Epithelial regulation by regionally defined populations of oral fibroblasts

Methods: Human gingival specimens were microdissected and enzymatically separated into epithelial and connective tissue (periodontal ligament and oral-gingival) components. Cultured cells were characterised by staining with a panel of regionally specific differentiating antibodies. These unique cellular phenotypes were then incorporated into organotypic co-cultures in both a heterotypic and homotypic manner. mRNA derived from cell populations was subjected to TaqMan™ Q-PCR to quantify expression levels of cytokines known to be involved in epithelial growth and differentiation.

Results: Immunofluorescent staining indicated persistence of the in vitro phenotypes comparable to those of the ex vivo samples. Organotypic cultures produced epithelia that proliferated, stratified and expressed differentiation markers similar to their tissues of origin. Regionally-differing cross-recombination of fibroblasts and epithelia resulted in a shift of the epithelial phenotype dependant upon the underlying connective tissue type. Q-PCR revealed marked differences in cytokine levels for both fibroblast and keratinocyte populations.

Conclusions: Individual phenotypic cell populations persisted in culture providing evidence that the methods of cell isolation and organotypic culture provide a) characterized cell populations that b) interact to reform tissues with phenotypic profiles similar to those of junctional and gingival epithelia in vivo. Marked organotypic growth variations may be attributable to diverse cytokine/receptor levels involved in epithelial-mesenchymal interactions. Such varying cytokine levels may represent intrinsic differences in sub-sets of the population that are at greater or lesser risk of developing periodontal disease.

This work was supported by the Wellcome Trust.