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K. J. Mandell, U. V. Jurkunas, D. M. Kim, R. Dana; Cultivation of Gingival Epithelium for Corneal Surface Reconstruction. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5727.
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Ocular disorders such as ocular cicatricial pemphigoid, Stevens-Johnson syndrome and alkali burns result in irreversible scarring of the cornea with depletion of corneal stem cells. Recent progress has been made in reconstruction of the corneal surface using oral mucosa, specifically that of buccal origin. Buccal tissue, however, is difficult to obtain from donors for experimental purposes, whereas gingival mucosa is routinely discarded during minor dental procedures and is readily available. In this study, we investigated the use of gingival epithelium as a potential source for reconstruction of the corneal surface.
Human gingival mucosa specimens were obtained as discarded tissue from routine dental procedures. Specimens were stored at 4º C in Dulbecco's Modified Eagle's Medium supplemented with antibiotic-antimycotic agents, 10% fetal bovine serum and L-glutamine. Using an established protocol, gingival epithelial cells were isolated by microdissection followed by digestion with dispase and trypsin/EDTA. Gingival epithelial cells were cultured for multiple passages on tissue culture-treated and collagen-coated polystyrene surfaces. For each passage, cell morphology and culture heterogeneity was assessed by phase contrast microscopy. After the third passage, gingival epithelial cells were cultured on glass coverslips, and expression of various cytokeratins was assessed by immunofluorescence confocal microscopy.
Epithelial cells were isolated from gingival mucosal specimens, and primary cultures were successfully established on both tissue culture substrates. Primary gingival epithelial cultures appeared to grow in well-organized clusters of flat, polygonal cells with well-defined margins. Immunohistochemistry demonstrated the expression of cytokeratin 3 and 13 in the epithelial cell clusters. Occasional staining with cytokeratin 12 was noted but was not confirmed in all the specimens. No major difference in cell morphology or culture heterogeneity was noted for gingival cultures grown on tissue culture-treated polystyrene as compared to collagen-coated polystyrene.
Primary cultures of gingival epithelium retained cell morphology and cytokeratin expression patterns typical of nonkeratinized oral epithelium. Cultivation of gingival mucosa provides a suitable model for study of epithelial cell ex vivo expansion for corneal surface reconstruction. Further experiments are required to optimize culture conditions in order to maximize epithelial expansion for transplant onto the corneal surface.
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