[Ocular surface reconstruction by tissue engineering].

Abstract

Ocular surface reconstruction by tissue engineering using somatic stem cells is a second-generation modality. In order to treat bilaterally affected, severe ocular surface disorders, we investigated the transplantation of two types of cultivated mucosal epithelia: allogenic corneal epithelial stem cells, and autologous oral mucosal epithelial cells. For this, first, we summarized the clinical results of allogenic keratoepithelioplasty and limbal transplantation. In addition, we showed that the immunological shift from Th1 to Th2 by using keyhole limpet hemocyanin was effective in suppressing the incidence of immunological rejection. Second, we investigated the transplantation of cultivated human corneal epithelial stem cells onto amniotic membrane. The cultivated sheet was created by co-culture with 3T3 fibroblasts, using the air-lift method, in cultivating the corneal epithelial stem cell on the amniotic membrane. These cultivated cells demonstrated positive keratin 3 and 12 specific to in vivo corneal epithelium, tight junction related proteins, and telomerase activity. The transplanted allogenic human corneal epithelial sheet survived on the corneal surface in all cases, and was quite effective for achieving ocular surface stability in the acute phase of Stevens-Johnson syndrome, ocular cicatricial pemphigoid, or chemical injury. However, a few cases developed immunological rejection or opportunistic infection. Third, to establish the transplantation of the autologous cultivated oral mucosal epithelial sheet, we performed animal experiments using rabbits. In vitro oral mucosal epithelial sheet showed histology similar to that of in vivo corneal epithelial sheet. It expressed positive keratin 3 as well. Since the autologous transplantation of this sheet survived on the ocular surface with the recovery of corneal transparency, a cultivated oral mucosal epithelium may become a substitute for corneal epithelium. Fourth, we created a cultivated human corneal endothelial cell sheet on amniotic membrane using a similar technique, and transplanted it to a rabbit eye as a xenograft. The transplanted corneal endothelial cell density was over 3,000 cells/mm2, and it was actively functioning even after the transplantation. Lastly, to explore cell markers for corneal epithelial stem cells, we established a technique using laser micro-capture, and introduced amplified fragment length polymorphism (AFLP), identifying several candidate molecules as stem cell markers.