Culture and characterization of rat junctional epithelium

Abstract

Junctional epithelium, which forms the interface between the gingival tissues and the teeth, is a unique nondifferentiating, nonkeratinizing simple epithelium with essentially no gradient of cytoplasmic alterations. The purpose of this study was to develop methods for obtaining and culturing junctional epithelial cells and characterizing these cells with monoclonal antibodies and by keratin analysis. Microsurgical methods were developed to obtain pure specimens of junctional epithelium from the interproximal molar regions of pathogen‐free rats. Specimens of palatal and free gingival epithelia were also taken. The tissue explants were cultured by three methods; an explant technique using 3T3 fibroblasts as feeder cells, a procedure which used cloning rings to isolate primary epithelial outgrowths, and growth in small chambers coated with solubilized basement membrane. Specimens from the three oral epithelia grew well by all methods and appeared homogenous by phase contrast microscopy. Immunocytochemical analysis of fresh specimens by antikeratin antibody staining showed differences in the staining patterns of the three oral epithelia and suggested the absence of 54 and 40 kD keratins from these tissues. Junctional, palatal and gingival epithelial cells grown by the cloning ring technique stained positively with the monoclonal antibody 34βE12 which stains all stratified epithelia and identifies 66 and 57 kD keratins, but failed to stain with antibodies to vimentin and desmin which are intermediate filament proteins in mesenchymal and muscle cells, respectively, Junctional epithelial cells grown on solubilized basement membrane showed positive staining with antidesmoplakin antibodies I and II, which identify desmosomal plaque proteins, and negative staining with the keratin‐specific monoclonal antibody AE2. Gingival cells grown by the same technique had the opposite staining pattern with these antibodies. Cells grown by the 3T3 feeder cell method were analyzed for their cytokeratin content by SDS polyacrylamide gel electrophoresis and immunoblotting using the keratin‐specific monoclonal antibodies AEl and AE3. The keratin patterns in freshly explanted tissues all differed, indicating that these oral epithelia are indeed different in vivo. Further, the keratin patterns in extracts of subconfluent and confluent cultured oral epithelial cells differed from those in the freshly isolated tissues, but were similar for the different epithelia. Specifically, subconfluent cultures of the three epithelia had identical keratin patterns, but differed from those of confluent cultures which were themselves identical. This report presents methods for obtaining and culturing junctional epithelium, and data which indicate that junctional cells differ from other oral epithelia in cytokeratin and desmosomal protein composition.