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 [...].

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.

Effect of repetitive up-and-down movements on torque/force generation, surface defects and shaping ability of nickel-titanium rotary instruments: an ex vivo study.

BACKGROUND: The screw-in effect is a tendency of a nickel-titanium (NiTi) rotary endodontic file to be pulled into the canal, which can result in a sudden increase in stress leading to instrument fracture, and over-instrumentation beyond the apex. To reduce screw-in force, repeated up-and-down movements are recommended to distribute flexural stress during instrumentation, especially in curved and constricted canals. However, there is no consensus on the optimal number of repetitions. Therefore, this study aimed to examine how repeated up-and-down movements at the working length affect torque/force generation, surface defects, and canal shaping ability of JIZAI and TruNatomy instruments. METHODS: An original automated root canal instrumentation device was used to prepare canals and to record torque/force changes. The mesial roots of human mandibular molars with approximately 30˚ of canal curvature were selected through geometric matching using micro-computed tomography. The samples were divided into three groups according to the number of up-and-down movements at the working length (1, 3, and 6 times; n = 24 each) and subdivided according to the instruments: JIZAI (#13/0.04 taper, #25/0.04 taper, and #35/0.04 taper) or TruNatomy (#17/0.02 taper, #26/0.04 taper, and #36/0.03 tape) (n = 12 each). The design, surface defects, phase transformation temperatures, nickel-titanium ratios, torque, force, shaping ability, and surface deformation were evaluated. Data were analyzed with the Kruskal-Wallis and Dunn's tests (α = 0.05). RESULTS: The instruments had different designs and phase transformation temperatures. The 3 and 6 up-and-down movements resulted in a smaller upward force compared to 1 movement (p < 0.05). TruNatomy generated significantly less maximum torque, force, and surface wear than JIZAI (p < 0.05). However, TruNatomy exhibited a larger canal deviation (p < 0.05). No statistical differences in shaping ability were detected between different up-and-down movements. CONCLUSIONS: Under laboratory conditions with JIZAI and TruNatomy, a single up-and-down movement at the working length increased the screw-in force of subsequent instruments in severely curved canals in the single-length instrumentation technique. A single up-and-down movement generated more surface defects on the file when using JIZAI. TruNatomy resulted in less stress generation during instrumentation, while JIZAI better maintained the curvature of root canals.

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.

Phase transformation and mechanical properties of heat-treated nickel-titanium rotary endodontic instruments at room and body temperatures.

BACKGROUND: The aim of this study was to evaluate the phase composition, phase transformation temperatures, bending property, and cyclic fatigue resistance of different heat-treated nickel-titanium (NiTi) rotary instruments with the same tip diameter and taper at room (RT; 25 ± 1 °C) and body (BT; 37 ± 1 °C) temperatures. METHODS: Five heat-treated NiTi rotary instruments, HyFlex EDM (EDM), HyFlex CM (CM), Vortex Blue (VB), RE file CT (RE) and JIZAI, and a non-heat-treated NiTi rotary instrument (Mtwo) with a size 40, 0.04 taper were investigated. Temperature-dependent phase transformation was examined with differential scanning calorimetry (DSC). The bending loads of the instruments at RT and BT were evaluated using a cantilever-bending test. Cyclic fatigue resistance at RT and BT was measured using a dynamic test, during which the instruments were rotated in combination with a 2-mm back-and-forth motion in an artificial curved canal, and the number of cycles to failure (NCF) was determined. The results were analyzed using two-way repeated measures analysis of variance, a simple main effect test, and the Bonferroni test (α = 0.05). RESULTS: DSC results indicated that EDM and Mtwo were primarily composed of martensite/R-phase and austenite, respectively, while the other heat-treated instruments were composed of a mix of martensite/R-phase and austenite at the tested temperatures. Regardless of the temperature setting, the bending loads of heat-treated instruments were significantly lower than those of Mtwo (p < 0.05). EDM showed the lowest bending loads and highest NCF at both temperatures (p < 0.05). CM, VB, and JIZAI showed significantly higher bending loads at BT than at RT (p < 0.05). The NCF of all the heat-treated instruments, except VB, was lower at BT than at RT (p < 0.05). At BT, the NCF of CM, VB, RE, and JIZAI were not significantly higher than that of Mtwo (p > 0.05). CONCLUSIONS: Heat-treated NiTi instruments exhibited lower bending loads and higher NCF values than Mtwo. However, this tendency was less pronounced at BT than at RT, especially in the NCF values of instruments with a mixture of martensite/R-phase and austenite phases at the tested temperatures.

Pulp inflammation induces Kv1.1 K+ channel down-regulation in rat thalamus.

OBJECTIVES: Signals from inflamed tooth pulp activate thalamic neurons to evoke central sensitization. We aimed to gain insights into the mechanisms mediating the early phase of pulpal inflammation-induced thalamic neural and glial activation. MATERIALS AND METHODS: Pulpal inflammation was induced via the application of mustard oil (MO) to the upper first molar of Wistar rats with local anesthesia (LA) or saline injection. After 0.5, 1, 2, and 24 hr, contralateral thalami were subjected to microarrays, a real-time polymerase chain reaction and immunohistochemistry to identify differentially expressed genes and assess potassium voltage-gated channel subfamily A member 1 (Kv1.1)-expressing axons and glial fibrillary acidic protein (GFAP)-expressing astrocytes. RESULTS: The Kv1.1 gene (Kcna1) was down-regulated and the density of Kv1.1-expressing axons decreased in non-anesthetized rats, but not in anesthetized rats 1 hr after the MO treatment. The density of GFAP-expressing astrocytes increased in both groups until 24 hr after the MO treatment, with a greater increase being observed in the saline-injection group than in the LA group. CONCLUSIONS: MO induced the transient down-regulation of Kcna1, transiently reduced the density of Kv1.1-expressing axons, and increased astrocytes in thalami within 1 hr of pulpal application. These results suggest central sensitization represented by neuronal hyperexcitability and astrocyte activation.

Effect of kinematics on the torque/force generation, surface characteristics, and shaping ability of a nickel-titanium rotary glide path instrument: An ex vivo study.

AIM: To evaluate the effect of various rotational motions on the torque/force generation, surface wear, and shaping ability of the ProGlider glide path instrument (Dentsply Sirona). METHODOLOGY: Mesiobuccal and mesiolingual canals of mandibular molars were selected based on the canal volume, length, angle of curvature (25°-40°), and radius of curvature (4-8 mm) after micro-computed tomographic scanning. The samples were randomly assigned to four groups (n = 13, each) according to movement kinematics [continuous rotation (CR; 300 rpm), optimum torque reverse motion (OTR; 180° forward and 90° reverse when torque >0.4 N cm), time-dependent reciprocal motion (TmR; 180° forward and 90° reverse), and optimal glide path motion (OGP; a combination of 90° forward, 90° reverse, 90° forward, and 120° reverse)]. Instrumentation was performed with an automated root canal instrument and torque/force analysing device. Maximum torque/force values, canal volume changes, and canal-centring ratios at 1, 3, 5, and 7 mm were evaluated. Surface defects (pits, grooves, microcracks, blunt cutting edges, and disruption of cutting edges) and spiral distortion on the ProGlider instrument were scored at the tip and 5 mm short of the tip before and after five consecutive uses with scanning electron microscopy. The Kruskal-Wallis test followed by Dunn's post-test with Bonferroni correction and Wilcoxon signed-rank test were used to analyse the data (α = 0.05). RESULTS: Optimal glide path motion generated significantly less clockwise torque and greater upward force than other groups (p < .05). OGP resulted in significantly fewer surface defects than CR (p < .05). In OGP and CR, the tip exhibited more surface defects than 5 mm short of the tip (p < .05). CR resulted in greater volume changes than OGP and TmR (p < .05) and greater centring ratios (i.e., more deviation) than OGP at 1 mm and OTR at 3 mm (p < .05). CONCLUSIONS: Under laboratory conditions using the ProGlider instrument, OGP generated significantly less clockwise torque and greater upward force than the other rotatory motions. OGP generated fewer superficial defects than CR, and the three modes of reciprocal rotation better maintained the apical curvature of root canals than CR with the ProGlider instrument.

Effect of different axial speed patterns on cyclic fatigue resistance of rotary nickel-titanium instruments.

BACKGROUND: To evaluate the effect of pecking motions with faster upward speed on the dynamic cyclic fatigue resistance of nickel-titanium rotary instruments with different metallurgy. METHODS: Forty each of ProTaper Universal F3 (PTU) and ProTaper Gold F3 (PTG) instruments (size #30/.09) were equally divided into four groups. The test was performed using an 18-mm-long stainless steel artificial canal with a 5-mm radius of curvature, a 45° canal curvature and a 2-mm canal diameter. A downward speed of 100 mm/min was employed, while the upward speed was set at 100, 150, 200 or 300 mm/min. Time to failure (Tf), number of cycles to failure (Nf) and number of pecking motions to failure (Np) were recorded. Statistical analysis was performed using Kruskal Wallis and Mann-Whitney U tests for Tf, Nf, and Np (α = 0.05). RESULTS: The 100/300 mm/min group showed significantly higher Np values than the 100/100 mm/min group (p < 0.05), whereas there were no significant differences in Tf and Nf among the tested speed groups (p < 0.05) in either PTU or PTG. PTG exhibited significantly higher Tf, Nf, and Np than PTU (p < 0.05). CONCLUSIONS: Under the tested conditions, the fastest upward speed group showed significantly higher cyclic fatigue resistance, as demonstrated by larger Np, than the same speed group. PTG had significantly higher cyclic fatigue resistance than PTU in all groups.

Polymorphonuclear Myeloid-Derived Cells That Contribute to the Immune Paralysis Are Generated in the Early Phase of Sepsis via PD-1/PD-L1 Pathway.

Immune paralysis is a protracted state of immune suppression following the early/acute inflammatory phase of sepsis. CD11b+ Gr-1+ cells induced during sepsis are heterogeneous myeloid-derived cells (MDCs). This study investigated the contribution of MDCs to immune paralysis. Treatment of mice with zymosan (ZM) induced a marked increase in the total number of splenocytes with an increase in the proportion of Gr-1hi cells and a decrease in the proportion of T cells on day 7; levels of these cells eventually return to levels similar to those of control mice on day 21. T-cell activation and gamma interferon (IFN-γ) expression by CD8+ T cells were clearly impaired in ZM-treated mice on day 21 (d21-ZM mice). Gr-1hi cells showed a CD11b+ Ly6Ghi polymorphonuclear phenotype. Injection of lipopolysaccharide (LPS) into d21-ZM mice impaired interleukin 6 (IL-6) production in serum, accompanied by accumulation of CD11b+ Gr-1hi cells in the peripheral blood. Transfer of Gr-1hi cells from d21-ZM mice into intact mice impaired IL-6 production, but similar transfer of Gr-1hi cells from PD-1/PD-L1-deficient d21-ZM mice showed no such suppressive effect. Conversely, either depletion of Gr-1hi cells by treatment with anti-Gr-1 monoclonal antibody (MAb) or neutralization of the PD-1/PD-L1 pathway by anti-PD-1 and anti-PD-L1 MAbs during the induction phase of sepsis ameliorated ZM-induced immune suppression. Our results suggest that the PD-1/PD-L1-mediated generation of Gr-1hi cells in the early phase of sepsis is required for the late phase of immune paralysis.

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.

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.

Crosstalk between dental pulp stem cells and endothelial cells augments angiogenic factor expression.

OBJECTIVES: We aimed to investigate whether the mesenchymal stem cell-endothelial cell crosstalk enhances angiogenic factor expression via nuclear factor-kappa B (NF-κB)-dependent mechanisms. MATERIALS AND METHODS: Human dermal microvascular endothelial cells (HDMECs) and stem cells from human exfoliated deciduous teeth (SHEDs) were cocultured for 96 hr, in the presence of NF-κB decoy oligodeoxynucleotides (ODNs) or scramble (control). Vascular endothelial cell growth factor (VEGF) and phospho-NF-κB p65 were measured with enzyme-linked immunosorbent assay. Angiogenesis-related gene expression was analyzed with microarray analysis followed by real-time polymerase chain reaction. Tube formation assay was conducted in the presence of NF-κB decoy. RESULTS: The VEGF and phospho-NF-κB p65 levels were significantly higher in the coculture with NF-κB decoy scramble than in single culture and coculture with NF-κB decoy ODN. Microarray analysis of SHEDs and HDMECs with NF-κB decoy scramble showed higher expression of proangiogenic genes, Bcl-2, NF-κB1, VEGFA, CXCL8, and CXCR1, and lower expression of proapoptotic genes, Bax and Caspase 9, compared to cells with NF-κB decoy ODN. Real-time PCR results for Bcl-2 and CXCL8 showed a similar trend. Tube formation assay showed more tube development in the presence of NF-κB decoy scramble. CONCLUSION: The SHED-HDMEC crosstalk enhanced proangiogenic factor expression via NF-κB-dependent pathways.

Comparison of torque, force generation and canal shaping ability between manual and nickel-titanium glide path instruments in rotary and optimum glide path motion.

This study aimed to analyze force/torque generation and canal volume changes of NiTi rotary glide path preparation using HyFlex EDM Glide Path File in comparison to manual stainless steel K-file instrumentation. Thirty extracted mandibular incisors with a minimally curved and narrow root canal were randomly divided into three groups (n = 10) according to the instrumentation kinematics: Optimum Glide Path motion (OGP) or continuous rotation (CR) with HyFlex EDM Glide Path Files using a custom-made automated-root-canal-preparation device and manual instrumentation with stainless steel K-files (SS) in watch-winding motion. Torque and force were monitored with a custom-made torque/force analyzing device. Canal volume changes and transportation values were measured on micro-computed tomographic images taken before and after the glide path preparation. The data were statistically evaluated using Kruskal-Wallis test and Mann-Whitney U test with Bonferroni correction, with a significance level set at 5%. Maximum upward apical force, representing the screw-in force, was lower in groups OGP and CR compared with that in group SS (P < 0.05). Group CR showed the highest maximum clockwise torque value and canal volume changes, followed by groups OGP and SS (P < 0.05). Canal transportation values at 1 and 3 mm from the apex were not significantly different among groups. Within the limitations of this study, rotary glide path preparation generated smaller screw-in force, larger torque and larger canal volume changes than manual preparation. OGP motion generated smaller torque and less canal volume changes than CR.

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.

Orthodontic force application upregulated pain-associated prostaglandin-I2/PGI2-receptor/TRPV1 pathway-related gene expression in rat molars.

This study aimed to analyze the mRNA expression and protein localization of prostaglandin I2 (PGI2) synthase (PGIS), the PGI2 receptor (IP receptor) and transient receptor potential cation channel, subfamily V, member 1 (TRPV1) in force-stimulated rat molars, toward the elucidation of the PGI2-IP receptor-TRPV1 pathway that is in operation in the pulp and possibly associated with orthodontic pain and inflammation. Experimental force was applied to the maxillary first and second molars by inserting an elastic band between them for 6-72 h. PGIS, PTGIR (the IP receptor gene), and TRPV1 mRNA levels in the coronal pulp were analyzed with real-time PCR. PGIS, IP receptor, and TRPV1 proteins were immunostained. The force stimulation induced significant upregulation of PGIS at 6-24 h, and PTGIR and TRPV1 at 6 and 12 h in the pulp. PGIS was immunolocalized in odontoblasts and some fibroblasts in the force-stimulated pulp. The IP receptor and TRPV1 immunoreactivities were detected on odontoblasts and some nerve fibers. It was concluded that PGIS, PTGIR, and TRPV1 in rat molar pulp were significantly upregulated shortly after the force application, and that the IP receptor was co-expressed on TRPV1-expressing nerves and odontoblasts. These findings suggest that the PGI2-IP receptor-TRPV1 pathway is associated with the acute phase of force-induced pulp changes involving odontoblasts and nerves.

Dental pulp tissue engineering of pulpotomized rat molars with bone marrow mesenchymal stem cells.

The major goal of dental pulp tissue engineering is to enable the healing of inflamed tissue or to replace necrotic pulp tissue with newly formed dental pulp tissue. Here, we report a protocol for pulp tissue engineering in vivo in pulpotomized rat teeth using constructs of rat bone marrow mesenchymal stem cells, preformed biodegradable scaffolds, and hydrogel. The constructs were implanted into pulpotomized pulp chambers for 3, 7, or 14 days. At 3 days, cells were located mainly along the preformed scaffolds. At 7 days, pulp tissue regeneration was observed in almost the entire implanted region. At 14 days, pulp tissue regeneration further progressed throughout the implanted region. In immunohistochemistry, at 3 days, a number of small and round macrophages immunoreactive to CD68 were predominantly distributed around the scaffolds. The density of CD68+ macrophages decreased until 14 days. On the other hand, nestin-expressing odontoblast-like cells beneath the dentin at the border of implanted region increased until 14 days. Quantitative gene expression analysis revealed that odontoblast differentiation marker dentin sialophosphoprotein mRNA in the implanted region gradually increased until 14 days. Together, the results suggested that regeneration of dental pulp tissue had occurred. Thus, our study provides a novel experimental rat model of dental pulp regeneration.

Anti-biofilm and bactericidal effects of magnolia bark-derived magnolol and honokiol on Streptococcus mutans.

Dental caries affects people of all ages and is a worldwide health concern. Streptococcus mutans is a major cariogenic bacterium because of its ability to form biofilm and induce an acidic environment. In this study, the antibacterial activities of magnolol and honokiol, the main constituents of the bark of magnolia plants, toward planktonic cell and biofilm of S. mutans were examined and compared with those of chlorhexidine. The minimal inhibitory concentrations of magnolol, honokiol and chlorhexidine for S. mutans were 10, 10 and 0.25 µg/mL, respectively. In addition, each agent showed bactericidal activity against S. mutans planktonic cells and inhibited biofilm formation in a dose- and time-dependent manner. Magnolol (50 µg/mL) had greater bactericidal activity against S. mutans biofilm than honokiol (50 µg/mL) and chlorhexidine (500 µg/mL) at 5 min after exposure, while all showed scant activity against biofilm at 30 s. Furthermore; chlorhexidine (0.5-500 µg/mL) exhibited high cellular toxicity for the gingival epithelial cell line Ca9-22 at 1 hr, whereas magnolol (50 µg/mL) and honokiol (50 µg/mL) did not. Thus; it was found that magnolol has antimicrobial activities against planktonic and biofilm cells of S. mutans. Magnolol may be a candidate for prevention and management of dental caries.

An ion extract obtained from mineral trioxide aggregate induced dentin remineralization and dentin tubule occlusion in artificially demineralized bovine dentin.

PURPOSE: To investigate the ability of a mineral trioxide aggregate (MTA) extract mixed with a phosphate buffered saline (PBS) system to induce remineralization and dentin tubule occlusion in artificially demineralized bovine dentin. METHODS: The MTA extract solution was prepared by mixing white ProRoot MTA with distilled water (1:2) for 48 hours, before subjecting it to centrifugation. The elemental composition of the MTA extract solution was analyzed with inductively coupled plasma atomic emission spectrometry. The deposits produced by the MTA extract-PBS mixture were chemically analyzed using electron probe microanalysis (EPMA) and X-ray diffraction (XRD). The effects of the two-step application of the mixture (MTA extract solution followed by PBS) to bovine dentin samples that had been artificially demineralized with phosphoric acid (10%, 10 seconds) were investigated with scanning electron microscopy and EPMA after the specimens had been stored in PBS for 1 or 7 days. RESULTS: The MTA extract solution contained calcium, silicone, and aluminum (Ca>Si>Al), and the deposits produced by the MTA extract-PBS mixture contained calcium, phosphorous, sodium, silicone, and aluminum (Ca>P>Na>Si>Al) as major mineral elements. XRD also revealed that the deposits contained hydroxyapatite. The two-step application process resulted in the formation of a 2-3 microm-thick "mineral infiltration layer", together with mineral tag-like structures in the dentin tubules. The MTA extract-treated specimens exhibited a significantly higher dentin tubule occlusion rate than the untreated specimens (P < 0.05).

Dentin tubule occluding ability of dentin desensitizers.

PURPOSE: To compare the dentin tubule-occluding ability of fluoroaluminocalciumsilicate-based (Nanoseal), calcium phosphate-based (Teethmate Desensitizer), resin-containing oxalate (MS Coat ONE) and diamine silver fluoride (Saforide) dentin desensitizers using artificially demineralized bovine dentin. METHODS: Simulated hypersensitive dentin was created using cervical dentin sections derived from bovine incisors using phosphoric acid etching followed by polishing with a paste containing hydroxyapatite. The test desensitizers were applied in one, two, or three cycles, where each cycle involved desensitizer application, brushing, and immersion in artificial saliva (n= 5 each). The dentin surfaces were examined with scanning electron microscopy, and the dentin tubule occlusion rate was calculated. The elemental composition of the deposits was analyzed with electron probe microanalysis. Data were analyzed by one-way ANOVA and the Tukey honestly significant different test. RESULTS: Marked deposit formation was observed on the specimens treated with Nanoseal or Teethmate Desensitizer, and tags were detected in the specimens' dentin tubules. These findings became more prominent as the number of application cycles increased. The major elemental components of the tags were Ca, F, and Al (Nanoseal) and Ca and P (Teethmate Desensitizer). The tubule occlusion rates of MS Coat ONE and Saforide were significantly lower than those of Nanoseal and Teethmate Desensitizer (P< 0.05).

Penetration kinetics of four mouthrinses into Streptococcus mutans biofilms analyzed by direct time-lapse visualization.

OBJECTIVE: The aim of this study was to determine whether different antiseptic mouthrinses show different penetration kinetics into Streptococcus mutans biofilms. MATERIALS AND METHODS: The biofilms, grown on glass-based dishes, were exposed to one of four mouthrinses containing chlorhexidine digluconate, essential oil, cetylpyridinium chloride, or isopropylmethylphenol. Then, penetration velocities were determined by monitoring fluorescence loss of calcein AM-stained biofilms with time-lapse confocal laser scanning microscopy. Bactericidal activity was assessed with fluorescent bacterial viable cell (Live/Dead) staining and viable cell counts. Bacterial detachment after the mouthrinse exposure was determined by measuring fluorescence reduction of SYTO9-stained biofilms. RESULTS: The essential oil-containing mouthrinse showed significantly faster penetration velocity than the other mouthrinses (ANCOVA and Bonferroni test, p < 0.05). However, even 5 min of exposure left the biofilm structure almost intact. After 30 s (consumer rinsing time) of exposure, the essential oil-containing mouthrinse showed the highest log reduction of viable cells (2.7 log CFU) measured by Live/Dead staining, and the mean reduction of total viable cells was 1.41 log CFU measured by viable cell count. CONCLUSIONS: The essential oil-containing mouthrinse showed the best penetration. Within 30 s of exposure, however, no mouthrinses injured all the microorganisms and all mouthrinses left the biofilm structure nearly intact. CLINICAL RELEVANCE: The mouthrinses tested showed different levels of biofilm penetration. The essential oil rinse was superior to other rinses by all three of the in vitro measurements performed.