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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.

Introduction: Periodontal ligament is regenerated in association with hard tissue regeneration. Tenomodulin (Tnmd) expression has been confirmed in periodontal ligament and it reportedly inhibits angiogenesis or is involved in collagen fibril maturation. The introduction of Tnmd by gene transfection in bone tissue regeneration therapy might inhibit topical hard tissue formation and induce the formation of dense fibrous tissue. Therefore, the effect of Tnmd introduction by gene transfection technique in vitro and in vivo was investigated in this study. Methods: Osteogenesis- and chondrogenesis-related gene expression levels in osteoblastic cells (MC3T3E1) and rat bone marrow derived cells were detected using qPCR three days after gene transfection with plasmid DNA (Tnmd) using non-viral gene transfection vectors: a calcium phosphate-based gene transfection vector (CaP(Tnmd)) or a cationic polymer-based reagent (JetPEI (Tnmd)). Next, an atelocollagen scaffold with or without CaP (Tnmd) or JetPEI (Tnmd) was implanted into a rat calvaria bone defect, and the remaining bone defect volume and the tissue reaction at 28 days after surgery were evaluated. Results: Runx 2 and SP7 mRNA was reduced by JetPEI (Tnmd) in both cells, but not in CaP(Tnmd). The volume of expressed Tnmd was at 9 ng/mL in both gene transfection vector. The remaining bone defect volume of JetPEI (Tnmd) was significantly bigger than that of the other groups and CaP (EGFP), and that of CaP (Tnmd) was significantly bigger than that of CaP (EGFP). Conclusions: Tnmd introduction treatment inhibits bone formation in artificial bone defect, however, the effect of that was dependent on non-viral gene transfection vector.

Wnt signaling plays a central role in many processes during embryonic development and adult homeostasis. At least 19 types of Wnt ligands, receptors, transducers, transcription factors, and antagonists have been identified in mammals. Two distinct Wnt signaling pathways, the canonical signaling pathway and the noncanonical signaling pathway, have been described. Some Wnt signaling pathway components are expressed in the dental epithelium and mesenchyme during tooth development in humans and mice. Functional studies and experimental analysis of relevant animal models confirm the effects of Wnt signaling pathway on the regulation of developing tooth formation and adult tooth homeostasis. Mutations in some Wnt signaling pathway components have been identified in syndromic and non-syndromic tooth agenesis. This review provides an overview of progress in elucidating the role of Wnt signaling pathway components in the tooth and the resulting possibilities for therapeutic development.

Periodontal diseases are the most prevalent infectious diseases with the irreversible loss of tooth supporting apparatus. To reestablish the stable health of periodontal tissues after healing, the concept of regenerative surgical procedures has been developed over the past three decades, including various grafting materials, guided tissue regeneration (GTR), and the use of enamel matrix derivatives (EMD) in a clinical setting. More recently, tissue engineering strategies have also been applied and developed for periodontal regeneration: (1) stem cell therapies; (2) recombinant human growth factor therapies; (3) combined use of cell and growth factors with matrix-based scaffolds. However, the complete and predictable reconstruction of healthy periodontal tissues still remains a challenging field. To overcome therapeutic limitations and develop stem cell-based strategies, it is necessary to optimize cell–scaffold combinations by understanding the cellular events during periodontal wound healing and regeneration. We reviewed: (1) the current status and strategies for periodontal regeneration; (2) a possible biomaterial design for the scaffold used in periodontal tissue engineering; (3) a possible interaction between scaffold materials and periodontal tissue cells.