HISTOMORPHOLOGICAL AND BIOCHEMICAL DIFFERENTIATION CAPACITY IN ORGANOTYPIC COCULTURES OF PRIMARY GINGIVAL CELLS

To establish a three-dimensional in vitro test system mimicking the ph ysiological situation of the oral cavity, organotypic co-cultures cons isting of primary gingival cells on a collagen matrix with fibroblasts were generated. The histomorphological development after 7 and 14 d r evealed close similarity with the non-keratinized gingiva epithelium. Furthermore, as epithelial specific markers synthesis and localization of keratins as well as the deposition of basement membrane components were assessed on frozen sections by immunofluorescence and keratin ex pression by in situ hybridization. Primary keratinocytes in convention al culture stained positive for keratin K14 and the mucosal differenti ation-specific keratins K4 and K13. while primary fibroblasts, isolate d from the same tissue source, and also some keratinocytes, were posit ive for vimentin. In organotypic co-cultures the keratinocytes formed a multilayered epithelium within 14 d containing basal cells and flatt ened cells in the uppermost layers. Comparable to native non-keratiniz ed gingiva keratin 14 gene expression was clearly detectable in the ba sal cell compartment but showed extending immunolocalization. In addit ion, particularly at the early stage (7 d), basally located keratinocy tes were also vimentin positive. According to morphological differenti ation K4 and K13 were detectable in suprabasal position at the RNA and protein level. The major basement membrane constituents collagen type IV and laminin increased with time revealing first an interrupted and later a fully extended staining underneath the basal cells. Maintenan ce of basal cell function was further demonstrated by cell proliferati on (BrdU incorporation) which was initially high (7 d) but declined to wards the later stages (14-21 d). The results demonstrate i) that this co-culture system leads to a stratified surface epithelium with morph ological and biochemical characteristics of the non-keratinized gingiv a epithelium and ii) that a state of physiological tissue balance was reached, thus rendering a suitable model for tissue compatibility stud ies.