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Background/purpose: Cementum shares many properties with bone; however, in contrast to bone, it is not innervated or vascularized and has a limited capacity for remodeling. Osteocytes located in the lacunae-canalicular system of bone tissue play a central role in bone remodeling by communicating with osteoblasts and osteoclasts. Although cementocytes are present in cellular cementum and are morphologically similar to osteocytes, it remains unclear whether they are involved in the dynamic functional regulation of metabolism in cementum. The present study focused on the extracellular vesicles (EVs) secreted by cementocytes and examined their effects on osteoclasts and osteoblasts. Materials and methods: EVs were extracted from the mouse cementocyte cell line, IDG-CM6. The effects of EVs on recombinant RANKL-induced osteoclastogenesis and recombinant Bone morphogenetic protein (BMP)-2-mediated osteoblastogenesis were investigated using the mouse osteoclast progenitor cell line, RAW264.7 and mouse pre-osteoblast cell line, MC3T3-E1, respectively. Results: EVs enhanced the formation of tartrate-resistant acid phosphatase activity-positive cells. Real-time PCR revealed that EVs up-regulated the expression of osteoclast-related genes. On the other hand, the cell culture supernatant of cementocytes significantly inhibited the differentiation of osteoclasts. Regarding osteoblastogenesis, EVs suppressed the expression of alkaline phosphatase, bone sialoprotein, and osteocalcin induced by recombinant BMP-2 at the gene and protein levels. Conclusion: A network of cementocytes, osteoblasts, and osteoclasts may exist in cellular cementum, which suggests the involvement of cementocytes in dynamic metabolism of cementum through EVs.

Background/purpose: Periodontal breakdown in periodontitis is exacerbated by pro-inflammatory responses of periodontal stromal cells such as periodontal ligament fibroblasts (PDLFs). Procyanidin B2 (PB2) is a ligand of the peroxisome proliferator-activated receptor (PPARγ). Herein, we investigated the expression of PPARγ isoforms in PDLFs and periodontal tissue, and examined the effects of PB2 on PPARγ isoform-dependent antiinflammatory responses. Materials and methods: PPARγ isoforms were examined by PCR. PPARγ isoform-dependent inflammatory functions and anti-inflammatory effects of PB2 in PDLFs were evaluated based on IL-6 expression. Co-immunoprecipitation analysis of fixed chromatin-tethered protein (CoIPfctp) was conducted to investigate the association of each PPARγ isoform with the NF-κB-transcriptional complex. The effects of PB2 on periodontitis progression were evaluated using a ligature-induced murine periodontitis model. Results: Three isoforms of PPARγ were expressed in PDLFs and periodontal tissues, consisting of the main full-length isoform (PPARγ) and two dominant negative isoforms that lack the ligand binding domain, namely the ubiquitously-expressed isoform (PPARγ-UBI) and unknown isoform (PPARγ-PDL). PPARγ and PPARγ-UBI were predominantly expressed. CoIP-fctp revealed that PPARγ-UBI was selectively associated with NF-κB p65, a key transcriptional factor of IL-6 expression. PB2 suppressed LPS-induced-IL-6 expression exacerbated by the over-expression of PPARγ-UBI. In the murine periodontitis model, topical application of PB2 significantly mitigated alveolar bone loss. Conclusion: These results suggest that the anti-inflammatory effects of PB2 in periodontal tissues/cells are distinct, and these effects arise from the inhibition of PPARγ-UBI; hence, the application of PB2 and modification of the splicing event in three PPARγ isoforms have therapeutic potential for preventing periodontitis.

Background/purpose: The acetylation of histone H3 proteins keeps local chromatin regions open and accessible, thereby facilitating transcriptional events. We recently reported integrative epigenomic and transcriptome analyses of differentiating dental pulp stem cells (DPSCs). A significant increase in the number of super-enhancers, which are local genomic locations marked by condensed open chromatin peaks that facilitate transcriptional events, in differentiating DPSCs were observed. However, it is still unclear whether histone deacetylase (HDACs) inhibitors (HDACis) have beneficial effects on the odontogenic differentiation of DPSCs and on the matrix mineralization-inducing ability of DPSCs. Materials and methods: DPSCs were cultured in an odontogenic induction medium for a prolonged period in the presence of HDACis, MS-275 and Trichostatin A (TSA). ATAC-seq and RNA-seq samples were collected from differentiating DPSCs to explore the epigenomic and transcriptomic alterations induced by HDACis and identify key target proteins that mediate HDACis-induced phenotypic changes. Results: MS-275 and TSA did not change whole-genome open chromatin accessibility or increase odontogenic differentiation, as assessed by alkaline phosphate activity. However, the matrix mineralization-inducing ability assessed by calcified nodule formation was significantly increased by MS-275 but not by TSA. FN1, which encodes fibronectin, was identified as upregulated by MS-275. The knockdown of fibronectin evidently suppressed MS-275-induced calcified nodule formation. Conclusion: MS-275 induced calcified nodule formation by the mechanistic upregulation of FN1, independent of epigenomic alterations. Hence, the application of MS-275 as direct capping materials has therapeutic potential for promoting reparative dentin formation by constructing a fibronectin-organizing physiological extracellular matrix environment that is adequate for matrix mineralization.