MicroRNA-27a-5p Downregulates Expression of Proinflammatory Cytokines in Lipopolysaccharide-Stimulated Human Dental Pulp Cells via the NF-κB Signaling Pathway.

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.

Biocompatibility and pro-mineralization effects of premixed calcium silicate-based materials on human dental pulp stem cells: An in vitro and in vivo study.

Premixed calcium silicate-based materials have recently been developed and are recommended for a wide range of endodontic procedures, including vital pulp therapy. This study investigated the in vitro biocompatibility and pro-mineralization effect and in vivo reparative dentin formation of EndoSequence Root Repair Material, EndoSequence BCRRM, Bio-C Repair, and Well-pulp PT. Both fresh and set extracts had no detrimental effect on the growth of human dental pulp stem cells. The fresh extracts had a higher calcium concentration than the set extracts and induced considerably greater mineralized nodule formation. EndoSequence Root Repair Material had the longest setting time, whereas Bio-C Repair had the shortest. When these materials were applied to exposed rat molar pulps, mineralized tissue deposition was found at the exposure sites after 2 weeks. These results indicate that the premixed calcium silicate-based materials tested could have positive benefits for direct pulp capping procedures.

Hypochlorous acid inactivates oral pathogens and a SARS-CoV-2-surrogate.

BACKGROUND: Droplets and aerosols produced during dental procedures are a risk factor for microbial and viral transmission. Unlike sodium hypochlorite, hypochlorous acid (HOCl) is nontoxic to tissues but still exhibits broad microbicidal effect. HOCl solution may be applicable as a supplement to water and/or mouthwash. This study aims to evaluate the effectiveness of HOCl solution on common human oral pathogens and a SARS-CoV-2 surrogate MHV A59 virus, considering the dental practice environment. METHODS: HOCl was generated by electrolysis of 3% hydrochloric acid. The effect of HOCl on human oral pathogens, Fusobacterium nucleatum, Prevotella intermedia, Streptococcus intermedius, Parvimonas micra, and MHV A59 virus was studied from four perspectives: concentration; volume; presence of saliva; and storage. HOCl solution in different conditions was utilized in bactericidal and virucidal assays, and the minimum inhibitory volume ratio that is required to completely inhibit the pathogens was determined. RESULTS: In the absence of saliva, the minimum inhibitory volume ratio of freshly prepared HOCl solution (45-60 ppm) was 4:1 for bacterial suspensions and 6:1 for viral suspensions. The presence of saliva increased the minimum inhibitory volume ratio to 8:1 and 7:1 for bacteria and viruses, respectively. Applying a higher concentration of HOCl solution (220 or 330 ppm) did not lead to a significant decrease in the minimum inhibitory volume ratio against S. intermedius and P. micra. The minimum inhibitory volume ratio increases in applications of HOCl solution via the dental unit water line. One week of storage of HOCl solution degraded HOCl and increased the minimum growth inhibition volume ratio. CONCLUSIONS: HOCl solution (45-60 ppm) is still effective against oral pathogens and SAR-CoV-2 surrogate viruses even in the presence of saliva and after passing through the dental unit water line. This study indicates that the HOCl solution can be used as therapeutic water or mouthwash and may ultimately reduce the risk of airborne infection in dental practice.

Three-dimensional cellular visualization in mouse apical periodontitis using combined whole-mount staining and optical tissue clearing.

Apical periodontitis is an inflammatory disease involving lesions located within the jawbone. Histological evaluations generally require decalcification and sectioning, which has limited our understanding of the three-dimensional (3D) organization and spatial distribution of different immune cell types in these lesions. A recently developed technique combining tissue clearing and whole-mount immunofluorescent labeling allows us to acquire such information from the deep tissue without sectioning. However, whole-mount immunofluorescent labeling in the jawbone requires further development. Here we provide a straightforward and efficient protocol to achieve 3D immunofluorescent imaging of murine periapical lesions.

Kinetics of LYVE-1-positive M2-like macrophages in developing and repairing dental pulp in vivo and their pro-angiogenic activity in vitro.

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.

Hypoxia-inducible factor 1α induces osteo/odontoblast differentiation of human dental pulp stem cells via Wnt/ß-catenin transcriptional cofactor BCL9.

Accelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/ß-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers (RUNX2, ALP, OC), BCL9, and Wnt/ß-catenin signaling target genes (AXIN2, TCF1) in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and ß-catenin expression and BCL9-ß-catenin co-localization. In addition, BCL9 formed a complex with ß-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/ß-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/ß-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury.

Revisiting the role of IL-1 signaling in the development of apical periodontitis.

Apical periodontitis (AP) develops as a result of an immune response to pulpal bacterial infection, and various cytokines are involved in the pathogenesis of AP, with Interleukin (IL)-1 being considered a key cytokine. The role of IL-1 in the pathogenesis of AP has been well studied. It is known that IL-1 expression in periapical lesions correlates closely with the development of AP. IL-1 is a potent bone-resorptive cytokine that induces osteoclast formation and activation. Hence, inhibiting its signaling with IL-1 receptor antagonist (IL-1RA) results in a reduction in periapical lesion size. On the other hand, IL-1 is also a central cytokine that combats bacterial infection by activating innate immune responses. Therefore, a complete loss of IL-1 signaling leads to a failure to limit bacterial dissemination and consequently exacerbates AP. In vivo, IL-1 expression is tightly regulated and its signaling is modulated to optimize the immune response. Obesity causes systemic low-grade chronic inflammation and increases the risk of cardiovascular, renal, and other disorders. In experimentally induced AP, obesity significantly increases periapical bone loss, albeit the underlying mechanism remains unclear. Recent technological innovations have enabled more comprehensive and detailed analyses than previously, leading to new insights into the role of IL-1RA in regulating IL-1 signaling, and modulating apical lesion progression in obesity. In this review, we provide a brief overview of the function of IL-1 in AP development, with special emphasis on the latest findings in normal weight and obese states.

Transient Receptor Potential Ankyrin 1 Is Up-Regulated in Response to Lipopolysaccharide via P38/Mitogen-Activated Protein Kinase in Dental Pulp Cells and Promotes Mineralization.

Increased expression of the transient receptor potential ankyrin 1 (TRPA1) channel has been detected in carious tooth pulp, suggesting involvement of TRPA1 in defense or repair of this tissue after exogenous noxious stimuli. This study aimed to elucidate the induction mechanism in response to lipopolysaccharide (LPS) stimulation and the function of TRPA1 in dental pulp cells. Stimulation of human dental pulp cells with LPS up-regulated TRPA1 expression, as demonstrated by quantitative RT-PCR and Western blotting. LPS stimulation also promoted nitric oxide (NO) synthesis and p38/mitogen-activated protein kinase (MAPK) phosphorylation. NOR5, an NO donor, up-regulated TRPA1 expression, whereas 1400W, an inhibitor of inducible nitric oxide synthase, and SB202190, a p38/MAPK inhibitor, down-regulated LPS-induced TRPA1 expression. Moreover, JT010, a TRPA1 agonist, increased the intracellular calcium concentration and extracellular signal-regulated kinase 1/2 phosphorylation, and up-regulated alkaline phosphatase mRNA in human dental pulp cells. Icilin-a TRPA1 agonist-up-regulated secreted phosphoprotein 1 mRNA expression and promoted mineralized nodule formation in mouse dental papilla cells. In vivo expression of TRPA1 was detected in odontoblasts along the tertiary dentin of carious teeth. In conclusion, this study demonstrated that LPS stimulation induced TRPA1 via the NO-p38 MAPK signaling pathway and TRPA1 agonists promoted differentiation or mineralization of dental pulp cells.

Author Correction: Strontium ranelate promotes odonto-/osteogenic differentiation/mineralization of dental papillae cells in vitro and mineralized tissue formation of the dental pulp in vivo.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

HIF1α inhibits LPS-mediated induction of IL-6 synthesis via SOCS3-dependent CEBPß suppression in human dental pulp cells.

Hypoxia-inducible factor 1 alpha (HIF1α) is a transcriptional factor that plays a key role in the regulation of various molecules expressed in hypoxic conditions. Ischemic/hypoxic conditions are regarded as a distinct characteristic of dental pulp inflammation due to the encasement of pulp tissue within the rigid tooth structure. This study was performed to examine the role of HIF1α in the regulation of interleukin (IL)-6, a proinflammatory cytokine expressed in inflamed dental pulp, in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). LPS stimulation promoted the expression of IL-6 in hDPCs, while HIF1α suppressed the expression of IL-6. Moreover, HIF1α induced suppressor of cytokine signaling 3 (SOCS3) expression in LPS-stimulated hDPCs, and SOCS3 activity led to downregulate expression of CCAAT enhancer-binding protein beta (CEBPß), an inducer of IL-6. LPS stimulation promoted HIF1α expression in hDPCs and mouse pulp tissue explants cultured under hypoxic conditions. These findings suggest that HIF1α negatively regulates IL-6 synthesis in LPS-stimulated hDPCs via upregulation of SOCS3 and subsequent downregulation of CEBPß.

Anti-inflammatory roles of microRNA 21 in lipopolysaccharide-stimulated human dental pulp cells.

microRNAs are small noncoding RNA molecules that regulate RNA silencing and posttranscriptional gene expression, and many microRNAs are involved in inflammatory processes. In particular, microRNA 21 (miR-21) is upregulated in inflammatory environment and reported to induce anti-inflammatory responses. However, the involvement of miR-21 in pulpal inflammation and the precise mechanisms of anti-inflammatory reactions induced by miR-21 remain unclear. We hypothesized that miR-21-5p expression is induced in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs) and that miR-21-5p downregulates the proinflammatory cytokine expression in LPS-stimulated hDPCs. We found that miR-21-5p was upregulated in LPS-stimulated hDPCs concomitant with elevated proinflammatory cytokine expression and nuclear factor-kappa B (NF-κB) phosphorylation. miR-21-5p and cytokine expression were downregulated by BAY11-7085 and caffeic acid phenylethyl ester (CAPE), specific and potent NF-κB inhibitors. Enforced expression of miR-21-5p downregulated the Toll-like receptor (TLR)/NF-κB signaling via reducing the expression of TNF receptor-associated factor 6 (TRAF6) and programmed cell death 4 (PDCD4), which further induced the decrease of proinflammatory cytokine expression. hDPCs forcibly overexpressing miR-21-5p downregulated the LPS-induced expression of TNF receptor-associated factor 6 (TRAF6; a component of the Toll-like receptor [TLR]/NF-κB signaling pathway), programmed cell death 4 (PDCD4, a positive regulator of the TLR/NF-κB signaling pathway), and proinflammatory cytokines. In contrast, miR-21-5p inhibitor-transfected hDPCs upregulated the expression of TRAF6, PDCD4, and inflammatory cytokines following LPS stimulation. These findings suggest that miR-21-5p expression was induced by the NF-κB signaling pathway, which was in turn negatively regulated by miR-21-5p via downregulation of TRAF6 and PDCD4 expression in LPS-stimulated hDPCs.

Strontium ranelate promotes odonto-/osteogenic differentiation/mineralization of dental papillae cells in vitro and mineralized tissue formation of the dental pulp in vivo.

This study examined the effects and mechanisms of strontium ranelate (SrRn)-a drug used to treat osteoporosis-on the proliferation and differentiation/mineralization of cloned dental pulp-like cells (mouse dental papillae cells; MDPs). It also determined whether topical application of SrRn to exposed dental pulp tissue promotes the formation of mineralized tissue in vivo. The MDPs were cultured with or without SrRn, and cell proliferation, odonto-/osteoblastic gene expression, mineralized nodule formation, and Akt phosphorylation were evaluated. The formation of mineralized tissue in SrRn-treated pulp tissue in rat upper first molars was evaluated histologically. The SrRn up-regulated cell proliferation and expression of Alp (alkaline phosphatase), Bsp (bone sialoprotein), Dmp (dentin matrix acidic phosphoprotein)-1, Dspp (dentin sialophosphoprotein), and Oc (osteocalcin) in a dose-dependent manner. Mineralized nodule formation was also enhanced by SrRn. NPS-2143, a calcium-sensing receptor (CaSR) antagonist, and siRNA against the CaSR gene blocked SrRn-induced proliferation, odonto-/osteoblastic gene expression, and mineralized nodule formation. SrRn induced Akt phosphorylation, and this was blocked by NPS-2143. Topical application of SrRn to exposed rat molar pulps induced the formation of osteodentin-like mineralized tissue. Our study revealed for the first time that SrRn promotes proliferation and odonto-/osteogenic differentiation/mineralization of MDPs via PI3K/Akt signaling activated by CaSR in vitro; mineralized tissue forms from the dental pulp in vivo.

Transient receptor potential melastatin (TRPM) 8 is expressed in freshly isolated native human odontoblasts.

OBJECTIVE: Cold-sensitive ion channels, such as transient receptor potential melastatin (TRPM) 8 and transient receptor potential ankyrin (TRPA) 1, may play a crucial role in the nociceptive function of odontoblasts, whereas expression of these TRP channels in human native odontoblasts remains to be elucidated. This study aimed to analyze the expression of TRPM8 and TRPA1 in freshly isolated native human odontoblasts. DESIGN: Odontoblasts were isolated from freshly extracted healthy human teeth (n=4); after removing the inner pulp tissues from the pulp chambers, odontoblasts remaining on the dentin surface were washed out with phosphate buffered saline and collected. Reverse transcription-polymerase chain reaction was employed to compare the expression levels of TRPM8, TRPA1, and dentin matrix acidic phosphoprotein 1 (DMP1) mRNAs between the isolated odontoblasts and the inner pulp tissues. The isolated cells were subjected to immunolocalization of TRPM8 and nestin. Paraformaldehyde-fixed, EDTA-demineralized frozen sections obtained from freshly extracted healthy human teeth (n=4) were also analyzed immunohistochemically using anti-nestin, TRPM8, and TRPA1 antibodies. RESULTS: Expression levels of TRPM8 and DMP1 in the isolated odontoblasts were significantly higher than those in the inner pulp tissues (p<0.05). Expression of TRPM8 and nestin was observed in the odontoblastic layer of the dental pulp tissue and isolated odontoblasts, while expression of TRPA1 was not detected. CONCLUSIONS: TRPM8, but not TRPA1, was detected in freshly isolated native human odontoblasts at the protein and mRNA levels, suggesting that odontoblasts play an important role in detecting external cold stimulation via TRPM8 in healthy condition.