The influence of oral fibroblasts on determination of regional specificity of oral epithelium

The oral mucosa shows remarkable regional variation in its morphology and pattern of differentiation. However, it is not clear if this regional specificity is regulated by fibroblasts in the sub-epithelial connective tissue or is an intrinsic property of the surface epithelium. Objectives: The goal of this study was to use organotypic tissue models, which mimic an in vivo oral mucosa, to further understand how interactions between keratinocytes and fibroblasts may determine the phenotype of the oral surface epithelium. Methods: To accomplish this, oral keratinocytes and fibroblasts were harvested from four intraoral sites (buccal mucosa, attached gingival, hard palate and floor of mouth) from five Caucasian male patients between the ages of 22 to 26. Mucosal keratinocytes were co-cultured with fibroblasts from 1 - the same site and the same individual (control cultures), 2 - different sites in the same individual, 3 - the same site in different individuals and 4 – different sites in different individuals. Results: Co-culture combinations were grown in a novel organotypic culture system which was constructed by seeding fibroblasts in a contracted Type I collagen gel. When this contracted collagen gel was then overlain with an acellular, cadaver-derived connective tissue (AlloDermTM), fibroblasts migrated from the gel into the upper dermis. Oral keratinocytes were then seeded directly onto this connective tissue which demonstrated preserved basement membrane organization. We are currently characterizing the phenotype of the epithelium grown in these organotypic co-cultures by immunohistochemical staining for markers of cell proliferation (BrdU incorporation), keratinocyte differentiation (keratins 1 and 4), apoptosis (TUNEL assay) and tissue morphology. Conclusion: By manipulating oral keratinocytes and fibroblasts in an improved organotypic culture model which simulates an in vivo oral mucosa, we will directly determine how the regional-specific properties of the oral epithelium at particular intraoral sites are dependent on the influence of local fibroblasts.