An in vitro oral mucosal model reconstructed from human normal gingival cells

Abstract

In order to develop in vitro models which are similar to native tissues, human oral mucosal models were reconstructed from gingival cells in three-dimensional cultures and the characteristics of the models were examined. Keratinocytes and fibroblasts from human normal gingival tissues were cultured in Keratinocyte Growth Medium (KGM) and in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) respectively. The models were reconstructed by seeding keratinocytes on contracted collagen gels containing fibroblasts (CCG), which corresponded to the lamina propria, and co-culturing in Reconstruction Medium composed of one part of DMEM supplemented with 10% FBS and three parts of 1.5mM CaCl2-added-KGM at the gas-liquid interface for 10 days. Stratified epithelial layers were formed on CCG, and terminal differentiation of the keratinocytes were recognized in the epithelial layers. Cell nuclei were still observed in the keratinized layers such as parakeratinized gingival tissues. Involucrin expression in the epithelial layers showed normal distribution by immunohistochemical detection. Transmission electron microscopic observation demonstrated that, in the epithelial layers, the formation of intercellular desmosomes and intermediate filaments in cytoplasms increased from the basal layer to the keratinized layers. These results suggest that oral mucosal models are well-differentiated and histologically similar to native tissues.