Notoginsenoside R1 alleviates TEGDMA-induced mitochondrial apoptosis in preodontoblasts through activation of Akt/Nrf2 pathway-dependent mitophagy.

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.

Downregulated microRNA-488 enhances odontoblast differentiation of human dental pulp stem cells via activation of the p38 MAPK signaling pathway.

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.

Downregulation of microRNA-143-5p is required for the promotion of odontoblasts differentiation of human dental pulp stem cells through the activation of the mitogen-activated protein kinases 14-dependent p38 mitogen-activated protein kinases signaling pathway.

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.

The effects of 2-hydroxyethyl methacrylate on matrix metalloproteinases 2 and 9 in human pulp cells and odontoblast-like cells in vitro.

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 ß-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.

Testicular acid phosphatase induces odontoblast differentiation and mineralization.

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 ß-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 ß-catenin and cyclin D1. In contrast, the overexpression of ACPT suppresses cell proliferation with a decrease in active ß-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 ß-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.

Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3.

A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1ß, tumor necrosis factor-α and interferon-γ) and IL-1ß-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.

Enzymatic isolation of viable human odontoblasts.

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.

Peroxisomes in dental tissues of the mouse.

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.

Caspase-7 participates in differentiation of cells forming dental hard tissues.

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.

The expression and role of Lysyl oxidase (LOX) in dentinogenesis.

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.

CpG ODN-induced matrix metalloproteinase-13 expression is mediated via activation of the ERK and NF-κB signalling pathways in odontoblast cells.

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.

Toxic effects of daily applications of 10% carbamide peroxide on odontoblast-like MDPC-23 cells.

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.

p38a MAPK is involved in BMP-2-induced odontoblastic differentiation of human dental pulp cells.

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.

Immunohistochemical expression of hard tissue related factors in the mouse dental pulp after immediate teeth separation.

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.

Increased matrix metalloproteinase-2 and bone sialoprotein response to human coronal caries.

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.

Plasma Membrane Ca2+-ATPase in Rat and Human Odontoblasts Mediates Dentin Mineralization.

Intracellular Ca2+ signaling engendered by Ca2+ influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca2+ is exported by the Na+-Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA) to maintain Ca2+ homeostasis. We previously demonstrated a functional coupling between Ca2+ 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 Ca2+ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca2+ concentration ([Ca2+]i). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca2+]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 [Ca2+]i in odontoblasts in both rat and human odontoblasts. The Ca2+ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca2+]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 [Ca2+]i homeostasis in odontoblasts by Ca2+ extrusion following [Ca2+]i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca2+ 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.

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts.

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.

MMP-2, MMP-9, and iNOS expression in human dental pulp subjected to orthodontic traction.

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.

Phosphatidylinositol-3-kinase inhibitor LY 294002 blocks Streptococcus mutans-induced interleukin (IL)-6 and IL-8 gene expression in odontoblast-like 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.

Laminin-1 acts as an adhesive for odontoblast-like cells and promotes their differentiation toward a hard tissue-forming phenotype.

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.