CULTURED AUTOLOGOUS ORAL MUCOSAL EPITHELIAL CELL SHEET GRAFTS FOR TREATMENT OF EXPERIMENTALLY-INDUCED LIMBAL STEM CELL DEFICIENCY

Abstract

Purpose: This pilot study investigates the effects of autologous oral mucosal cell sheet grafts for experimentally-induced limbal stem cell deficiency (LSCD) in rabbits

Methods: Severe LSCD was created by superficial surgical excision of all limbal tissue. Three months later, when rabbits showed stable corneal vascularization and opacification, oral mucosal biopsies were performed. Autologous oral mucosal cell sheets were engineered and transplanted onto the surface of the corneas. Follow-up exams were performed on transplanted cornea (n=5) and sham cornea (n=4), every 4-6 weeks for 6 months after transplantation, using slit lamp observation and photography

Results: Three out of five grafted corneas showed a decrease in superficial vascularization, while almost all the sham corneas did not show similar decrease. H&E staining showed similar squamous epithelium in the grafted corneas compared to a normal eye. The sham corneas showed more attenuated and abnormal epithelium with numerous areas of thinning, and other areas with erosions and complete absence of normal squamous epithelium. The sham cornea also showed very significant infiltration of mononuclear inflammatory cells. Progenitor stem cell marker deltaNp63 stained markedly in the grafted cornea and to a lesser extent in the sham cornea. Proliferating cell nuclear antigen (PCNA) and Ki-67 staining were much greater in the sham corneas compared to the grafted and normal corneas. K3 and K13 staining results show that oral mucosa epithelial cell sheet transplantation resulted in a regenerated epithelium in the grafted corneas compared to the sham corneas. Mu5ac was decreased in the central region of grafted corneas, compared to sham corneas. Very little alpha smooth muscle actin staining (a-SMA, which indicates the presence of myofibroblast cells) was present in grafted corneas, while there was more a-SMA staining in sham corneas. Staining for anti-angiogenic factor TIMP-3 was increased, and pro-angiogenic factor MMP-3 was decreased in grafted corneas compared to sham corneas.

Conclusions: In conclusion, our results indicate that autologous oral mucosa epithelial cell sheet grafts resulted in improved epithelialization of the corneal surface and decreased vascular fibrosis of the cornea

Keywords: 482 cornea: epithelium