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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.

Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is constitutively produced by endothelial cells and plays a vital role in maintaining vascular homeostasis. Chronic periodontitis is an inflammatory disease characterized by bleeding of periodontal tissues that support the tooth. In this study, we aimed to determine the role of PAI-1 produced by endothelial cells in response to infections caused by the primary periodontal pathogen Porphyromonas gingivalis. We demonstrated that P. gingivalis infection resulted in significantly reduced PAI-1 levels in human endothelial cells. This reduction in PAI-1 levels could be attributed to the proteolysis of PAI-1 by P. gingivalis proteinases, especially lysine-specific gingipain-K (Kgp). We demonstrated the roles of these degradative enzymes in the endothelial cells using a Kgp-specific inhibitor and P. gingivalis gingipain-null mutants, in which the lack of the proteinases resulted in the absence of PAI-1 degradation. The degradation of PAI-1 by P. gingivalis induced a delayed wound healing response in endothelial cell layers via the low-density lipoprotein receptor-related protein. Our results collectively suggested that the proteolysis of PAI-1 in endothelial cells by gingipains of P. gingivalis might lead to the deregulation of endothelial homeostasis, thereby contributing to the permeabilization and dysfunction of the vascular endothelial barrier.

Cementum resorption, unlike bone resorption, is clinically known to occur only with limited pathological stimuli, such as trauma, orthodontic forces, and large apical periodontitis; however, the molecular mechanisms that control osteoclast formation on the cementum surface remain unclear. In this study, we focused on extracellular vesicles (EVs) secreted by cementoblasts and analyzed their effects on osteoclast differentiation. EVs were extracted from the conditioned medium (CM) of the mouse cementoblast cell line OCCM-30. Transmission electron microscopy (TEM) analysis confirmed the presence of EVs with a diameter of approximately 50–200 nm. The effect of the EVs on osteoclast differentiation was examined using the mouse osteoclast progenitor cell line RAW 264.7 with recombinant receptor activator of nuclear factor (NF)-κB ligand (rRANKL) stimulation. EVs enhanced the formation of tartrate-resistant acid phosphatase (TRAP) activity-positive cells upon rRANKL stimulation. EVs also enhanced the induction of osteoclast-associated gene and protein expression in this condition, as determined by real-time PCR and Western blotting, respectively. On the other hand, no enhancing effect of EVs was observed without rRANKL stimulation. A Western blot analysis revealed no expression of receptor activator of NF-κB ligand (RANKL) in EVs themselves. The effect on rRANKL-induced osteoclast differentiation was examined using the CM of cementoblasts in terms of TRAP activity-positive cell formation and osteoclast-associated gene expression. The conditioned medium partly inhibited rRANKL-induced osteoclast differentiation and almost completely suppressed its enhancing effect by EVs. These results indicate that cementoblasts secreted EVs, which enhanced RANKL-induced osteoclast differentiation, and simultaneously produced soluble factors that neutralized this enhancing effect of EVs, implicating this balance in the regulation of cementum absorption. A more detailed understanding of this crosstalk between cementoblasts and osteoclasts will contribute to the development of new therapies for pathological root resorption.

Abstract Mechanical stress maintains tissue homeostasis by regulating many cellular functions including cell proliferation, differentiation, and inflammation and immune responses. In inflammatory microenvironments, macrophages in mechanosensitive tissues receive mechanical signals that regulate various cellular functions and inflammatory responses. Macrophage function is affected by several types of mechanical stress, but the mechanisms by which mechanical signals influence macrophage function in inflammation, such as the regulation of interleukin-1β by inflammasomes, remain unclear. In this review, we describe the role of mechanical stress in macrophage and monocyte cell function.

In the oral mechanical environment, periodontal ligament cells (PDL cells) contribute to maintaining periodontal tissue homeostasis. Recent studies showed that exosomes, which are small vesicles secreted by various types of cells, play a pivotal role in cell-to-cell communication in biological processes. We examined the secretion of exosomes from PDL cells stimulated with cyclic stretch and their role in the inflammatory response of macrophages using the human macrophage cell line THP-1 and human primary monocytes/macrophages. We prepared supernatants from human PDL cells (PDL-sup) stimulated with cyclic stretch. The treatment of macrophages with PDL-sup, but not PDL-sup from unstimulated PDL cells, inhibited the production of IL-1β in LPS/nigericin-stimulated macrophages. The pretreatment of PDL cells with GW4869, an inhibitor of exosome secretion, or siRNA for Rab27B, which controls exosome secretion, abrogated the inhibitory effects of PDL-sup. A transmission electron microscopy analysis demonstrated the existence of exosomes with diameters ranging between 30 and 100 nm in PDL-sup, suggesting that exosomes in PDL-sup contribute to this inhibition. An immunofluorescence microscopy analysis revealed that exosomes labeled with PKH67, a fluorescent dye, were incorporated by macrophages as early as 2 h after the addition of exosomes. Purified exosomes inhibited IL-1β production in LPS/nigericin-stimulated macrophages and the nuclear translocation of NF-κB as well as NF-κB p65 DNA-binding activity in LPS-stimulated macrophages, suggesting that exosomes suppress IL-1β production by inhibiting the NF-κB signaling pathway. Our results indicate that PDL cells in mechanical environments contribute to the maintenance of periodontal immune/inflammatory homeostasis by releasing exosomes.

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.

Macrophages are immune cells of hematopoietic origin that play diverse roles in host defenses and tissue homeostasis. In mechanical microenvironments, macrophages receive mechanical signals that regulate various cellular functions. However, the mechanisms by which mechanical signals influence the phenotype and function of macrophages in the process of inflammation have not yet been elucidated in detail. We herein examined the effects of cyclic stretch (CS) on NLR family, pyrin domain-containing 3 (NLRP3) inflammasome activation in J774.1, a murine macrophage cell line, and mouse primary bone marrow-derived macrophages. We showed that cyclic stretch inhibited adenosine triphosphate (ATP)-stimulated interleukin (IL)-1β secretion in lipopolysaccharide (LPS)-primed macrophages using ELISA and Western blot analyses. Cyclic stretch did not affect the degradation of the Inhibitor of κB or the nuclear translocation/transcriptional activity of nuclear factor (NF)-κB, suggesting that cyclic stretch-mediated inhibition was independent of the NF-κB signaling pathway. Consistent with these results, cyclic stretch did not affect the LPS-induced expression of inflammasome components, such as pro-IL-1β and NLRP3, which is known to require the activation of NF-κB signaling. We showed that the cyclic stretch-mediated inhibition of IL-1β secretion was caused by the suppression of caspase-1 activity. The addition of compound C, a specific inhibitor of adenosine monophosphate-activated protein kinase (AMPK), to LPS-primed macrophages inhibited IL-1β secretion as well as caspase-1 activation, suggesting that AMPK signaling is involved in ATP-triggered IL-1β secretion. Furthermore, the phosphorylation of AMPK induced by ATP in LPS-primed macrophages was significantly suppressed by cyclic stretch, indicating that cyclic stretch negatively regulates IL-1β secretion through the inhibition of caspase-1 activity by attenuating the AMPK pathway. Our results suggest that mechanical stress functions to maintain homeostasis through the prevention of excessive inflammasome activation in macrophages in mechanical microenvironments.

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

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.

Tissue-engineered grafts using a scaffold can be used in treating periodontal disease; however, previous scaffolds for the cultivations of the periodontal ligament cells have been structurally incompatible with the morphological requirements of human periodontal tissue. Here, we describe a self-organized honeycomb-patterned film (honeycomb film) that acted as an appropriate scaffold for periodontal tissue regeneration. The honeycomb films were prepared from biodegradable poly(ε-caprolactone) with highly regular three-dimensional micropatterned surface topography by casting a polymer solution of water-immiscible solvent under humid conditions. To evaluate its performance in activating the proliferation and organizing of cells, we have demonstrated specific behaviors of the cultured periodontal ligament cells on the self-organized honeycomb structures in vitro. Fibroblast-like cells derived from the periodontal ligament of extracted human molar teeth were cultivated on three types of honeycomb films with 5-, 10-, and 15-µm pore sizes for 4 h to 42 d. Morphological observation of the cultured tissues at 4—72 h revealed that the pseudopodiums of cell bodies were attached to the pillars in the honeycomb structure. A certain number of cells shifted their cell bodies into the honeycomb structural lumen through the oscula of 10- and 15-µm pores. After 28 and 42 d, the cells were observed to have formed multiple layers; further, each cell had penetrated through the 10- and 15-µm pores in the honeycomb film. The morphological examination of the honeycomb film along with the pillar structures revealed that the scaffold was clusteringly arrayed with interconnected structures, remarkably enhanced proliferation, and extension of the cultured cells. We consider that the film can be applied in periodontal therapy for use as a scaffold for periodontal tissue regeneration.