Kento Tazawa

Introduction: Apical periodontitis, caused by bacterial infection through the root canals, is characterized by chronic inflammation and bone resorption around the root apex. Metformin, a first-line therapeutic drug for type 2 diabetes mellitus, has attracted attention for its potential anti-inflammatory properties and role in regulating bone homeostasis. The hypothesis in this study was that metformin inhibits bone destruction in apical periodontitis by suppressing macrophage-mediated inflammatory responses. The aim of this study was to evaluate the effect of systemic metformin administration on experimentally induced apical periodontitis development in an animal model and clarify the underlying anti-inflammatory mechanism of metformin in lipopolysaccharide-stimulated mouse macrophages.Methods: Evaluations on the effects of metformin on the progression of periapical lesions were conducted in experimentally induced mouse apical periodontitis in vivo, and its anti-inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages in vitro were analyzed.Results: Metformin significantly reduced periapical bone destruction on postoperative days 21 and 28, and decreased the number of osteoclasts on the periapical alveolar bone on postoperative day 28. It also suppressed pro-inflammatory cytokine expression and nuclear factor kappa B signaling in lipopolysaccharide-stimulated RAW264.7. RNA-sequencing data revealed the downregulation of the mammalian target of rapamycin signaling after metformin treatment, which was confirmed by the downregulation of the mammalian target of rapamycin phosphorylation by metformin. Furthermore, metformin activated adenosine monophosphate-activated protein kinase, a potent negative regulator of mammalian target of rapamycin complex 1. The suppression of inflammatory cytokine expression by metformin was abolished by compound C, a potent adenosine monophosphate-activated protein kinase inhibitor.Discussion: This study revealed that metformin suppressed inflammatory bone destruction in periapical lesions. The mechanism partially involves inhibiting the mammalian target of rapamycin/nuclear factor-kappa B signaling in macrophages through adenosine monophosphate-activated protein kinase signaling activation. Findings from this study show that metformin has therapeutic potential in inflammatory bone destruction, such as apical periodontitis.

Aim: The anatomy and functions of lymphatic vessels (LV) in mammals remain poorly understood compared to the blood vascular system. In particular, whether or not LV exist in the dental pulp is still controversial. This study aims to identify the existence of LV in the mouse dental pulp using a Prox1 (Prospero homeobox 1)‐eGFP transgenic mouse model combined with a tissue‐clearing technique. Methodology: Mandible or mandibular first molars of Prox1‐eGFP mice were extracted, cleared for whole‐mount observation and imaged using confocal microscopy. Dylight 594‐lectin was injected intracardially to differentiate Prox1‐eGFP+ LV from the blood vascular network. To further determine if pulpal LV act as an interstitial fluid drainage system, we examined ink absorption in surgically exposed dental pulp of mandibular first molars. Results: At the early stage of tooth development, abundant Prox1‐eGFP+ LV distinct from lectin‐labelled blood vessels were present in a dental pulp. However, after the initiation of root development, the expression of Prox1‐eGFP in dental pulp decreased. In mature dental pulp, Prox1‐eGFP+ LV was scattered with a discontinuous lumen. In response to a non‐infectious transient pulp stimulation (TPS), the Prox1‐eGFP+ LV increased in number and diameter with continuous lumen reaching the apical foramen. Ink particles applied to exposed dental pulp are distributed throughout the dental pulp via interstitial spaces and vessel‐like structures. Histological evaluation revealed that ink particles were mainly present in the cell‐free zone. However, due to TPS, ink particles were taken up into Prox1‐eGFP+ LV. Conclusion: Our findings suggest the presence of LV in the mature dental pulp that contributes to fluid drainage in this tissue together with the extravascular pathway.

MicroRNAs (miRNAs) play a crucial role in cell differentiation through epigenetic regulation of gene expression. In human dental pulp cells, we have identified miRNA-27a being upregulated under inflammatory conditions. Here, we aimed to examine whether (i) overexpression of miRNA-27a in human dental pulp stem cells (hDPSCs) enhances their odonto/osteoblastic differentiation via Wnt and bone morphogenetic protein signaling; and (ii) hDPSCs overexpressing miRNA-27a promote new bone formation in vivo. hDPSCs were cultured in osteogenic medium to promote differentiation. To examine the role of miRNA-27a, hDPSCs were transfected with either a miRNA-27a mimic to enhance or an inhibitor to suppress miRNA-27a expression. Odonto/osteoblastic differentiation was assessed by evaluating the expression of specific markers, Wnt and bone morphogenetic protein (BMP) signaling molecules, and mineralization capacity using RT-qPCR, western blotting, Alizarin Red S (ARS) staining, and alkaline phosphatase (ALP) activity. Potential miRNA-27a binding sites in the 3'UTRs of DKK3 and SOSTDC1 were identified via bioinformatics analysis and validated through the luciferase reporter assay. In vivo, miRNA-27a-overexpressing hDPSCs were seeded into collagen honeycomb scaffolds and implanted into mouse calvarial bone cavities to assess new bone formation. MiRNA-27a was highly upregulated in hDPSCs committed to odonto/osteoblastic differentiation. Overexpression of miRNA-27a led to increased expression of odonto/osteoblastic markers and enhanced mineralization capacity, while inhibition of miRNA-27a had the opposite effect. MiRNA-27a targeted DKK3, promoting β-catenin nuclear translocation and inhibiting SOSTDC1, which enhanced SMAD1/5 phosphorylation. Binding sites for miRNA-27a were identified in the 3'UTRs of DKK3 and SOSTDC1. In vivo, miRNA-27a-overexpressing hDPSCs promoted new bone formation in mouse calvaria bone cavities. Transfection of miRNA-27a in hDPSCs enhanced their odonto/osteoblastic differentiation by targeting DKK3 and SOSTDC1, thereby promoting the Wnt and BMP signaling. Transplantation of miRNA-27a-overexpressing hDPSCs promoted new bone formation in vivo. These findings deepen our understanding of the effects of miRNA on Wnt and BMP pathways and suggest a potential clinical application for miRNA-27a in promoting hard tissue regeneration, offering a promising therapeutic target for dental and craniofacial tissue reconstruction.

MicroRNA-27a-5p (miR-27a-5p) was significantly upregulated in dental pulp inflammation, yet its underlying mechanisms remain unclear. This study investigated the effect of miR-27a-5p on the expression of proinflammatory cytokines in human dental pulp cells (hDPCs) stimulated by lipopolysaccharide (LPS). LPS-stimulated hDPCs showed concurrent increases in the expression of miR-27a-5p and proinflammatory cytokines (IL-6, IL-8, and MCP1), and the increased expression was suppressed by NF-κB inhibitor BAY 11-0785. Transfection of the miR-27a-5p mimic downregulated the expression of proinflammatory cytokines, NF-κB activity, and the expression of NF-κB signaling activators (TAB1, IRAK4, RELA, and FSTL1) in LPS-stimulated hDPCs. Luciferase reporter assays revealed that miR-27a-5p bound directly to the 3’-UTR of TAB1. siTAB1 downregulated NF-κB activity and proinflammatory cytokine expression. Downregulation of proinflammatory cytokine expression, NF-κB activity, and NF-κB signaling activator expression (TAB1, IRAK4, and RELA) was also found in LPS-stimulated rat incisor pulp tissue explants following transfection with the miR-27a-5p mimic ex vivo. MiR-27a-5p, whose expression was induced by NF-κB signaling, negatively regulated the synthesis of proinflammatory cytokines via targeting NF-κB signaling. In particular, TAB1, a potent NF-κB activator, was targeted by miR-27a-5p. These results provide insights into the negative regulatory effects of miR-27a-5p, particularly those targeting the TAB1-NF-κB signaling pathway, on pulp inflammation.

Aim: To investigate the effect of microRNA-27a-5p (miR-27a-5p) on the expression of proinflammatory cytokines in human dental pulp cells (hDPCs) stimulated by lipopolysaccharide (LPS). Methodology: Expression of miR-27a-5p was evaluated in LPS-stimulated hDPCs isolated from third molars of healthy patients by a TaqMan microRNA assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and a cytometric bead array were used to measure mRNA and protein expression levels, respectively, of proinflammatory cytokines interleukin (IL)-6, IL-8, and monocyte chemotactic protein 1 (MCP1). Luciferase assays and western blotting were conducted to assess nuclear factor κB (NF-κB) activity. Gene/protein expression of NF-κB signaling activators, i.e., transforming growth factor beta-activated kinase 1 binding protein 1 (TAB1), IL-1 receptor-associated kinase 4 (IRAK4), NF-κB p65 (RELA), and follistatin-like 1 (FSTL1), was evaluated by RT-qPCR and western blotting. Transfection of an miR-27a-5p mimic was performed using Lipofectamine RNAiMax reagent. Wildtype and mutated 3□-untranslated region (UTR) of TAB1-contained luciferase reporter vectors were co-transfected with the miR-27a-5p mimic. Small interfering RNA against TAB1 (siTAB1) was designed to block its expression. One-way analysis of variance and Student’s t -test were used to determine statistical significance (α = .05). Results: LPS-stimulated hDPCs showed concurrent increases in the expression of miR-27a-5p and proinflammatory cytokines (IL-6, IL-8, and MCP1), and the increased expression was suppressed by NF-κB inhibitor BAY 11-0785. Transfection of the miR-27a-5p mimic downregulated expression of proinflammatory cytokines, NF-κB activity, and expression of NF-κB signaling activators (TAB1, IRAK4, RELA, and FSTL1) in LPS-stimulated hDPCs. Luciferase reporter assays revealed that miR-27a-5p bound directly to the 3□-UTR of TAB1. siTAB1 downregulated NF-κB activity and proinflammatory cytokine expression. Downregulation of proinflammatory cytokine expression, NF-κB activity, and NF-κB signaling activator expression (TAB1, IRAK4, and RELA) was also found in LPS-stimulated rat incisor pulp tissue explants following transfection with the miR-27a-5p mimic ex vivo . Conclusions: MiR-27a-5p, whose expression was induced by NF-κB signaling, negatively regulated the synthesis of proinflammatory cytokines via targeting NF-κB signaling. In particular, TAB1, a potent NF-κB activator, was targeted by miR-27a-5p. These results provide insights into the negative regulatory effects of miR-27a-5p, particularly those targeting the TAB1-NF-κB signaling pathway, on pulp inflammation.

Tissue-resident macrophages expressing lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) are found in multiple tissues and organs. We aimed to evaluate the dynamics and biological functions of LYVE-1⁺ macrophages in dental pulp during post-injury tissue remodeling. Immunofluorescence staining of mouse embryos revealed that LYVE-1⁺ macrophages colonized dental pulp before birth. In mature rat molar dental pulp, LYVE-1⁺ macrophages were the main subset of macrophages expressing CD163, an M2 marker, and were distributed throughout the tissue. In response to dental pulp injury induced by cavity preparation, LYVE-1⁺ macrophages quickly disappeared from the affected area of the pulp and gradually repopulated during the wound healing process. RAW264.7 mouse macrophages cultured with a mixture of macrophage colony-stimulating factor, interleukin-4, and dexamethasone increased LYVE-1 expression, whereas lipopolysaccharide-stimulation decreased LYVE-1 expression. Enforced expression of Lyve1 in RAW264.7 cells resulted in increased mRNA expression of matrix metalloproteinase 2 (Mmp2), Mmp9, and vascular endothelial growth factor A (Vegfa). Lyve1-expressing macrophages promoted the migration and tube formation of human umbilical vein endothelial cells. In conclusion, LYVE-1⁺ tissue-resident M2-like macrophages in dental pulp showed dynamism in response to pulp injury, and possibly play an important role in angiogenesis during wound healing and tissue remodeling.

Aim: To elucidate mechanisms by which mineral trioxide aggregate (MTA) suppresses pro‐inflammatory cytokine mRNA expression in lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophages.Methodology: Mineral trioxide aggregate extracts were prepared by immersing set ProRoot MTA in culture medium. RAW264.7 cells were cultured in the presence of LPS and MTA extracts. mRNA expression levels of interleukin (IL)‐1α, IL‐6, early growth response 2 (Egr2), suppressor of cytokine signalling 3 (Socs3) and IL‐10 were quantified with reverse transcription‐quantitative polymerase chain reaction. Phosphorylation of nuclear factor‐kappa B (NF‐κB) p65 in RAW264.7 cells was analysed by Western blotting. Intracellular calcium imaging was performed with Fluo‐4 AM. The activity of nuclear factor of activated T cells (NFAT) was determined by luciferase assays. Enforced expression and silencing of Egr2 in RAW264.7 cells were carried out using an expression vector and specific RNAi, respectively. In vivo kinetics of Egr2⁺ cells in MTA‐treated rat molar pulp tissues were examined using immunohistochemistry. Data were analysed by one‐way analysis of variance, followed by the Tukey–Kramer test (P < 0.05).Results: Exposure to MTA extracts resulted in reduced mRNA expression levels of IL‐1α and IL‐6, as well as reduced expression of phosphorylated NF‐κB, in LPS‐stimulated RAW264.7 cells. Exposure to MTA extracts induced Ca²⁺ influx, which was blocked by NPS2143, an antagonist of calcium‐sensing receptor (CaSR); Ca²⁺ influx then triggered activation of calcineurin/NFAT signalling and enhanced mRNA expression of Egr2. Enforced expression of Egr2 in RAW264.7 cells promoted the expression of both IL‐10 and Socs3. In vivo application of MTA onto rat molar pulp tissue resulted in the appearance of Egr2‐expressing cells that coexpressed CD163, a typical M2 macrophage marker.Conclusions: Mineral trioxide aggregate extracts induced downregulation of IL‐1α and IL‐6 in LPS‐stimulated RAW264.7 cells via CaSR‐induced activation of calcineurin/NFAT/Egr2 signalling and subsequent upregulation of IL‐10 and Socs3.