and porcine corneal matrix was evaluated by histopathological examination after 2,4,8 weeks. The expression of cytokeratin 12(CK12), a marker for corneal epithelial cells, on MSCs was identified by immunohistochemistry and immunofluorescent stain.Results Cultured MSCs showed the positive response for CD29 (95.97%), CD44 (96.49%), CD90 (92.79%), CD 105(94.66%), CD34(0.59%), CD45(0.36%). Differentiated osteoblasts presented the black stain for AgNO3 and adipocytes displayed the orange red color for Oil red O. MSCs grew well on the porcine corneal matrix. The corneal grafts survived well without rejection till the fourth week after transplantation in experimental eyes and the rejection of corneal graft occurred in control eyes. Lots of CK12 positive cells were found in transplanted cornea in experimental eyes and absent expression of CK12 was in control eyes.Conclusion The porcine corneal matrix seeded with MSCs can survive well in transplanted corneal stroma of rabbits, reduce the neovascularization and improved corneal transparency in experimental group. The MSCs can differentiate into corneal epithelium-like cells. It is a feasible model of constructing tissue engineered cornea.