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.

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.

Correction: Han et al. MicroRNA-146b-5p Suppresses Pro-Inflammatory Mediator Synthesis via Targeting TRAF6, IRAK1, and RELA in Lipopolysaccharide-Stimulated Human Dental Pulp Cells. Int. J. Mol. Sci. 2023, 24, 7433.

In the original publication, there was a mistake in Figure 4J,K as published [...].

Fibroblast growth factor signalling regulates the development of tooth root.

Fibroblast growth factor (FGF) signalling plays a crucial role in the morphogenesis of multiple tissues including teeth. While the role of the signal has been studied in tooth crown development, little is known about root development. Of several FGF ligands involved in hard tissue formation, we suggest that FGF18 regulates the development of murine tooth roots. We implanted FGF18-soaked heparin beads into the lower first molar tooth buds at postnatal day 6 (P6), followed by transplantation under the kidney capsule. After 3 weeks, FGF18 significantly facilitated root elongation and periodontal tissue formation compared to the control. In situ hybridisation showed that Fgf18 transcripts were initially localised in the dental pulp along Hertwig's epithelial root sheath at P6 and P10 and subsequently in the dental follicle cells at P14. Fgf receptors were expressed in various dental tissues during these stages. In vitro analysis using the dental pulp stem cells revealed that FGF18 inhibited cell proliferation and decreased expression levels of osteogenic markers, Runx2, Alpl and Sp7. Consistently, after 1 week of kidney capsule transplantation, FGF18 application did not induce the expression of Sp7 and Bsp, but upregulated Periostin in the apical region of dental mesenchyme in the grafted molar. These findings suggest that FGF18 facilitates molar root development by regulating the calcification of periodontal tissues.

Improvement of the setting properties of mineral trioxide aggregate cements using cellulose nanofibrils.

Cellulose nanofibrils (CNFs) exhibit excellent mechanical properties and are used to reinforce various composites. The effects of incorporating CNFs into commercial mineral trioxide aggregate (MTA) cements (NEX MTA (NEX) and ProRoot® MTA (PR)) on the underwater setting properties, compressive strength, and flowability were estimated in this study. NEX mixed without CNFs disintegrated after water immersion. NEX mixed with CNF-suspended solutions showed good setting properties under water immersion and a similar compressive strength, which was kept in air (100% relative humidity). PR did not degrade after water immersion, regardless of the presence of CNFs, and no significant difference in the compressive strength caused by CNFs incorporation was detected. The relative flowability of the NEX mixture decreased with increasing CNFs content up to 1.0 w/v%. The application of CNF-incorporated MTA in various dental cases is promising because CNFs prevent the water-immersion-dependent collapse of some MTA cements immediately after mixing.

Hydraulic calcium silicate-based root canal sealers mitigate proinflammatory cytokine synthesis and promote osteogenesis in vitro.

Background/purpose: The mineralized tissue-inductive ability and anti-inflammatory properties of hydraulic calcium silicate-based (HCSB) sealers have not been fully elucidated. This study aimed to evaluate the effects of the HCSB sealers Bio-C sealer (BioC), Well-Root ST (WST), and EndoSequence BC sealer (BC), on osteoblastic differentiation/mineralization and proinflammatory cytokine synthesis by macrophages. Materials and methods: Diluted extracts of set sealers or calcium chloride solutions of approximately equivalent Ca2+ concentrations were applied to a mouse osteoblastic cell line (Kusa-A1 cells) and lipopolysaccharide-stimulated mouse macrophage cell line (RAW264.7 cells). Expressions of osteoblastic markers in Kusa-A1 cells and proinflammatory cytokines in RAW264.7 cells were evaluated by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Mineralized nodules were detected by Alizarin red S staining. Cell proliferation was assessed by WST-8 assay and cell attachment on set sealers was examined by scanning electron microscopy. Results: The three sealer extracts significantly upregulated osteocalcin and osteopontin mRNA, and promoted significant mineralized nodule formation in Kusa-A1 cells. The three sealer extracts significantly downregulated the mRNA expressions of interleukin (IL)-1α, IL-1ß, IL-6, and tumor necrosis factor (TNF)-α and protein levels of IL-6 and TNF-α in RAW264.7 cells. Calcium chloride solutions induced osteoblastic differentiation/mineralization. AH Plus Jet (a control sealer) extract did not. The three HCSB sealers did not interfere with the growth and attachment of Kusa-A1 cells. Conclusion: BioC, WST, and BC were biocompatible, upregulated osteoblastic differentiation/mineralization, and downregulated proinflammatory cytokine expression. Ca2+ released from HCSB sealers might be involved, at least in part, in the induction of osteoblastic differentiation/mineralization.

Autologous concentrated growth factor mediated accelerated bone healing in root-end microsurgery: A multicenter randomized clinical trial.

Introduction: Concentrated growth factor (CGF) is a new-generation autologous platelet concentrate that promotes tissue regeneration and has anti-inflammatory properties. This randomized multicenter trial aimed to evaluate the effects of CGF on bone healing in combination with root-end microsurgery. Methods: Healthy adult patients indicated for root-end microsurgery were randomly assigned to either the CGF or control (no CGF implantation) groups. CGF was implanted into the bone cavity after root-end filling with mineral trioxide aggregate. Clinical and periapical radiographic evaluations were conducted at 1, 3, 6, and 12 months postoperatively, with follow-up cone-beam computed tomography (CBCT) at 6 months. The lesion volume reduction rate was calculated based on data from the preoperative and follow-up CBCT images. Results: A total of 24 patients were enrolled. The treatment success rate was 91.7% and 83.3% on 12-month periapical radiography and 6-month CBCT, respectively, without a significant difference between the two groups. The lesion volume reduction rate in the CGF group (75.6%) was significantly higher than that in the control (61.0%) group. Conclusions: Autologous CGF in conjunction with root-end microsurgery accelerated lesion reduction as observed on CBCT. Administering autologous blood products to stimulate healing in addition to removing the source of infection appears to be a promising treatment option for root-end microsurgery.

MicroRNA-146b-5p Suppresses Pro-Inflammatory Mediator Synthesis via Targeting TRAF6, IRAK1, and RELA in Lipopolysaccharide-Stimulated Human Dental Pulp Cells.

MicroRNA-146b-5p (miR-146b-5p) is up-regulated during and to suppress the inflammation process, although mechanisms involved in the action of miR-146b-5p have not been fully elucidated. This study examined the anti-inflammation effects of miR-146b-5p in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). An increase in human miR-146b-5p (hsa-miR-146b-5p) expression following the mRNA expression of pro-inflammatory cytokines was observed in LPS-stimulated hDPCs. The expression of hsa-miR-146b-5p and pro-inflammatory cytokines was down-regulated by a nuclear factor-kappa B (NF-κB) inhibitor, and the expression of hsa-miR-146b-5p was also decreased by a JAK1/2 inhibitor. Enforced expression of hsa-miR-146b-5p abolished phosphorylation of NF-κB p65 and down-regulated the expression of pro-inflammatory cytokines and NF-κB signaling components, such as interleukin-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), and REL-associated protein involved in NF-κB (RELA). Expression of rat miR-146b-5p (rno-miR-146b-5p) and pro-inflammatory cytokine mRNA was also up-regulated in experimentally-induced rat pulpal inflammation in vivo, and rno-miR-146b-5p blocked the mRNA expression of pro-inflammatory mediators and NF-κB signaling components in LPS-stimulated ex vivo cultured rat incisor pulp tissues. These findings suggest that the synthesis of miR-146b-5p is controlled via an NF-κB/IL6/STAT3 signaling cascade, and in turn, miR-146b-5p down-regulates the expression of pro-inflammatory mediators by targeting TRAF6, IRAK1, and RELA in LPS-stimulated hDPCs.

Isolation of a Nocardiopsis chromatogenes strain that degrades PLA (polylactic acid) in pig waste-based compost.

A new Nocardiopsis species that degrades polylactic acid (PLA) was isolated from pig dung-based compost from a municipal composting facility in Japan. To obtain strains capable of efficient PLA degradation, the effect of non-enzymatic degradation of PLA was minimized by maintaining the temperature at or below 37 °C. Screening 15 animal waste-based compost samples, consisting of pig dung, cow dung, horse dung, or chicken droppings, revealed that compost derived from pig dung was most efficient for degradation of PLA films. Hence, pig waste-based compost was used to isolate PLA-degrading microorganisms by screening for PLA-degrading microorganisms in compost using an agar plate-based method in which an emulsifier was omitted to avoid selecting strains that assimilated the emulsifier instead of PLA in the medium. Repeated enrichment obtained six strains. The one that exhibited stable PLA degradation on agar plates was subjected to genomic analysis and identified as Nocardiopsis chromatogenes, an actinomycete.

Biocompatibility and pro-mineralization effect of tristrontium aluminate cement for endodontic use.

Background/purpose: Tristrontium aluminate (S3A) is a hydraulic cement with setting behavior similar to that of mineral trioxide aggregate (MTA). This study examined the biological effects of S3A on mouse dental papilla cells (MDPs) in vitro and on rat exposed pulps in vivo. Materials and methods: Extracts of S3A and MTA were prepared by immersing each cement in ultrapure water. MDPs were cultured with S3A or MTA extracts, and cell proliferation was evaluated with a tetrazolium-salt assay. Attachment of MDPs on the set cements was examined with scanning electron microscopy (SEM). mRNA expression of bone morphogenic protein (Bmp2), osteocalcin (Oc) and osteopontin (Opn) in MDPs exposed to S3A or MTA extracts was determined with reverse transcription-quantitative polymerase chain reaction. Mineralized nodule formation was evaluated with Alizarin Red S staining. Simulated body fluid (SBF)-dipped S3A was examined with SEM and energy dispersive X-ray analysis (EDX). Exposed molar pulps of male Wistar rats capped with S3A or MTA were histologically examined. Results: S3A extract did not inhibit proliferation of MDPs. Set S3A and MTA exhibited attachment of MDPs on their surface. S3A extract showed significantly higher mineralized nodule formation and mRNA expression of Bmp2, Oc, and Opn than did MTA extract. SBF-dipped S3A exhibited formation of surface precipitates, which were composed of Ca, P, Sr, and Al. Direct pulp capping with S3A and with MTA induced mineralized tissue repair of the exposed pulp. Conclusion: S3A possesses biocompatibility and pro-mineralization effects comparable to those of MTA.

Angiogenesis during coronal pulp regeneration using rat dental pulp cells: Neovascularization in rat molars in vivo and proangiogenic dental pulp cell-endothelial cell interactions in vitro.

Background/purpose: Angiogenesis is considered a crucial event for dental pulp regeneration. The purpose of this study was to demonstrate neovascularization during coronal pulp regeneration in rat molars using rat dental pulp cells (rDPCs) and to examine whether rDPC-endothelial cell interactions promote proangiogenic capacity in vitro. Materials and methods: Maxillary first molars of Wistar rats (n = 42) were pulpotomized and rDPCs isolated from incisors were implanted with a porous poly (l-lactic acid) (PLLA) scaffold and hydrogel (Matrigel). After 3, 7, and 14 days, coronal pulp tissues were examined histologically and by nestin and CD146 immunohistochemistry. rDPCs and rat dermal microvascular endothelial cells (rDMECs) were cocultured for 4 days and vascular endothelial growth factor (VEGF) synthesis and angiogenic factor gene expression were determined by enzyme-linked immunosorbent assays and real-time polymerase chain reaction, respectively. Effects of cocultured medium on tube formation by rDMECs were also evaluated. Results: Implantation of rDPC/PLLA/Matrigel induced coronal pulp regeneration with dentin bridge formation and arrangement of nestin-positive odontoblast-like cells at 14 days. PLLA/Matrigel without rDPCs did not induce pulp regeneration. CD146-positive blood vessels increased in density in the remaining pulp tissues at 3 and 7 days, and in the regenerated pulp tissue at 14 days. rDPC/DMEC coculture significantly promoted VEGF secretion and mRNA expression of nuclear factor-kappa B, angiogenic chemokine CXCL1, and chemokine receptor CXCR1. Cocultured medium significantly promoted tube formation. Conclusion: Coronal pulp regeneration with rDPC/PLLA/Matrigel was accompanied by neovascularization. rDPC-rDMEC interactions may promote angiogenic activity represented by proangiogenic factor upregulation and tube formation in vitro.

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.

Evaluation of the anti-inflammatory effects of surface-reaction-type pre-reacted glass-ionomer filler containing root canal sealer in lipopolysaccharide-stimulated RAW264.7 macrophages.

A prototype surface-reaction-type pre-reacted glass-ionomer (S-PRG) filler containing root canal sealer (S-PRG sealer) exhibits bioactive potential by releasing multiple ions. This study explored the suppressive effects and modes of action of S-PRG sealer extracts on proinflammatory cytokine expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Expression of proinflammatory cytokines was evaluated by RT-qPCR and ELISA. Expression of phosphorylated nuclear factor-kappa B (p-NF-kB) p65 was evaluated by western blotting. S-PRG sealer extracts significantly downregulated mRNA expression levels of interleukin (IL)-1α, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells; the extracts also reduced the levels of IL-6 protein and p-NF-kB. In order to verify that Zn2+ was responsible for downregulation of proinflammatory cytokine expression, N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) was used as a heavy metal chelator with strong affinity for Zn2+. These effects were mitigated by TPEN. The application of ZnCl2 reproduced the actions of S-PRG sealer extracts. These data suggest that S-PRG sealer has anti-inflammatory potential involving heavy metal ions such as Zn2+.

Development of an automated system to measure ion channel currents using a surface-modified gold probe.

Artificial lipid bilayer single-channel recording technique has been employed to determine the biophysical and pharmacological properties of various ion channels. However, its measurement efficiency is very low, as it requires two time-consuming processes: preparation of lipid bilayer membranes and incorporation of ion channels into the membranes. In order to address these problems, we previously developed a technique based on hydrophilically modified gold probes on which are immobilized ion channels that can be promptly incorporated into the bilayer membrane at the same time as the membrane is formed on the probes' hydrophilic area. Here, we improved further this technique by optimizing the gold probe and developed an automated channel current measurement system. We found that use of probes with rounded tips enhanced the efficiency of channel current measurements, and introducing a hydrophobic area on the probe surface, beside the hydrophilic one, further increased measurement efficiency by boosting membrane stability. Moreover, we developed an automated measurement system using the optimized probes; it enabled us to automatically measure channel currents and analyze the effects of a blocker on channel activity. Our study will contribute to the development of high-throughput devices to identify drug candidates affecting ion channel activity.

Evaluation of the cytocompatibility of methacrylate resin-based root canal sealers with osteoblast-like cells.

This study compared the cytocompatibilities of three methacrylate resin-based root canal sealers [MetaSEAL Soft (MSS), Hybrid Root SEAL (HRS), and Superbond Sealer (SBS)] in either freshly mixed or set conditions using the Kusa A1 osteoblastic cell line. The three sealers and an epoxy resin-based sealer (AH Plus; AHP) were extracted in culture medium; cell growth and osteogenic properties were analyzed. Cell adhesion on set sealers was analyzed with scanning electron microscopy. The respective extents of cell growth were as follows in freshly mixed and set sealer extracts: SBS>MSS>AHP>HRS and SBS=AHP>MSS>HRS. Light irradiation of MSS and HRS increased the cell growth of set sealer extracts. Set SBS, MSS, and AHP did not alter expression of osteogenic genes or formation of mineralized nodules. Attached cells were observed only on SBS. In conclusion, the four sealers exhibited varying degrees of compatibility to osteoblasts; SBS and HRS were the most and least compatible, respectively.

Distrontium Cerate as a Radiopaque Component of Hydraulic Endodontic Cement.

This study aimed to synthesize distrontium cerate (2SrO·CeO2: S2Ce) and evaluate its properties as an alternative component of the endodontic cement. S2Ce cement was prepared through calcination of strontium hydroxide and cerium carbonate. Subsequently, the crystal phase was confirmed using X-ray diffraction. S2Ce cement exhibited a rapid setting time (121 min) and achieved a high compressive strength (72.1 MPa) at 1 d after mixing, comparable to the compressive strength of a commercial mineral trioxide aggregate (MTA) cement (ProRoot MTA) after 28 d post mixing. However, the compressive strength decreased after 28 d of storage when the W/P ratio was 0.30-0.40 (p < 0.05). Ion dissolution test of the S2Ce cement showed that strontium ions were released after immersion in water (5.27 mg/mL after 1 d), whereas cerium dissolution was not detected. S2Ce exhibited approximately three times higher radiopacity (9.0 mm aluminum thickness equivalent) compared to the commercial MTA (p < 0.05). These findings suggest that S2Ce is a possible component for hydraulic endodontic cement that demonstrates a rapid setting and high radiopacity.

Preparation and properties of tristrontium aluminate as an alternative component of mineral trioxide aggregate (MTA) cement.

This study evaluated tristrontium aluminate (S3A) and its viability as a component for tricalcium silicate (C3S) cements. The properties of S3A, C3S, and S3A/C3S mixtures were evaluated in terms of setting time, compressive strength, flowability, and radiopacity. X-ray diffraction (XRD) pattern verified the powder synthesized in the laboratory as S3A, consequently, confirming the preparation method. S3A exhibited the lowest setting time, followed by C3S and S3A/C3S mixtures. Compressive strength of C3S was significantly higher than S3A. The S3A/C3S mixture showed comparable compressive strength to C3S for 1-day post initial mixing. There was no significant difference in flowability between S3A/C3S and mineral trioxide aggregate (MTA). S3A showed comparable radiopacity to MTA, whereas that of the S3A/C3S mixture was significantly lower comparatively; however, it achieved sufficient radiopacity (3 mm aluminum thickness equivalent). Further studies are needed to improve the manufacturing process of S3A and evaluate the bioactive effect of strontium.

A Novel Bioactive Endodontic Sealer Containing Surface-Reaction-Type Prereacted Glass-Ionomer Filler Induces Osteoblast Differentiation.

Surface­reaction­type prereacted glass-ionomer (S­PRG) fillers exhibit bioactive properties by the release of multiple ions. This study examined whether a novel endodontic sealer containing S­PRG fillers (PRG+) has the capacity to induce osteoblast differentiation. Kusa­A1 osteoblastic cells were cultured with extracts of PRG+, PRG- (an experimental sealer containing S­PRG­free silica fillers), AH Plus (an epoxy-resin­based sealer), and Canals N (a zinc-oxide noneugenol sealer). Cell viability and mineralized nodule formation were determined using WST­8 assay and Alizarin red staining, respectively. Osteoblastic-marker expression was analyzed with RT­qPCR and immunofluorescence. Phosphorylation of extracellular signal­regulated kinase (ERK) and p38 mitogen­activated protein kinase (MAPK) was determined with Western blotting. Extracts of freshly mixed PRG+, PRG-, and AH Plus significantly decreased cell growth, but extracts of the set samples were not significantly cytotoxic. Set PRG+ significantly upregulated mRNAs for alkaline phosphatase and bone sialoprotein (IBSP) compared to set PRG-, and upregulation was blocked by NPS2143, a calcium­sensing receptor antagonist. Set PRG+ significantly accelerated IBSP expression, mineralized nodule formation, and enhanced the phosphorylation of ERK and p38 compared with set PRG-. In conclusion, PRG+ induced the differentiation and mineralization of Kusa­A1 cells via the calcium-sensing receptor-induced activation of ERK and p38 MAPK.

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.