UBC Theses and Dissertations
In vitro model of periodontal epithelium Pan, Peter
To develop a model for junctional epithelium, porcine periodontal ligament epithelial cells (PLE cells) were cultured from the epithelial rests of Malassez. The obtained epithelial cell lines were grown on a porous membrane that allows a good cell adhesion and access of the nutrients from both basal and apical directions, and which enhanced the spatial development of the epithelium. The epithelial cells that were cultured for 7 days or longer formed 4 to 6 cell layers that had distinct characteristics of basal cells and stratified surface cells. Generally, one of the most important tools used to characterize the phenotype of epithelium is the analysis of cytokeratins. The expression of keratins by PLE cells in culture was similar to that of junctional epithelium in vivo. In older cultures, though, the cells expressed cornification markers and a cytokeratin pattern similar to that of sulcular epithelium in vivo. The biochemical and ultrastructural characterization of the extracellular matrix of epithelial cultures revealed the presence of a basal lamina that contained laminin but not type IV collagen, similar to the composition of the internal basal lamina (IBL) of junctional epithelium in vivo. Permeability of the epithelium was studied by assaying transepithelial resistance and penetration of different tracer molecules through the epithelium. It was found that the maximum permeability barrier, about 10 times that of the filter without the cells, was obtained when cells were plated at a high density and cultured for about 10 days in alpha-ME supplemented with 15% fetal calf serum. Larger molecules such as dextran 70 diffused more slowly through the epithelium than smaller molecules, e.g. methionine and phenol red. The permeability could be modulated by several means. These included treatment with phorbolmyristate acetate and hyaluronidase, and alteration of serum concentration in the culture medium. My model for junctional epithelium allowed the study of the effects of one possible periodontopathogen, Treponema denticola, on the epithelium. T. dent/cola caused rapid loss of extracellular spaces, vacuolization of cells, sloughing off of part of the surface cells and increased transepithelial permeability. These effects were found to result, at least partly, from effects of T. denticola surface proteins that rapidly diffused into the epithelial cells. The epithelial model was found to mimic reasonably well junctional epithelium. It provides a new means to study the behaviour of junctional epithelium in a controlled, reproducible and relatively easy way.
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