Search

Intracellular Ca 2+ signaling engendered by Ca 2+ influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca 2+ is exported by the Na + -Ca 2+ exchanger (NCX) and plasma membrane Ca 2+ -ATPase (PMCA) to maintain Ca 2+ homeostasis. We previously demonstrated a functional coupling between Ca 2+ extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under physiological conditions. We also examined the role of PMCA in the Ca 2+ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca 2+ concentration ([Ca 2+ ] i ). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca 2+ ] i was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor teeth and human odontoblasts. We detected mRNA encoding PMCA1-4 in human odontoblasts. The application of hypotonic or alkaline solutions transiently increased [Ca 2+ ] i in odontoblasts in both rat and human odontoblasts. The Ca 2+ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca 2+ ] i increase was decreased by PMCA inhibitors in both cell types. Alizarin red and von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did not affect DMP-1 or DSPP mRNA levels at pH 7.4-8.8 and under isotonic and hypotonic conditions, respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These findings indicate that PMCA participates in maintaining [Ca 2+ ] i homeostasis in odontoblasts by Ca 2+ extrusion following [Ca 2+ ] i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca 2+ to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of high-pH dental materials.

Incomplete polymerization or biodegradation of dental resin materials results in the release of resin monomers such as triethylene glycol dimethacrylate (TEGDMA), causing severe injury of dental pulp cells. To date, there has been no efficient treatment option for this complication, in part due to the lack of understanding of the mechanism underlying these phenomena. Here, for the first time, we found that notoginsenoside R1 (NR1), a bioactive ingredient extracted from Panax notoginseng, exerted an obvious protective effect on TEGDMA-induced mitochondrial apoptosis in the preodontoblast mDPC6T cell line. In terms of the mechanism of action, NR1 enhanced the level of phosphorylated Akt (protein kinase B), resulting in the activation of a transcriptional factor, nuclear factor erythroid 2-related factor 2 (Nrf2), and eventually upregulating cellular ability to resist TEGDMA-related toxicity. Inhibiting the Akt/Nrf2 pathway by pharmaceutical inhibitors significantly decreased NR1-mediated cellular antioxidant properties and aggravated mitochondrial oxidative damage in TEGDMA-treated cells. Interestingly, NR1 also promoted mitophagy, which was identified as the potential downstream of the Akt/Nrf2 pathway. Blocking the Akt/Nrf2 pathway inhibited mitophagy and abolished the protection of NR1 on cells exposed to TEGDMA. In conclusion, these findings reveal that the activation of Akt/Nrf2 pathway-mediated mitophagy by NR1 might be a promising approach for preventing resin monomer-induced dental pulp injury., (Copyright © 2021 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) play critical roles in various biological processes including cell differentiation. Some researchers suggested that the p38 mitogen-activated protein kinases (MAPK) signaling pathway had an effect on regulating the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). This study focuses on the effects of miR-143-5p on hDPSCs by regulating the p38 MAPK signaling pathway. The targeting relationship of MAPK14 and miR-143-5p targets were verified by TargetScan and dual-luciferase reporter gene assay. Through overexpression of miR-143-5p or silencing of miR-143-5p, expressions of miR-143-5p, MAPK14, Ras, MAPK kinase (MKK) 3/6, dentin sialophosphoprotein (DSPP), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by reverse transcription quantitative polymerase chain reaction. Protein expressions of MAPK14, Ras, and MKK3/6 were determined by western blot analysis. ALP and alizarin red S staining were used to detect mineralization. Initially, MAPK14 was found to be negatively regulated by miR-143-5p. Meanwhile, the upregulated miR-143-5p decreased the p38 MAPK signaling pathway related genes (MAPK14, Ras, and MKK3/6) and odontoblastic differentiation markers (ALP, DSPP, and OCN) expression. On the contrary, the downregulated miR-143-5p increased the p38 MAPK signaling pathway related genes (MAPK14, Ras, and MKK3/6) and odontoblastic differentiation markers (ALP, DSPP, and OCN) expression. Furthermore, ALP activity and mineralized nodules increased after downregulation of miR-143-5p, and after its upregulation, ALP activity and mineralized nodules decreased. Our data suggest that poor expression of miR-143-5p promotes hDPSCs odontoblastic differentiation through the activation of the p38 MAPK signaling pathway by upregulating MAPK14., (© 2018 Wiley Periodicals, Inc.)

Human dental pulp stem cells (hDPSCs) are primarily derived from the pulp tissues of permanent third molar teeth. They were widely used in human bone tissue engineering. It was previously indicated that microRNA (miR) expressions are closely associated with hDPSCs development. However, the specific effect of miR-488 on hDPSCs still remains unclear. In this study, we aimed to investigate effects of miR-488 on the differentiation of hDPSCs into odontoblast cells through the p38 mitogen-activated protein kinases (MAPK) signaling pathway by binding to MAPK1. The hDPSCs were isolated and cultured in vitro. Dual-luciferase reporter gene assay was performed to test the relationship between MAPK1 (p38) and miR-488. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to detect the mRNA and protein expressions of p38 MAPK signaling pathway-related genes (MAPK1, Ras, and Mitogen-activated protein kinase kinase 3/6 [MKK3/6]), along with expressions of dentin Sialophosphoprotein (DSPP), alkaline phosphatase (ALP), and osteonectin (OCN). ALP staining and alizarin red staining were conducted to detect ALP activity and degree of mineralization. Initially, we found that MAPK1 was the target gene of miR-488. Besides, downregulation of miR-488 was observed to stimulate the p38 MAPK signaling pathway and to increase the messenger RNA and protein expressions of DSPP, ALP, and OCN. Furthermore, ALP activity and formation of a mineralized nodule in hDPSCs were enhanced upon downregulation of miR-488. The aforementioned findings provided evidence supporting that downregulation of miR-488 promotes odontoblastic differentiation of hDPSCs through the p38 MAPK signaling pathway by targeting MAPK1, paving the basis for further study about hDPSCs., (© 2018 Wiley Periodicals, Inc.)

The present study was designed to investigate the effect of laminin-1 (LN-1 or LN-111) on an odontoblast-like cell line, MDPC-23. Wells of non-treated polystyrene plates were coated with various concentrations of LN-1 (0.1, 1, 10, and 100 µg/mL) and left to dry for 2 days. Water-coated surfaces were used as controls. MDPC-23 cell proliferation, differentiation and mineralization were evaluated in terms of the CCK-8 assay, ALP activity, real-time RT-PCR and Alizarin red staining. The data indicated that LN-1 promoted the proliferation of MDPC-23 cells in a concentration-dependent manner. Moreover, it enhanced ALP activity and expression of key odontogenic genes (DMP-1 and DSPP) upon addition of mineralization reagents, leading to significant promotion of calcification by the cells. These results demonstrate that LN-1 acts as an adhesive for odontoblast-like cells, allowing up-regulation of odontogenic genes and accelerating matrix mineralization. In the context of the present study, the optimal LN-1 coating concentration for MDPC-23 cells was suggested to be 100 µg/mL.

Objectives: Ten-eleven translocation 1 (TET1) is a DNA methylcytosine (mC) dioxygenase discovered recently that can convert 5-mC into 5-hydroxymethylcytosine (5hmC). We previously reported that TET1 promotes odontoblastic differentiation of human dental pulp cells (hDPCs). The gene encoding the family with sequence similarity 20, member C (FAM20C) protein, is a potential TET1 target and showed demethylation during odontoblastic differentiation of hDPCs in our previous study. This study aimed to explore whether TET1-mediated hydroxymethylation could activate the FAM20C gene, thereby regulating hDPC differentiation., Materials and Methods: The expression pattern of FAM20C and its potential changes during odontoblastic induction of hDPCs were assessed by Western blotting. Lentivirus-mediated transduction with short hairpin RNA (shRNA) was used to knock down FAM20C and TET1 expression in hDPCs. The mineralization potential of hDPCs was evaluated with an ALPase activity assay and by observing the mineralized matrix deposition and the expression of odontoblast-related markers DSPP and DMP1. Recombinant human FAM20C protein (rhFAM20C) was reintroduced into shTET1 cells in a rescue experiment. The dynamic hydroxymethylation status of the FAM20C gene promoter was examined using hydroxymethylated DNA immunoprecipitation (IP)-PCR. Chromatin IP-PCR and agarose gel electrophoresis were utilized to validate the recruitment of TET1 to its target loci in the FAM20C promoter., Results: FAM20C protein level was upregulated after the odontoblastic induction of hDPCs. shRNA-mediated FAM20C suppression reduced the expression of DSPP and DMP1 after odontoblastic induction for 7 and 14 days. ALPase activity was reduced on day 7, and the formation of mineralized nodules was attenuated on day 14 after odontoblastic induction in FAM20C-inhibited hDPCs. Genomic 5hmC levels significantly decreased, and total 5mC levels increased in TET1-deficient hDPCs. In addition, a significant reduction in FAM20C also emerged. The rhFAM20C treatment of shTET1 cells attenuated the mineralization abnormalities caused by TET1 depletion. TET1 depletion prompted a decline in 5hmC levels in several regions on the FAM20C promoter. Enhanced TET1 recruitment was detected at the corresponding loci in the FAM20C promoter during odontoblastic induction., Conclusion: TET1 knockdown suppressed odontoblastic differentiation by restraining its direct binding to FAM20C promoter, and hence inhibiting FAM20C hydroxymethylation and subsequent transcription. These results suggest that TET1 potentially promotes the cytodifferentiation potential of hDPCs through its DNA demethylation machinery and upregulation of FAM20C protein expression., (© 2017 John Wiley & Sons Ltd.)

Aim: To assess the effects of 2-hydroxyethyl methacrylate (HEMA) on proliferation and migration of human pulp cells, as well as on matrix metalloproteinase (MMP-2 and MMP-9) expression in human odontoblast-like cells, contributing to the goal of determining the relationship between resin materials and MMP activity in pulp-dentine complexes., Methodology: Dental pulp cell cultures were established from pulp tissue of human teeth extracted for orthodontic purposes. Pulp cell differentiation was characterized in the presence of dentine sialophosphoprotein, bone sialoprotein and alkaline phosphatase by reverse transcription polymerase chain reaction. MMP activity was assessed by gelatine zymography with media containing HEMA. Cell viability was evaluated using methyl thiazolyl tetrazolium assay for 24-72 h. Cell migration was tested using Transwell migration assay. Western blotting was used to visualize MMP expression with the nontoxic HEMA concentrations (0-400 μg mL -1 ) for 48 h., Results: Pulp cell proliferation decreased with HEMA exposure in a time- and concentration-dependent manner. HEMA concentrations ≤400 μg mL -1 did not induce changes in cell viability at 48 h (P < 0.05). Pulp cells were induced to differentiate into odontoblast-like cells in media containing 5 mg mL -1 ascorbic acid and 10 mmol L -1 β-sodium glycerophosphate for 3-4 weeks. After incubation with HEMA, dose-dependent inhibition was observed; HEMA had a strong inhibitory effect on MMP activity. Compared with the control group, cell migration and MMP expression were inhibited significantly with increasing HEMA concentration at noncytotoxic doses (P < 0.05)., Conclusions: Cell viability was not affected at HEMA concentrations ≤400 μg mL -1 . Within this range, HEMA inhibited MMP-2 and MMP-9 expression and activity, which may protect against type I collagen degradation effectively during dentine adhesive procedures., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Protease-activated receptors (PARs) are G protein-coupled receptors, which are activated by proteolytical cleavage of the amino-terminus and act as sensors for extracellular proteases. We hypothesized that PAR-1 and PAR-2 can be modulated by inflammatory stimulus in human dental pulp cells. PAR-1 and PAR-2 gene expression in human pulp tissue and MDPC-23 cells were analyzed by quantitative polymerase chain reaction. Monoclonal PAR-1 and PAR-2 antibodies were used to investigate the cellular expression of these receptors using Western blot, flow cytometry, and confocal microscopy in MDPC-23 cells. Immunofluorescence assays of human intact and carious teeth were performed to assess the presence of PAR-1 and PAR-2 in the dentin-pulp complex. The results show for the first time that human odontoblasts and MDPC-23 cells constitutively express PAR-1 and PAR-2. PAR-2 activation increased significantly the messenger RNA expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MMP-14 in MDPC-23 cells ( P < 0.05), while the expression of these enzymes decreased significantly in the PAR-1 agonist group ( P < 0.05). The high-performance liquid chromatography and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis showed the presence of MMP-13 activity cleaving PAR-1 at specific, noncanonical site TLDPRS 42 ↓F 43 LL in human dental pulp tissues. Also, we detected a presence of a trypsin-like activity cleaving PAR-2 at canonical site SKGR 20 ↓S 21 LIGRL in pulp tissues. Confocal microscopy analysis of human dentin-pulp complex showed intense positive staining of PAR-1 and PAR-2 in the odontoblast processes in dentinal tubules of carious teeth compared to intact ones. The present results support the hypothesis of activation of the upregulated PAR-1 and PAR-2 by endogenous proteases abundant during the inflammatory response in dentin-pulp complex.

Objective: Petrodentine, the core of the lungfish tooth plate, is a well-mineralized tissue similar to mammalian enamel and analogous to enameloid in fish teeth. Petrodentine is formed solely by petroblasts, which are specialized odontoblasts, whereas enameloid is a composite tissue produced by both odontoblasts and dental epithelial cells. To clarify the details of petrodentine formation, petroblasts were investigated using histochemical and immunohistochemical techniques., Methods: Extant lungfish (Lepidosiren paradoxa) were used in this study. Tooth plates during the stage of petrodentine formation were observed by means of histochemistry and immunohistochemistry. Commercial kits were used to detect enzyme activity. Correlative sections were immunostained using antibodies against selected peptides. Routine staining such as periodic acid-Schiff (PAS) reaction to identify glycogen and Elastica van Gieson staining for the detection of elastic fibers in histological sections were performed. In addition, conventional transmission electron microscopy was used for observing the fine structure., Results: Petroblasts showed marked acid and alkaline phosphatase activities, and positive immunoreactivities against anti-nestin, anti-V-ATPase, and anti-Ca 2+ -ATPase, during the maturation stage, but in the matrix formation stage, reactions were much weaker than that of the maturation stage. During the maturation stage, petroblasts showed intense PAS reactivity, and glycogen particles were observed in petroblasts by transmission electron microscopy. Glucose transporter 1-immunoreactivity was observed in petroblasts in the matrix formation stage and the initial to mid part of the maturation stage., Conclusions: The results in this study suggested that petroblasts have two functional stages, matrix formation and maturation, and glycogen plays an important role in the modulation of petroblasts., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Aim: To improve an enzymatic method previously used for isolation of rat odontoblasts to isolate viable mature human odontoblasts., Methodology: Collagenase I, collagenase I/hyaluronidase mixture and hyaluronidase were used to extract mature human odontoblasts from the pulp chamber. Detachment of odontoblasts from dentine was determined with field emission scanning electron microscopy (FESEM) and to analyse the significance of differences in tubular diameter, and the t-test was used. MTT-reaction was used to analyse cell viability, and nonparametric Kruskal-Wallis and Mann-Whitney post hoc tests were used to analyse the data. Immunofluorescent staining of dentine sialoprotein (DSP), aquaporin-4 (AQP4) and matrix metalloproteinase-20 (MMP-20) and quantitative PCR (qPCR) of dentine sialophosphoprotein (DSPP) were used to confirm the odontoblastic nature of the cells., Results: MTT-reaction and FESEM demonstrated collagenase I/hyaluronidase resulted in more effective detachment and higher viability than collagenase I alone. Hyaluronidase alone was not able to detach odontoblasts. Immunofluorescence revealed the typical odontoblastic-morphology with one process, and DSP, AQP4 and MMP-20 were detected. Quantitative PCR of DSPP confirmed that the isolated cells expressed this odontoblast-specific gene., Conclusion: The isolation of viable human odontoblasts was successful. The cells demonstrated morphology typical for odontoblasts and expressed characteristic odontoblast-type genes and proteins. This method will enable new approaches, such as apoptosis analysis, for studies using fully differentiated odontoblasts., (© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Odontoblasts differentiate from dental mesenchyme during dentin formation and mineralization. However, the molecular mechanisms controlling odontoblast differentiation remain poorly understood. Here, we show that expression of testicular acid phosphatase (ACPT) is restricted in the early stage of odontoblast differentiation in proliferating dental mesenchymal cells and secretory odontoblasts. ACPT is expressed earlier than tissue-nonspecific alkaline phosphatase (TNAP) and partly overlaps with TNAP in differentiating odontoblasts. In MDPC-23 odontoblastic cells, expression of ACPT appears simultaneously with a decrease in β-catenin activity and is abolished with the expression of Phex and Dsp. Knockdown of ACPT in MDPC-23 cells stimulates cell proliferation together with an increase in active β-catenin and cyclin D1. In contrast, the overexpression of ACPT suppresses cell proliferation with a decrease in active β-catenin and cyclin D1. Expression of TNAP, Osx, Phex and Dsp is reduced by knockdown of ACPT but is enhanced by ACPT overexpression. When ACPT is blocked with IgG, alkaline phosphatase activity is inhibited but cell proliferation is unchanged regardless of ACPT expression. These findings suggest that ACPT inhibits cell proliferation through β-catenin-mediated signaling in dental mesenchyme but elicits odontoblast differentiation and mineralization by supplying phosphate during dentin formation. Thus, ACPT might be a novel candidate for inducing odontoblast differentiation and mineralization for dentin regeneration.

We have previously reported that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation in mouse embryonic stem cell (ESC)-derived odontoblast-like cells, suggesting that MMP-3 plays a potentially unique physiological role in regeneration by odontoblast-like cells. MMPs are able to process virtually any component of the extracellular matrix, including collagen, laminin and bioactive molecules. Because odontoblasts produce dentin matrix protein-1 (DMP-1), we examined whether the degraded products of DMP-1 by MMP-3 contribute to enhanced proliferation in odontoblast-like cells. IL-1β increased mRNA and protein levels of odontoblastic marker proteins, including DMP-1, but not osteoblastic marker proteins, such as osteocalcin and osteopontin. The recombinant active form of MMP-3 could degrade DMP-1 protein but not osteocalcin and osteopontin in vitro. The exogenous degraded products of DMP-1 by MMP-3 resulted in increased proliferation of odontoblast-like cells in a dose-dependent manner. Treatment with a polyclonal antibody against DMP-1 suppressed IL-1β-induced cell proliferation to a basal level, but identical treatment had no effect on the IL-1β-induced increase in MMP-3 expression and activity. Treatment with siRNA against MMP-3 potently suppressed the IL-1β-induced increase in DMP-1 expression and suppressed cell proliferation (p < 0.05). Similarly, treatment with siRNAs against Wnt5a and Wnt5b suppressed the IL-1β-induced increase in DMP-1 expression and suppressed cell proliferation (p < 0.05). Rat KN-3 cells, representative of authentic odontoblasts, showed similar responses to the odontoblast-like cells. Taken together, our current study demonstrates the sequential involvement of Wnt5, MMP-3, DMP-1 expression, and DMP-1 degradation products by MMP-3, in effecting IL-1β-induced proliferation of ESC-derived odontoblast-like cells.

A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background Information: Stem cells from apical papilla (SCAPs) are a potent candidate for the apexogenesis/apexification due to their multiple differentiation capacity. During the orthodontic treatment of developing teeth, SCAPs in vivo are usually subjected to the cyclic stress induced by compression forces. However, it remains unclear whether mechanical stress can affect the proliferation and differentiation of human SCAPs., Results: Human SCAPs were isolated and stimulated by 200 g mechanical stimuli for 30 min and their proliferation and differentiation capacity were evaluated in vitro at different time points. MTT and FCM results demonstrated that cell proliferation was enhanced, while TEM findings showed the morphological and ultrastructural changes in stress-treated SCAPs. ALP activity and mineralization capacity of stress-treated SCAPs were upregulated . In the meantime, higher odontogenic and osteogenic differentiation were found in stress-treated SCAPs by real-time RT-PCR and Western blot, as indicated by the expression of related markers at both mRNA and protein levels. Moreover, the protein expressions of pJNK and pERK MAPK pathways were upregulated., Conclusion: Together, these findings suggest that mechanical stress is an important factor affecting the proliferation and differentiation of SCAPs via the activation of ERK and JNK signaling pathway.

Patients with mild forms of peroxisomal biogenesis disorders show facial dysmorphism and exhibit dentition problems accompanied by enamel hypoplasia. However, no information is available on the role of peroxisomes in dental and paradontal tissues. Therefore, we studied the distribution of these organelles, their protein composition and the expression of corresponding genes during dental development and in mature decalcified teeth in mice. Perfusion-fixed heads of mice of different developmental stages (E13.5 to adult) were cut in sagittal direction into two halves and embedded in paraffin for serial sectioning and subsequent peroxidase-based immunohistochemistry or double-immunofluorescence preparations. Frozen, unfixed heads of newborn mice were used for cryosectioning and subsequent laser-assisted microdissection of ameloblasts and odontoblasts, RNA isolation and RT-PCR analysis. Our results revealed the presence of peroxisomes already in the bud stage of dental development. An increase in peroxisome abundance was noted during differentiation of ameloblasts and odontoblasts with the highest number of organelles in Tomes' processes of mature ameloblasts. A strong heterogeneity of peroxisomal enzyme content developed within differentiated dental cell types. A drastic down-regulation of catalase in maturing ameloblasts was noted in contrast to high levels of lipid metabolizing enzymes in peroxisomes of these cells. As known from the literature, differentiated ameloblasts are more prone to oxidative damage which could be explained by the low catalase levels inside of this cell type.

Background: bleaching has been widely studied, mainly due to the possible undesirable effects that can be caused by this esthetic procedure. The cytotoxicity of the bleaching agents and its components to pulp cells has been demonstrated in several researches. The aim of this study was to evaluate the toxic effects of successive applications of 10% carbamide peroxide (CP) gel on odontoblast-like cells., Materials and Methods: Enamel-dentin discs obtained from bovine incisors were adapted to artificial pulp chambers (APCs). The groups were formed as follows: G1: Without treatment (control group); G2: 10% carbamide peroxide, CP (five applications/one per day); G3: 10% CP (one unique application); and G4: 35% hydrogen peroxide, HP (three applications of 15 min each). After treatment, cell metabolism (MTT), alkaline phosphatase (ALP) activity and plasma membrane damage (flow cytometry) were analyzed., Results: Reductions in cell metabolism and alkaline phosphatase activity along with severe damage of the cytoplasmic membrane were noted in G2. In G3, no damage was observed, compared to the control group. Intermediary values of toxicity were obtained after 35% HP application., Conclusion: It can be concluded that one application of 10% CP did not cause toxic effects in odontoblast-like cells, but the successive application of this product promoted severe cytotoxic effects. The daily application of the bleaching agents, such as used in the at-home bleaching technique, can increase the damages caused by this treatment to the dental pulp cells.

Phosphatases are involved in bone and tooth mineralization, but their mechanisms of action are not completely understood. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) regulates inhibitory extracellular pyrophosphate through its pyrophosphatase activity to control mineral propagation in the matrix; mice without TNAP lack acellular cementum, and have mineralization defects in dentin, enamel, and bone. PHOSPHO1 is a phosphatase found within membrane-bounded matrix vesicles in mineralized tissues, and double ablation of Alpl and Phospho1 in mice leads to a complete absence of skeletal mineralization. Here, we describe mineralization abnormalities in the teeth of Phospho1(-/-) mice, and in compound knockout mice lacking Phospho1 and one allele of Alpl (Phospho1(-/-);Alpl(+/-) ). In wild-type mice, PHOSPHO1 and TNAP co-localized to odontoblasts at early stages of dentinogenesis, coincident with the early mineralization of mantle dentin. In Phospho1 knockout mice, radiography, micro-computed tomography, histology, and transmission electron microscopy all demonstrated mineralization abnormalities of incisor dentin, with the most remarkable findings being reduced overall mineralization coincident with decreased matrix vesicle mineralization in the Phospho1(-/-) mice, and the almost complete absence of matrix vesicles in the Phospho1(-/-);Alpl(+/-) mice, whose incisors showed a further reduction in mineralization. Results from this study support prominent non-redundant roles for both PHOSPHO1 and TNAP in dentin mineralization.

Aim: To investigate the effects of CpG ODN (CpG oligodeoxynucleotides) to model the action of bacterial challenge on pulpal matrix metalloproteinase-13 (MMP-13) expression and elucidate the associated intracellular signalling pathways., Methodology: Real-time PCR was used to detect the effects of CpG ODN on MMP-13 mRNA expression levels in a murine odontoblast-lineage cell line (OLCs). The possible involvement of TLR9/MyD88, NF-κB or MAPK pathways involved in the CpG ODN-induced MMP-13 expression was examined by real-time PCR, transient transfection, luciferase activity assay and ELISA. Western blotting was performed to assay the phosphorylation of ERK at a range of time points., Results: MMP-13 was constitutively expressed in OLCs, and their exposure to CpG ODN significantly increased MMP-13 expression. Pre-treatment of OLCs with the inhibitory peptide MyD88, or chloroquine, attenuated the CpG ODN-induced expression of MMP-13. Treatment of the OLCs with CpG ODN increased NF-κB-luciferase activity. This activity was decreased by the over-expression of a nondegrading mutant of IκBα (IκBαSR), although enhanced by the over-expression of NF-κB p65. MMP-13 expression induced by CpG ODN was markedly suppressed by NF-κB inhibitors (pyrrolidine dithiocarbamate, PDTC), IκBα phosphorylation inhibitors (Bay 117082) or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone, TPCK). The inhibitor of ERK1/2, U0126, but not inhibitors of p38 MAPK and JNK, SB203580 and SP600125, decreased CpG ODN-mediated MMP-13 expression., Conclusion: The CpG ODN-induced MMP-13 expression in OLCs is mediated through TLR9, NF-κB and the ERK pathway indicating that potentially the recognition of CpG ODN by TLR9 on odontoblasts may regulate the remodelling of injured dental pulp and hard tissues by inducing MMP-13 expression., (© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Sodium fluoride (NaF) is widely used for the treatment of dental caries and dentin hypersensitivity. However, its pro-apoptotic effect on odontoblasts may lead to harmful side-effects. The purpose of this study was to evaluate the pro-apoptotic effects of NaF in odontoblasts and elucidate the possible underlying molecular mechanisms. NaF generated cytotoxic effects in odontoblast-lineage cell (OLC) in a dose- and time-dependent manner. Exposure of cells to 4mM NaF for 24h induced caspase-3 activation, ultrastructural alterations, and resulted in the translocation of Bax to the mitochondria and the release of cytochrome c from the mitochondrial inter-membrane space into the cytosol, indicating that fluoride-mediated apoptosis is mitochondria-dependent. Fluoride treatment also increased phosphorylation of JNK and ERK, but not p38, and apoptosis induced by fluoride was notably or partly suppressed by treatment with JNK or ERK inhibitors, respectively. Taken together, these findings suggest that NaF induces apoptosis in OLC odontoblasts through a JNK-dependent mitochondrial pathway., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Apoptosis during tooth development appears dependent on the apoptotic executioner caspase-3, but not caspase-7. Instead, activated caspase-7 has been found in differentiated odontoblasts and ameloblasts, where it does not correlate with apoptosis. To further investigate these findings, the mouse incisor was used as a model. Analysis of caspase-7-deficient mice revealed a significant thinner layer of hard tissue in the adult incisor. Micro computed tomography scan confirmed this decrease in mineralized tissues. These data strongly suggest that caspase-7 might be directly involved in functional cell differentiation and regulation of the mineralization of dental matrices., (© 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.)

Aim: To establish whether eliminating Lysyl oxidase (LOX) gene would affect dentine formation., Methodology: Newborn wild-type (wt) and homo- and heterozygous LOX knock-out (Lox(-/-) and Lox(+/-) , respectively) mice were used to study developing tooth morphology and dentine formation. Collagen aggregation in the developing dentine was examined histochemically with picrosirius red (PSR) staining followed by polarized microscopy. Because Lox(-/-) die at birth, adult wt and Lox(+/-) mouse tooth morphologies were examined with FESEM. Human odontoblasts and pulp tissue were used to study the expression of LOX and its isoenzymes with Affymetrix cDNA microarray., Results: No differences between Lox(-/-) , Lox(+/-) and wt mice developing tooth morphology were seen by light microscopy. Histochemically, however, teeth in wt mice demonstrated yellow-orange and orange-red polarization colours with PSR staining, indicating thick and more densely packed collagen fibres, whilst in Lox(-/-) and Lox(+/-) mice, most of the polarization colours were green to green-yellow, indicating thinner, less aggregated collagen fibres. Fully developed teeth did not show any differences between Lox(+/-) and wt mice with FESEM. Human odontoblasts expressed LOX and three of four of its isoenzymes., Conclusions: The data indicate that LOX is not essential in dentinogenesis, even though LOX deletion may affect dentine matrix collagen thickness and packing. The absence of functional LOX may be compensated by LOX isoenzymes., (© 2012 International Endodontic Journal.)

Objective: The aim of this study is to clarify the effects of zinc chloride (ZnCl2) exposure on the induction of heme oxygenase-1 (HO-1) expression and its regulatory mechanisms in MDPC-23 mouse odontoblast-like cells., Methods: MDPC-23 cells were incubated with ZnCl2, and the levels of HO-1 protein, phosphorylated forms of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38, and phosphorylated forms of amino kinase terminal (Akt) and nuclear factor-κB (NF-κB) p65 were determined with western immunoblotting. The level of HO-1 mRNA was determined with RT-PCR analysis. After pretreatment with inhibitors of ERK, JNK, p38, phosphoinositide-3 kinase (PI3K), and NF-κB, HO-1 protein level was determined in MDPC-23 cells exposed to ZnCl2., Results: Following exposure to 500μM ZnCl2, the levels of both HO-1 mRNA and protein were markedly increased. The phosphorylated forms of ERK, JNK, and p38 increased after ZnCl2 exposure. Furthermore, the expression of HO-1 was markedly suppressed by treatment with the p38 inhibitor, SB203580, and mildly suppressed by the MAPK/ERK kinase inhibitor, U0126. However, treatment with the JNK inhibitor, SP600125, did not suppress ZnCl2-induced HO-1 expression. In addition, the phosphorylated forms of Akt, a downstream kinase of PI3K, and NF-κB p65 increased after ZnCl2 exposure. Treatment with the PI3K inhibitor, LY294002, and the NF-κB inhibitor, Bay11-7082, suppressed ZnCl2-induced HO-1 expression., Conclusion: These results suggest that ZnCl2 exposure induces HO-1 expression via multiple intracellular signalling pathways, including p38, ERK, PI3K/Akt, and NF-κB, in this odontoblast-like cell line., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Sphingomyelin phosphodiesterase 3 (Smpd3) encodes a membrane-bound enzyme that cleaves sphingomyelin to generate several bioactive metabolites. A recessive mutation called fragilitas ossium (fro) in the Smpd3 gene leads to impaired mineralization of bone and tooth extracellular matrix (ECM) in fro/fro mice. In teeth from fro/fro mice at various neonatal ages, radiography and light and electron microscopy showed delayed mantle dentin mineralization and a consequent delay in enamel formation as compared with that in control +/fro mice. These tooth abnormalities progressively improved with time. Immunohistochemistry showed expression of SMPD3 by dentin-forming odontoblasts. SMPD3 deficiency, however, did not affect the differentiation of these cells, as shown by osterix and dentin sialophosphoprotein expression. Using a transgenic mouse rescue model (fro/fro; Col1a1-Smpd3) in which Smpd3 expression is driven by a murine Col1a1 promoter fragment active in osteoblasts and odontoblasts, we demonstrate a complete correction of the tooth mineralization delays. In conclusion, analysis of these data demonstrates that Smpd3 expression in odontoblasts is required for tooth mineralization.

To investigate the functions of recombinant human dentin phosphoprotein (rhDPP), we examined cell adhesion, viability and the odontoblastic differentiation activity of human dental pulp cells (hDPCs). Firstly, rhDPP was constructed using pBAD-HisA plasmid in Escherichia coli. Cell adhesion and viability of hDPCs by rhDPP was examined using a crystal violet assay and a MTT assay, ALP, mineralization activity and odontoblastic differentiation-related mRNA levels of hDPCs were measured to elucidate the odontoblastic differentiation effect of rhDPP on hDPCs. Initially, rhDPP significantly and dose-dependently increased hDPCs adhesion versus the untreated control (p < 0.05). Cell viability was also significantly increased by rhDPP at 5 days (p < 0.001). Furthermore, the odontoblastic differentiation effect of rhDPP was verified by measuring ALP activity, mineralization activity and the mRNA levels of odontoblastic differentiation markers. Taken together, rhDPP is expected to play an important role on hDPCs, thereby suggesting its potential use for tooth repair and regeneration.

Introduction: The equilibrium of inhibitors and inducers plays an important role in the mineralization of dentin during dentinogenesis. High-temperature requirement protein A1 (HtrA1) is a novel bone mineralization inhibitor involved in physiological and pathological bone formation. However, the expression of HtrA1 in tooth mineralization is unknown. The purpose of the present study was to investigate the localization and quantity of HtrA1 and its possible related molecules during induced reparative dentin formation., Methods: Rats were randomly sacrificed after direct pulp capping on days 0, 7, 14, and 21. Maxillary segments were obtained and routinely prepared for histological analysis, immunohistochemistry, quantum dots-based double immunofluorescence, and CRi's Nuance imaging system (CRI, Woburn, MA)-based quantitative determination., Results: The square measure values of reparative dentin significantly increased on day 7 and continued to increase until day 21. HtrA1, matrix Gla protein (MGP), nestin, and bone sialoprotein were positively stained and colocalized in the odontoblasts and/or odontoblast-like cells zone and reparative dentin during induced reparative dentin formation. The expressions of HtrA1 and MGP were significantly enhanced after direct pulp capping on day 7 and did not significantly change between days 7, 14, and day 21. Both expressions of HtrA1 and MGP were positively correlated with the square measure values of reparative dentin; however, no correlation was found between the expressions of HtrA1 and MGP., Conclusions: HtrA1 could be observed and might possibly be involved in the process of reparative dentin formation associated with MGP., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The lysyl oxidase (LOX) family is an emerging family of amine oxidases responsible for the formation of collagen fibrils in the extracellular matrix. To date, 5 LOX family genes have been identified in humans, encoding LOX and LOX-like proteins (LOXL, LOXL2, LOXL3, and LOXL4). The goal of this study was to evaluate the expression and function of the LOX family genes in odontoblastic differentiation of human dental pulp (HDP) cells., Methods: Expression of the LOX family genes was assessed by reverse transcriptase polymerase chain reaction analysis, and the amine oxidase activity of HDP cells was evaluated by peroxidase-coupled fluorometric assays. Mineral nodule formation and expression of odontoblastic marker genes were assessed in the presence and absence of specific small interfering RNAs (siRNAs) of the LOX family genes., Results: Among the LOX family genes, only LOX and LOXL showed prominent expression during odontoblastic differentiation of HDP cells. Suppression of LOX and LOXL expression by siRNA-induced interference substantially decreased the amine oxidase activity of the differentiating HDP cells. Furthermore, interference of LOX and LOXL expression inhibited mineral nodule formation and expression of odontoblastic marker genes during odontoblastic differentiation of HDP cells., Conclusions: These findings show for the first time that the LOX- and LOXL-mediated organization of collagen fibrils in extracellular matrices of HDP cells might be an important regulator for odontoblastic differentiation of HDP cells., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Dental pulpal nerve fibers express ionotropic adenosine triphosphate (ATP) receptors, suggesting that ATP signaling participates in the process of dental nociception. In this study, we investigated if the principal enzymes responsible for extracellular ATP hydrolysis, namely, nucleoside triphosphate diphosphohydrolases (NTPDases), are present in human dental pulp. Immunohistochemical and immunofluorescence experiments showed that NTPDase2 was predominantly expressed in pulpal nerve bundles, Raschkow's nerve plexus, and in the odontoblast layer. NTPDase2 was expressed in pulpal Schwann cells, with processes accompanying the nerve fibers and projecting into the odontoblast layer. Odontoblasts expressed the gap junction protein, connexin43, which can form transmembrane hemichannels for ATP release. NTPDase2 was localized close to connexin43 within the odontoblast layer. These findings provide evidence for the existence of an apparatus for ATP release and degradation in human dental pulp, consistent with the involvement of ATP signaling in the process of dentin sensitivity and dental pain.

Aim: To investigate whether the p38α mitogen-activated protein kinases (MAPK) is involved in bone morphogenetic protein (BMP)-2-induced odontoblastic differentiation of human dental pulp cells (HDPCs)., Methodology: Recombinant retrovirus encoding shRNA against p38α MAPK was constructed to investigate the role of p38α MAPK on BMP-2-induced odontoblastic differentiation of HDPCs. HDPCs were transfected with retrovirus expressing sh-p38α. Activation of p38α MAPK was detected by Western blot. The effects of p38α MAPK on BMP-2-induced odontoblastic differentiation of HDPCs were measured by alkaline phosphatase (ALP) activity, and the expression of odontoblastic markers was identified by quantitative real-time polymerase chain reaction analysis. The effect of SD-282, a p38a-specific inhibitor, on BMP-2-induced odontoblastic differentiation was also investigated., Results: BMP-2 dose- and time-dependently upregulated phosphorylation of p38α of HDPCs. Compared with BMP-2-treatment group, gene knock-down of p38α MAPK significantly inhibited ALP activity and the formation of mineralized nodules in HDPCs. Moreover, suppression of p38α MAPK repressed the odontoblastic differentiation in HDPCs. Consistently, inhibition of p38α by SD-282 also decreased odontoblastic differentiation., Conclusions: p38α MAPK is involved in BMP-2-induced odontoblastic differentiation of HDPCs., (© 2011 International Endodontic Journal.)

We examined change of Runx2 and ALP expression in mouse tooth pulp which exposed to teeth separation experiment by immunohistochemistry as a model for conservative dentistry treatment. 8-week-old 36 male ddY mice were used and wedge was inserted between upper 1st and 2nd molars. The wedge was removed 30 minutes as well as 3 hours after the insertion and the samples were prepared extending up to 1 week of time period for regular histopathological and immunohistochemical examinations for ALP and Runx2 expression. The opposite sides without wedge insertion were taken as controls. In the control group pulp, weak expressions of Runx2 and ALP in the vessel endothelial cells as well as the pulp cells were revealed, suggesting the appearance of these genes upon mechanical stress induced by mastication and tongue pressure etc. On the other hand in the experiment group, Runx2 expression increased both in 30-minute and 3-hour teeth separation group. The expression became maximum at 24 hours. Then it gradually decreased and became similar level with the control group at 1-week after the wedge insertion. Similarly ALP expression increased after the wedge insertion and was maximum at 24 hours and then gradually decreased to the levels similar with the control group. These results suggest that when immunohistochemical expression of Runx2 as well as ALP was used as an index, no severe damage occur upon clinical application of wedge insertion.

Objectives: Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) play important roles in dentine formation, caries progression and hybrid layer degradation. This study tested the hypothesis that the distribution and concentrations of MMP-2, MMP-9, TIMP-1 and TIMP-2 are different at different depths of human coronal dentine, including odontoblasts., Methods: Protein localization was performed using immunohistochemistry. Co-localization of the MMPs and their inhibitors was conducted using immunofluorescence double labelling. Protein concentrations were measured by ELISA and gelatinolytic potential was assessed with gelatine zymography., Results: MMP-2 was the main gelatinase in dentine and was concentrated in the odontoblasts, deep dentine and the dentinoenamel junction. TIMP-2 was co-localized with MMP-2 mainly in the odontoblasts but its concentration was low. Both MMP-9 and TIMP-1 showed a decreasing distribution from the deep to the superficial dentine layers; however, the concentration of TIMP-1 was much higher than that of MMP-9. The gelatinolytic potential of dentine protein extracts decreased gradually from deep to superficial dentine., Conclusions: The concentrations and distribution patterns of MMP-2, MMP-9, TIMP-1 and TIMP-2, and the gelatinolytic potential of dentine matrix are variable along different dentine depths. Thus, differential collagen degradation potentials may be expected depending upon the depth in which dentine is exposed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In tooth development, transforming growth factor beta (TGF-β) and bone morphogenetic protein (BMP) are involved in cell differentiation and matrix protein production. TGF-β and BMP have two signaling pathways: the Smad pathway and the non-Smad pathway. However, only a few studies have focused on the non-Smad pathway in tooth development. TGF-β-activated kinase 1 (TAK1) is activated by TGF-β or BMP and binds to TAK1-binding protein (TAB1), activating p38 or c-Jun N-terminal kinase (JNK), forming the non-Smad signaling pathway. In this study, we examined the distribution of these kinases, TGF-β receptor 1 (TGF-β-R1), BMP receptor-1B (BMPR-1B) and Smad4 in cells of the rat molar germ histochemically, in order to investigate the signaling pathway in each type of cell. Immunostaining for TGF-β-R1, BMPR-1B, Smad4, TAK1, TAB1 and phosphorylated-p38 (p-p38) showed similar reactions. In the cervical loop, reactions were clearer than in other enamel epithelium. In the inner enamel epithelium, signal increased with differentiation into ameloblasts, became strongest in the secretory stage, and decreased rapidly in the maturation stage. Signal also increased upon differentiation from preodontoblasts to odontoblasts. In Hertwig's epithelial sheath, with the exception of BMPR-1B, reactions were stronger in the later stage, showing more enamel protein secretion than in the early stage. However, no clear reaction corresponding to phosphorylated-JNK was observed in any type of cell. These results suggest that TGF-β or BMP is involved in the induction of differentiation of inner enamel epithelium cells into ameloblasts, and preodontoblast differentiation into odontoblasts, the regulation of cervical loop cell proliferation, the elongation or regulation of the epithelial sheath, and the secretion of enamel protein and dentin matrix protein through the non-Smad signaling pathway via TAK1, TAB1 and p38 as well as Smad signaling pathways in the rat molar germ.

Matrix metalloproteinases (MMPs) are important in dentinal caries, and analysis of recent data demonstrates the presence of other collagen-degrading enzymes, cysteine cathepsins, in human dentin. This study aimed to examine the presence, source, and activity of cysteine cathepsins in human caries. Cathepsin B was detected with immunostaining. Saliva and dentin cysteine cathepsin and MMP activities on caries lesions were analyzed spectrofluorometrically. Immunostaining demonstrated stronger cathepsins B in carious than in healthy dentin. In carious dentin, cysteine cathepsin activity increased with increasing depth and age in chronic lesions, but decreased with age in active lesions. MMP activity decreased with age in both active and chronic lesions. Salivary MMP activities were higher in patients with active than chronic lesions and with increasing lesion depth, while cysteine cathepsin activities showed no differences. The results indicate that, along with MMPs, cysteine cathepsins are important, especially in active and deep caries.

The nitric oxide (NO) receptor enzyme soluble guanylate cyclase (sGC) contains one prosthetic heme group as an αβ heterodimer, and two heterodimer isoforms (α(1)β(1), α(2)β(1)) were characterized to have enzyme activity. To test the irreversible inflammation-dependent regulation of sGC in odontoblasts, we incubated decalcified frozen sections of healthy and inflamed human third molars with antibodies against β-actin, nitrotyrosine, inducible nitric oxide synthase (iNOS), α(1)-, β(1)-, and α(2)-subunits of sGC and analyzed them at protein levels by quantitative immunohistochemistry. The irreversible inflammation induced an increase in the signal intensities for nitrotyrosine and iNOS and a decrease for the α(1)-, β(1)-, and α(2)-subunits of sGC in odontoblasts. Inflammatory mediators, reactive oxygen, and nitrogen species may impair the expression of the α(1)-, β(1)-, and α(2)-subunits in odontoblasts. The decrease of sGC at the protein level in inflamed odontoblasts is compatible with a critical role for sGC to mediate biological effects of NO in health.

Dentin sialophosphoprotein (Dspp) is critical for proper dentin biomineralization because genetic defects in DSPP cause dentin dysplasia type II and dentinogenesis imperfecta types II and III. Dspp is processed by proteases into smaller subunits; the initial cleavage releases dentin phosphoprotein (Dpp). We incubated fluorescence resonance energy transfer (FRET) peptides containing the amino acid context of the Dpp cleavage site (YEFDGKSMQGDDPN, designated Dspp-FRET) or a mutant version of that context (YEFDGKSIEGDDPN, designated mutDspp-FRET) with BMP-1, MEP1A, MEP1B, MMP-2, MMP-8, MMP-9, MT1-MMP, MT3-MMP, Klk4, MMP-20, plasmin, or porcine Dpp and characterized the peptide cleavage products. Only BMP-1, MEP1A, and MEP1B cleaved Dspp-FRET at the G-D peptide bond that releases Dpp from Dspp in vivo. We isolated Dspp proteoglycan from dentin power and incubated it with the three enzymes that cleaved Dspp-FRET at the G-D bond. In each case, the released Dpp domain was isolated, and its N-terminus was characterized by Edman degradation. BMP-1 and MEP1A both cleaved native Dspp at the correct site to generate Dpp, making both these enzymes prime candidates for the protease that cleaves Dspp in vivo. MEP1B was able to degrade Dpp when the Dpp was at sufficiently high concentration to deplete free calcium ion concentration. Immunohistochemistry of developing porcine molars demonstrated that astacins are expressed by odontoblasts, a result that is consistent with RT-PCR analyses. We conclude that during odontogenesis, astacins in the predentin matrix cleave Dspp before the DDPN sequence at the N-terminus of Dpp to release Dpp from the parent Dspp protein., (© 2011 American Society for Bone and Mineral Research.)

Background: It has been suggested that host matrix metalloproteinase-2 (MMP-2) present in dentin may be involved in caries progression, however, its response to caries is not known. Bone sialoprotein (BSP) has been implicated in dentin mineralization and MMP-2 modulation., Objective: To identify and compare the distribution of MMP-2 and BSP in healthy human coronal dentin and those with early caries., Methods: Freshly extracted 3rd molars and premolars with and without early caries were fixed, demineralized and subjected to immunohistochemistry using a monoclonal anti-MMP-2 antibody and monoclonal anti-BSP antibody with an avidin-biotin complex method. Immunoreactivity was visualized with 3,3'-diaminobenzidine substrate and observed under light microscopy., Results: Immunohistochemical analysis revealed that MMP-2 and BSP are not detected in the tubule lumens of healthy dentin. However, intense immunoreactivity for MMP-2 and BSP was detected in association with the full length of the caries-affected dentinal tubules. The MMP-2 and BSP at the dentino-enamel junction appeared unaltered., Conclusion: The results indicate that MMP-2 and BSP may be actively secreted by odontoblasts in response to carious insult. MMP-2 and BSP accumulation in the caries-affected dentinal tubules may indicate their potential involvement in the host defense mechanism which results in calcification of regions affected by the carious process., (Copyright © 2011 S. Karger AG, Basel.)

The two distinct molecular forms of cholinesterase (ChE) are acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Our previous studies have reported that ChE is involved in tooth development. However, further experiments are needed to understand the precise action of ChE in tooth development. This study aimed to localise types of ChE in human tooth germs, and identify their distribution pattern. ChE were localised in frozen sections of jaws which were prepared from dead fetuses, neonates and stillborns who were free from visible abnormalities by Karnovsky and Root method. AChE was identified in the inner and outer enamel epithelia including the cervical loop region, stratum intermedium and preameloblasts of tooth germs at bell stage. Secretory ameloblasts were free from staining. The bud and cap stages of permanent tooth germs showed AChE activity on the lingual aspect and top surface of the epithelial ingrowths, respectively. BuChE activity was localised in the degenerating dental lamina. Our study reported the first evidence of localisation of ChE in human tooth development and identified the possible molecular form of ChE in tooth germs as AChE. Also, our results have provided strong evidence to speculate the action of AChE is on the cells of enamel organ during tooth development.

Introduction: It was recently reported that heme oxygenase-1 (HO-1) activity is related to stem cell differentiation; however, the involvement of HO-1 in pulp cell growth and differentiation has not been well explored. The purpose of this study was to investigate the role of HO-1 in the growth and differentiation of human dental pulp cells (HDPCs)., Methods: We evaluated cell growth by MTT assay, mineralization by alizarin red staining, and differentiation marker mRNA expression by reverse transcriptase polymerase chain reaction., Results: HO-1 induction by cobaltic protoporphyrin IX (CoPP) in HDPCs increased cell growth and mineralization and up-regulated the messenger RNA expression of such odontoblastic markers as alkaline phosphatase, osteopontin, bone sialoprotein, dentin matrix protein-1, and dentin sialophosphoprotein. Carbon monoxide scavenger, iron chelator, HO-1 inhibitor, and HO-1 small interfering RNA (siRNA) attenuated HDPC growth and differentiation., Conclusions: CoPP treatment results in dental pulp cell proliferation and odontoblast differentiation that appears partly mediated by HO-1. Our results suggest that odontoblastic differentiation and growth are positively regulated by HO-1 induction and negatively regulated by HO-1 inhibition. Thus, pharmacologic HO-1 induction might represent a potent therapeutic approach for pulp capping and the regeneration of HDPCs., (Copyright 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.

A dentin primer incorporating an antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) shows strong antibacterial effects, and may provide better prognosis for direct capping of infected pulp exposed by caries removal compared with conventional adhesives. However, influences of MDPB on healing of the pulp have not yet been fully elucidated. The purpose of this study was to compare the influences of unpolymerized MDPB on proliferation, differentiation and mineralization of odontoblast-like MDPC-23 cells with those of other resin monomers, Bis-GMA, MDP, TEGDMA and HEMA. The inhibitory effects of MDPB on the proliferation of MDPC-23 were lower than those of Bis-GMA. While MDPB strongly affected the differentiation compared with the other monomers, it was less inhibitory than Bis-GMA and MDP on the mineralization ability of odontoblast-like cells. These findings indicate that MDPB has superior biocompatibility than Bis-GMA in terms of hard tissue formation by odontoblastic cells, suggesting its possible less negative influences on dentinogenesis., ((c) 2009 Elsevier Ltd. All rights reserved.)

Introduction: Collagen-degrading matrix metalloproteinases (MMPs) are expressed by odontoblasts and present in dentin. We hypothesized that odontoblasts express other collagen-degrading enzymes such as cysteine cathepsins, and their activity would be present in dentin, because odontoblasts are known to express at least cathepsin D. Effect of transforming growth factor beta (TGF-beta) on cathepsin expression was also analyzed., Methods: Human odontoblasts and pulp tissue were cultured with and without TGF-beta, and cathepsin gene expression was analyzed with DNA microarrays. Dentin cathepsin and MMP activities were analyzed by degradation of respective specific fluorogenic substrates., Results: Both odontoblasts and pulp tissue demonstrated a wide range of cysteine cathepsin expression that gave minor responses to TGF-beta. Cathepsin and MMP activities were observed in all dentin samples, with significant negative correlations in their activities with tooth age., Conclusions: These results demonstrate for the first time the presence of cysteine cathepsins in dentin and suggest their role, along with MMPs, in dentin modification with aging., (Copyright (c) 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: We have previously shown that the p38 gene is highly expressed in odontoblasts during active primary dentinogenesis, but is drastically down-regulated as cells become quiescent in secondary dentinogenesis. Based on these observations, we hypothesized that p38 expression might be upregulated, and the protein activated by phosphorylation, when odontoblasts are stimulated such as during tertiary reactionary dentinogenesis., Methods: We stimulated immortalized, odontoblast-like MDPC-23 cells, alone or in combination, with heat-inactivated Streptococcus mutans, EDTA-extracted dentine matrix proteins (DMPs), or growth factors, including transforming growth factor (TGF)-beta1, tumor necrosis factor-alpha (TNF-alpha), and adrenomedullin (ADM). We used ELISA to measure the resulting phosphorylation of the p38 protein, as well as its degree of nuclear translocation., Results: Our results suggest that the p38-MAPKinase pathway is activated during odontoblast stimulation in tertiary dentinogenesis by both p38 phosphorylation and enhanced nuclear translocation., Conclusions: Data indicate that odontoblast behaviour therefore potentially recapitulates that during active primary dentinogenesis., (Copyright 2010 American Association of Endodontists. All rights reserved.)

Aims: Although heme oxygenase-1 (HO-1) is involved in osteoblastic differentiation, the HO-1- and odontoblastic differentiation-inducing effects of mechanical stress (MS) have not been clarified in human dental pulp cells (HDPCs). In this study, we examined the effects of MS on the odontoblastic differentiation of immortalized HDPCs and on the primary intracellular signaling pathways, including the HO-1 pathway, implicated in this differentiation., Main Methods: A Flexercell strain unit was used to generate cyclic tensile strain in HDPCs. Expressions of mRNAs encoding HO-1 and HDPC differentiation markers, such as osteopontin (OPN), bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), and dentin matrix-protein-1 (DMP-1), were evaluated using the reverse transcription-polymerase chain reaction. Expression of the NF-E2-related transcription factor 2 (Nrf2) protein was analyzed by Western blotting., Key Findings: MS significantly increased the expression of HO-1, OPN, BSP, DSPP, and DMP-1 mRNAs in HDPCs. HO-1 silencing and inhibitors of HO-1, p38 MAPK, ERK, phosphoinositide 3-kinase, and nuclear factor-kappaB (NF-kappaB) all attenuated MS-stimulated differentiation. The MS-induced nuclear translocation of Nrf2 was suppressed by inhibitors of PI3K and NF-kappaB., Significance: Collectively, these results provide the first evidence that MS stimulates odontoblastic differentiation of HDPCs via modulation of the Nrf2-mediated HO-1 pathway., (Copyright 2009 Elsevier Inc. All rights reserved.)

Objective: To test the hypothesis that some metalloproteinases (MMP-2, MMP-9) and inducible nitric oxide synthetase (iNOS) enzymes in dental pulp samples do not vary when subjected to orthodontic treatment., Materials and Methods: Human dental pulps were taken from male and female patients (N=10; age 10-14 years). A straight wire technique was used with nickel-titanium or steel archwires. The increase of pressure applied on teeth was gradual. Five patients were subjected to premolar extractions after 14 months of treatment and one after 24 months. Samples were Bouin-fixed, paraffin-embedded, and afterwards processed for immunohistochemistry using anti-MMP-2, anti-MMP-9, and anti-iNOS antibodies., Results: A reduction of MMP-2, MMP-9, and iNOS expression occurred in treated samples. This became more evident with increased treatment time., Conclusion: The hypothesis is rejected. The reduction of expression of those proteins revealed a time-dependent relationship.

Unlabelled: The odontoblast is the secretory cell responsible for primary, secondary and tertiary reactionary dentinogenesis. We provide evidence that the changes in secretory activity of odontoblasts reflect differential transcriptional control and that common regulatory processes may exist between dentine and bone., Introduction: Based on the hypothesis that differential dentine secretion (primary and secondary dentinogenesis) is associated with changes in the transcriptional control within the cell, we have investigated the transcriptome of odontoblasts at young and mature stages and subsequently used this information to identify key regulatory intracellular pathways involved in this process., Materials and Methods: We used microarray analysis to compare the transcriptome of early stage (primary dentinogenesis) and late stage (secondary dentinogenesis) odontoblasts from 30 month old bovine teeth. Secondarily, we used post-array sqRT-PCR to confirm the differential expression of 23 genes in both populations of odontoblasts. Finally, immunohistochemistry was performed on bovine and murine tissues with antibodies to DMP1 and anti-phospho p38 proteins., Results: DMP-1 and osteocalcin gene expression were up-regulated in the mature odontoblasts, whereas collagen I, DSPP, TGF-beta1 and TGF-beta1R gene expression were down-regulated. Microarray analysis highlighted 574 differentially regulated genes (fold change>2 - p<0.05). This study supports further existing similarities between pulp cells and bone cells. Using post-array Sq-RT-PCR we characterized transcript levels of genes involved in the p38 MAP kinase pathway (PTPRR, NTRKK2, MAPK13, MAP2K6, MKK3). Differential p38 gene activation was confirmed by immunohistochemistry for p38 protein in murine teeth. Finally, immunohistochemistry for DMP1 indicated that odontoblasts involved in primary and secondary dentinogenesis may coexist in the same tooth., Conclusion: As established in bone cells, the transcriptome of the odontoblast was shown here to evolve with their stage and functional maturity. Identification of the involved signalling pathways, as highlighted for p38, will enable the deciphering of physiology and pathology of mineralised tissue formation.

The cytotoxic effects of fluoride on odontoblasts are not clear. In this study, we examined whether NaF induces apoptosis in MDPC-23 odontoblast-like cells and the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in NaF-induced apoptosis. MDPC-23 cells incubated with 5 mM NaF for 24 hrs exhibited caspase-3 activation, cleavage of poly(ADP-ribose) polymerase, DNA fragmentation, and an increase in cytoplasmic nucleosomes. Prior to the induction of apoptosis, all MAPKs examined were phosphorylated, but in a different manner. In contrast to the sustained phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38, NaF exposure induced a biphasic phosphorylation of extracellular signal-regulated protein kinase (ERK). NaF-induced apoptosis was markedly suppressed by treatment with the JNK inhibitor, SP600125, and mildly suppressed by the MAPK/ERK kinase inhibitor, U0126. Inhibition of p38 activity did not protect cells from apoptosis. Thus, exposure to NaF induces apoptosis in odontoblast-like cells, depending on JNK and, less significantly, ERK pathways.

In adhesive restorations, one major problem is hybrid layer degradation. At present, this deterioration is explained by the activation of the endogenous matrix metalloproteinases (MMPs) present in dentin due to the acidic property of adhesive systems. We hypothesized that self-etching adhesive should also stimulate the expression of MMPs in odontoblasts. In cultured tooth slices, we evaluated the changes in MMP-2 and proMMP-9 expression in the dentin-pulp complex after self-etching adhesive treatment on dentin cavities in immunochemistry and by zymography. The treatment resulted in increased MMP-2 expression in odontoblasts, as shown by immunohistochemistry. Zymography showed increased proMMP-9 and MMP-2 in dentin under self-etching treatment when pulp was present. These results showed that self-etching adhesive stimulates the secretion of MMPs from the dentin-pulp complex and, more precisely, by odontoblasts, suggesting that odontoblasts participate in hybrid layer degradation.

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have been implicated in the inflammation-dependent sensitization of nociceptors, and the inflammatory mediator bradykinin (BK) led to a reduced threshold in the nociceptor terminals, activating intracellular signaling by phosphorylating receptors and ion channels. The effects of BK on the non-transcriptional modulation of the ERK1/2 in the peripheral nociceptor terminals, including in nerve endings of the dentin-pulp complex, are unknown. The time-dependent effects of BK (10(-7) M) on the ERK1/2 phosphorylation in nerve terminals of the dentin-pulp complex were investigated by quantitative and double immunolabeling with organ bath experiments. In nerve terminals, total and p-ERK1/2 were detected. In comparison with the controls, the numbers of p-ERK1/2-positive nerve endings increased after 1 and 3 min and decreased after 10 min of BK treatment. Analysis of the data indicates that BK induces phosphorylation-mediated local activation of ERK1/2 in nerve terminals modulating nociception in the dentin-pulp complex.

Objective: This study examined the molecules implicated in the cytodifferentiation of dental hard tissue cells., Methods: Rat pups at postnatal days 4, 7 and 10 were used. Differential display-polymerase chain reaction (DD-PCR), Western blot and immunofluorescent localisation were performed to search differentially expressed genes in tooth development., Results: Leukocyte-common antigen-related tyrosine phosphatase (lar-tp) was differentially detected between the rat maxillary 2nd and 3rd molar germs on postnatal day 10, which were at the dental hard tissue formation and cap/early bell developmental stages, respectively. Both the mRNA and protein expression levels of lar-tp were higher in the 3rd molar germs than in the 2nd. In addition, the levels in the 2nd molar germs at postnatal days 4, 7 and 10, which corresponded to the early/late bell, crown and root stages, respectively, decreased in a time dependent manner. The immunoreactivity against intracellular P-subunit of lar-tp was detected in the ameloblasts and odontoblasts as well as in the undifferentiated inner enamel epithelia and dental papilla cells. However, strong immunoreactivity against extracellular E-subunit was observed only in the undifferentiated inner enamel epithelia and dental papilla cells in the 3rd molar germs and in the stratum intermedium in the 2nd molar germs., Conclusion: This is the first identification of lar-tp in the molar tooth development and suggests that this molecule may be involved in the cytodifferentiation of dental hard tissue cells.

Aim: To investigate the involvement of the phosphatidylinositol-3-kinase (PI3K) in interleukin-6 (IL-6) and interleukin-8 (IL-8) gene expression in odontoblast-like cells when exposed to heat-killed Streptococcus mutans., Methodology: Cultured human odontoblast-like cells (Dulbecco's modified Eagle's medium) were pre-treated with a specific inhibitor for PI3K (LY 294002) and subsequently stimulated with heat-killed S. mutans for 6 and 24 h. After stimulation, RNA was extracted from the cells and cDNA synthesis was performed. Gene expression of IL-6 and IL-8 was analysed by real-time polymerase chain reaction and normalized to the gene expression of beta-actin. Cell survival was determined for stimulation experiments., Results: The gene expression of IL-6 and IL-8 was significantly increased in response to heat-killed S. mutans (P = 0.002 and P < 0.001, respectively). After 6 h, the mRNA expression of IL-6 and IL-8 was significantly higher than after 24 h of stimulation (P = 0.019 and P < 0.001, respectively). Pre-treatment with the inhibitor LY 294002 blocked the induced gene expression of IL-6 and IL-8 (P = 0.002 and P < 0.001, respectively). No differences in viable cell counts were found after stimulation with heat-killed S. mutans and/or pre-treatment with LY 294002 compared with the unstimulated control., Conclusion: Gene expression of IL-6 and IL-8 was induced by heat-killed S. mutans via signalling pathways mediated by PI3K. These findings indicate that odontoblasts respond to cariogenic bacteria with increased gene expression of pro-inflammatory mediators and hence participate in immune processes.

Nitric oxide (NO) stimulates osteoblast differentiation, but whether NO contributes to odontoblast differentiation during dentin repair is unknown. By using reverse transcription/polymerase chain reaction and immunostaining, we investigated the gene expression and/or immunolocalization of endothelial NO synthase (eNOS), inducible NOS (iNOS), and nitrotyrosine (a biomarker for NO-derived peroxinitrite), and alkaline phosphatase (ALP) and osteocalcin (early and terminal differentiation markers of odontoblasts, respectively) in dental pulp tissue after rat tooth preparation. At the early stage (1-3 days) post-preparation, markedly increased expression of iNOS and nitrotyrosine was found in odontoblasts and pulp cells beneath the cavity, whereas eNOS expression was significantly decreased. ALP mRNA expression was significantly increased after 1 day but decreased after 3 days, whereas ALP activity was weak in the dentin-pulp interface under the cavity after 1 day but strong after 3 days. Osteocalcin mRNA expression was significantly increased at this stage. At 7 days post-preparation, tertiary dentin was formed under the cavity. All the molecules studied were expressed at control levels in odontoblasts/pulp cells beneath the cavity. These findings show that abundant NO is released from odontoblasts and pulp cells at an early stage after tooth preparation and indicate that, after tooth preparation, the up-regulation of iNOS and nitrotyrosine in odontoblasts is synchronized with increased cellular expression of ALP and osteocalcin. Therefore, the NO synthesized by iNOS after tooth preparation probably participates in regulating odontoblast differentiation during tertiary dentinogenesis.