Differential regulation of epithelial growth by gingival and periodontal fibroblasts in vitro.

Abstract

To investigate the underlying molecular mechanisms by which gingival and periodontal ligament (PDL) fibroblasts regulate epithelial phenotype. Fibroblast populations regulate the epithelial phenotype through epithelial-mesenchymal interactions (EMI). Previous studies have proposed that maintenance of the junctional epithelium (JE) is dependent on the differential effects from gingival and PDL tissues. However, these cell populations are undefined and the signalling mechanisms which may regulate JE are unknown. Immunohistochemical analyses were performed on formalin-fixed paraffin-embedded sections of dentogingival tissues to identify phenotypic differences in fibroblast populations. The effect of distinct fibroblasts on epithelial phenotype was studied via 3D organotypic cultures, consisting of an H400 epithelium supported by human gingival fibroblasts (HGF) or human periodontal ligament fibroblasts (HPDLF), embedded in collagen gel. To investigate the involvement of Wnt signalling in EMI, the Wnt antagonist rhDKK1 was added to HGF constructs. The gene expression of Wnt antagonists and agonists was tested via RNA extraction and qPCR. Specific gene silencing using RNA interference was performed on HPDLF/HGF constructs. Gingival fibroblasts were characterized by Sca1 expression, and PDL fibroblasts, characterized by Periostin and Asporin expression. Through the construction of 3D organotypic cultures, we showed that HGF supported epithelial multilayering, whilst HPDLF failed to support epithelial cell growth. Furthermore, HGF constructs treated with rhDKK1 resulted in a profound reduction in epithelial thickness. We identified SFRP4 to be highly specifically expressed in HPDLF, at both the mRNA and protein levels. A knockdown of SFRP4 in HPDLF constructs led to an increase in epithelial growth. The study demonstrates the presence of phenotypically distinct fibroblast populations within dentogingival tissues and that these specific populations have different influences on the epithelium. Our data suggest that a downregulation of Wnt signalling within PDL may be important in maintaining the integrity and anatomical position of the JE.