Establishment and application of human cultivated oral mucosal epithelial sheet for ocular surface reconstruction.

Abstract

Abstract: : Purpose: To report the establishment of cultivated oral mucosal epithelial sheet and the clinical outcome of its transplantation in severe ocular surface diseases. Methods: 2–3 mm² of oral biopsy specimens were obtained from the oral cavity and incubated with 1.2IU Dispase and 0.25% Trypsin–EDTA to form a single cell suspension. The oral epithelium suspension was co–cultured with inactivated 3T3 fibroblast for 2 weeks on a denuded amniotic membrane (AM), and air–lifted at the end of culture period. Immunohistochemical examination using anti–keratin 4, 13, and 3 antibodies and electron microscopy were performed to characterize the cultivated oral epithelial sheet. Cultivated oral mucosal epithelial sheets were clinically applied to reconstruct the ocular surface and conjunctival fornix in severe ocular surface diseases. Subjects consisted of 12 cases, 14 eyes (four eyes with Stevens–Johnson syndrome, five with a chemical injury, two with ocular cicatricial pemphigoid and three with idiopathic). Survival of transplanted oral mucosal epithelium was monitored by slit–lamp examination with fluorescein staining. Results: Cultivated oral mucosal epithelial sheet grown on denuded AM showed 5–6 layers of non–keratinized stratification which expressed keratin 4/13 as well as cornea–specific keratin 3. Electron microscopic examination revealed differentiated junctional structures and microvillis. Two days after transplantation, all cases showed the survival of transplanted oral mucosal epithelium on ocular surface and its phenotype was distinguishable from the conjunctival epithelium by fluorescein staining. Ectopic transplanted oral epithelium on the corneal surface showed no evidence of returning to an in vivo oral tissue phenotype however, mild peripheral neovascularization during postoperative period was observed. Conclusions: The tissue engineered procedure to generate transplantable cultivated oral mucosal epithelial sheet was established. Ocular surface was reconstructed using this non–ocular epithelium without returning to an in vivo oral tissue phenotype. Clinical application of autologous oral mucosal epithelial transplantation reduces post–operative complications associated with immunosuppressions and is beneficial for severe cases.

Keywords: cornea: clinical science • conjunctiva • cornea: epithelium