Mapping of DNA methylation-sensitive cellular processes in gingival and periodontal ligament fibroblasts in the context of periodontal tissue homeostasis.

Interactions between gingival fibroblasts (GFs) and oral pathogens contribute to the chronicity of inflammation in periodontitis. Epigenetic changes in DNA methylation are involved in periodontitis pathogenesis, and recent studies indicate that DNA methyltransferase (DNMT) inhibitors may protect against epithelial barrier disruption and bone resorption. To assess the impact of DNMT inhibition on GFs, cells were cultured with decitabine (5-aza-2'-deoxycytidine, DAC) for 12 days to induce DNA hypomethylation. We observed several potentially detrimental effects of DAC on GF biological functions. First, extended treatment with DAC reduced GF proliferation and induced necrotic cell death. Second, DAC amplified Porphyromonas gingivalis- and cytokine-induced expression and secretion of the chemokine CCL20 and several matrix metalloproteinases (MMPs), including MMP1, MMP9, and MMP13. Similar pro-inflammatory effects of DAC were observed in periodontal ligament fibroblasts. Third, DAC upregulated intercellular adhesion molecule-1 (ICAM-1), which was associated with increased P. gingivalis adherence to GFs and may contribute to bacterial dissemination. Finally, analysis of DAC-induced genes identified by RNA sequencing revealed increased expression of CCL20, CCL5, CCL8, CCL13, TNF, IL1A, IL18, IL33, and CSF3, and showed that the most affected processes were related to immune and inflammatory responses. In contrast, the genes downregulated by DAC were associated with extracellular matrix and collagen fibril organization. Our observations demonstrate that studies of DNMT inhibitors provide important insights into the role of DNA methylation in cells involved in periodontitis pathogenesis. However, the therapeutic potential of hypomethylating agents in periodontal disease may be limited due to their cytotoxic effects on fibroblast populations and stimulation of pro-inflammatory pathways.

Immunoexpression of RANK, RANKL and OPG in sporadic odontogenic keratocysts and their potential association with recurrence.

BACKGROUND: Odontogenic keratocysts (OKCs) are clinically aggressive lesions with relatively high recurrence rates. Dysregulation of functional equilibrium in the RANK/RANKL/OPG system is responsible for osteolysis associated with the development of OKCs. Previously published findings imply that immunoexpression of these 3 proteins may correlate with bone resorption activity in OKCs. OBJECTIVES: The rationale behind this study was to assess the potential for receptor activator of nuclear factor kappa-B (RANK), receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) expression, as well as RANKL/OPG expression ratio, to serve as prognostic indicators for OKC recurrence. MATERIAL AND METHODS: We investigated the immunoexpression patterns of RANK, RANKL and OPG, and their correlation with recurrence rates, in 41 patients with OKCs treated with enucleation. RESULTS: We found no statistically significant differences between recurrent and non-recurrent cysts in terms of either: epithelial (p = 0.404) and stromal (p = 0.469) immunoreactivity of RANK; epithelial (p = 0.649) and stromal (p = 0.198) immunoreactivity of RANKL; or epithelial (p = 1) and stromal (p = 0.604) immunoreactivity of OPG. We also did not find significant differences in the distribution of cases with respect to ratios of RANKL/OPG immunostaining scores between recurrent and non-recurrent OKCs, both in the epithelium and in the connective tissue (p = 1 and p = 0.237, respectively). CONCLUSIONS: Our results suggest that immunoexpression levels of RANK, RANKL and OPG at the time of pathological diagnosis, as well as the RANKL/OPG ratio, are not useful as prognostic markers for OKC recurrence.