Odontogenic Markers Research Articles

Effects of transforming growth factor-β1 on odontoblastic differentiation in dental papilla cells is determined by IPO7 expression level

Transforming growth factor β1 (TGF-β1) is one of the broad-spectrum growth-promoting factors that participate in tooth development. The influence of TGF-β1 on the odontoblastic differentiation is still controvercy. Mouse primary dental papilla cells (mDPCs) as well as an immortalized mouse dental papilla cell line (mDPC6Ts) were treated with exogenous TGF-β1 during odontoblastic differentiation. RT-qPCR, Western blot, alizarin red staining and ALP staining were carried out to investigate the influence of TGF-β1 on odontoblastic differentiation. IPO7, important for SMAD complex translocation was also detected in mDPCs and mDPC6Ts in response to TGF-β1. After silencing IPO7 by transfection, the translocation process of P-SMAD2 was investigated by nuclear and cytoplasmic extraction as well as co-immunoprecipitation assay. The odontogenic markers, mineralization and IPO7 expression were significantly up-regulated in TGF-β1-treated mDPCs while down-regulated in mDPC6Ts. The total level of P-SMAD2 was not influenced by IPO7 in mDPCs, however, IPO7 could bind to P-SMAD2 and affect the nuclear-cytoplasm-shuttling of P-SMAD2. Our data demonstrated that TGF-β1 plays opposite roles in odontoblast differentiation in mDPCs and immortalized mouse dental papilla cell line (mDPC6Ts), which is determined by IPO7.

RICK regulates the odontogenic differentiation of dental pulp stem cells through activation of TNF-α via the ERK and not through NF-κB signaling pathway.

Dental pulp stem cells (DPSCs) are capable of both self-renewal and multilineage differentiation, which play a positive role in dentinogenesis. Studies have shown that tumor necrosis factor-α (TNF-α) is involved in the differentiation of DPSCs under pro-inflammatory stimuli, but the mechanism of action of TNF-α is unknown. Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) is a biomarker of an early inflammatory response that plays a key role in modulating cell differentiation, but the role of RICK in DPSCs is still unclear. In this study, we identified that RICK regulates TNF-α-mediated odontogenic differentiation of DPSCs via the ERKsignaling pathway. The expression of the biomarkers of odontogenic differentiation dental matrix protein-1 (DMP-1), dentin sialophosphoprotein (DSPP), biomarkers of odontogenic differentiation, increased in low concentration (1-10 ng/ml) of TNF-αand decreased in high concentration (50-100 ng/ml). Odontogenic differentiation increased over time in the odontogenic differentiation medium. In the presence of 10 ng/L TNF-α, the expression of RICK increased gradually over time, along with odontogenic differentiation. Genetic silencing of RICK expression reduced the expression of odontogenic markers DMP-1 and DSPP. The ERK, but not the NF-κB signaling pathway, was activated during the odontogenic differentiation of DPSCs. ERK signaling modulators decreased when RICK expression was inhibited. PD98059, an ERK inhibitor, blocked the odontogenic differentiation of DPSCs induced by TNF-α. These results provide a further theoretical and experimental basis for the potential use of RICK in targeted therapy for dentin regeneration.

Polymodal Activation and Desensitization of TRPV1 Receptor in Human Odontoblasts-Like Cells with Eugenol

Dentinal hypersensitivity is a frequent reason for dental consultation, and its pathophysiology has not been fully clarified. Previous findings have made it possible to establish a relationship between the cellular sensory capacity and the activation of the polymodal transient receptor potential vanilloid 1 (TRPV1), which is responsible for the nociceptive response and whose desensitization could cause analgesia. Thus, the objective of this study was to determine the expression, localization, and functional activity of TRPV1 in human odontoblasts-like-cells (hOLCs) and the effect of eugenol (EUG) on its activation and desensitization. Human dental pulp stem cells (hDPSCs) were obtained from third molars and were characterized using flow cytometry, and their differentiation potential toward the osteoblastic, chondrogenic, and adipogenic lineages was investigated. Subsequently, the hDPSCs underwent odontogenic differentiation for 7, 14, and 21 days, and their phenotype (odontogenic markers dentin matrix protein-1 (DMP-1) and dentin sialoprotein (DSP)) was evaluated using immunofluorescence. The TRPV1 gene expression in hOLCs was estimated using RT-qPCR, and its localization was analyzed using immunofluorescence. Half-maximal effective concentration (EC50) from both eugenol (EUG) and capsaicin (CAP) was determined; in addition, receptor activation was evaluated against chemical, thermal, and pH stimuli. For the statistical analysis, a one-way ANOVA with a Tukey post hoc test (p < 0.05) was used. After establishing the in vitro model of hOLCs and the membrane location of TRPV1, its chemical activation with EUG and CAP was demonstrated, as well as its thermal activation at ≥ 43°C and with an acidic (<6) or basic pH (between 9 and 12). Receptor desensitization was achieved after 20 min of exposure to two concentrations of EUG (603.5 and 1000 µM). These findings represent a stepping-stone for the construction of a pulp pain study model oriented toward a therapeutic alternative for the treatment of dentinal hypersensitivity.

Autophagy-Related Protein MAP1LC3C Plays a Crucial Role in Odontogenic Differentiation of Human Dental Pulp Cells.

Autophagy plays important roles in odontogenic differentiation of dental pulp cells (DPCs) in the developmental stage of tooth bud. Few studies have reported the role of autophagy during reparative dentin formation process. The objective of this study was to discover gene expression pattern correlated to autophagy and their role during odontogenic differentiation process in DPCs. After tooth cavities were prepared on the mesial surface of lower first molar crown of rats. Odontogenic differentiation and reparative dentin formation were assessed based on detection of morphology change with hematoxylin and eosin staining. After tooth cavities were prepared on the mesial surface of lower first molar crown of rats, odontogenic differentiation and reparative dentin formation were assessed based on detection of morphology change with hematoxylin and eosin staining and dentin sialophosphoprotein (DSPP), whereas autophagy inhibitor 3-methyladenine (3MA) reversed. Results of quantitative polymerized chain reaction array of autophagosome formation related genes revealed that GABARAPL2 was prominently upregulated while expression of other ATG8 family members were moderately increased after tooth cavity preparation. In addition, human DPCs incubated in differentiation medium predominantly upregulated MAP1LC3C, which selectively decreased by 3MA but not by autophagy enhancer trehalose. Knock-down of MAP1LC3C using shRNA resulted in strong downregulation of dentin matrix protein 1 and DSPP as well-known odontogenic marker compared to knock-down of MAP1LC3B during odontogenic differentiation process of human DPCs. Our results suggest that MAP1LC3C plays a crucial role in odontogenic differentiation of human DPCs via regulating autophagic flux.

Early Odontogenic Differentiation of Dental Pulp Stem Cells Treated with Nanohydroxyapatite-Silica-Glass Ionomer Cement.

This study aimed to investigate the effects of nanohydroxyapatite–silica–glass ionomer cement (nanoHA–silica–GIC) on the differentiation of dental pulp stem cells (DPSCs) into odontogenic lineage. DPSCs were cultured in complete Minimum Essential Medium Eagle—Alpha Modification (α-MEM) with or without nanoHA–silica–GIC extract and conventional glass ionomer cement (cGIC) extract. Odontogenic differentiation of DPSCs was evaluated by real-time reverse transcription polymerase chain reaction (rRT–PCR) for odontogenic markers: dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), osteocalcin (OCN), osteopontin (OPN), alkaline phosphatase (ALP), collagen type I (COL1A1), and runt-related transcription factor 2 (RUNX2) on day 1, 7, 10, 14, and 21, which were normalized to the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Untreated DPSCs were used as a control throughout the study. The expressions of DSPP and DMP1 were higher on days 7 and 10, that of OCN on day 10, those of OPN and ALP on day 14, and that of RUNX2 on day 1; COL1A1 exhibited a time-dependent increase from day 7 to day 14. Despite the above time-dependent variations, the expressions were comparable at a concentration of 6.25 mg/mL between the nanoHA–silica–GIC and cGIC groups. This offers empirical support that nanoHA–silica–GIC plays a role in the odontogenic differentiation of DPSCs.

The influence of experimental bioactive glasses on pulp cells behavior in vitro

ObjectivesTo assess in vitro the effect of experimental mesoporous BAG, on human dental pulp cells (hDPCs) behavior in terms of cytocompatiblity and bioactivity via mineralization potential. MethodsFine (FP) and large particles (LP) of a fixed BAG composition named 0NaMBG have been elaborated by a sol-gel process. In vitro assessment was achieved on cultured primary hDPCs using indirect contact. The effect of the concentration of 800μg/mL on cell metabolic activity and cytotoxicity were examined by performing Alamar blue and crystal violet assays. Alizarin Red staining was used to detect and quantify the formation of mineralized nodules and ALP activity was colourimetrically quantified. The expression of Odontogenic markers: DMP-1 and osteopontin (OPN) expression and cell morphology was evaluated by confocal microscopy. ResultsAccording to the Alamar blue and crystal violet assay, 0NaMBG samples were non-cytotoxic. Cells treated with 0NaMBG particles expressed higher metabolic activity than control cells, especially for LP. Both FP and LP significantly increased both extra and intra cellular ALP activity. hDPCs exhibited good cell spreading and adhesion in the presence of FP and LP extracts by confocal imaging. Further, Alizarin red S assay demonstrated more mineralization nodules and significant enhancement of the extracellular calcium deposition when cells were interfaced with both FP and LP compared to the control cells. Moreover, LP extracts enhanced the production and secretion of odontogenic markers: dentin matrix protein 1 (DMP-1) and osteopontin. SignificanceLP have a higher surface area and pore volume, which could explain their greater bioactivity in contact with pulp cells. The clinical relevance of these findings implicate that 0NaMBG could be used as fillers in dental therapeutic materials suitable for dentin and/or pulp tissues preservation.

Simvastatin-Enriched Macro-Porous Chitosan-Calcium-Aluminate Scaffold for Mineralized Tissue Regeneration.

The present study evaluated the odontogenic potential of human dental pulp cells (HDPCs) exposed to chitosan scaffolds containing calcium aluminate (CHAlCa) associated or not with low doses of simvastatin (SV). Chitosan scaffolds received a suspension of calcium aluminate (AlCa) and were then immersed into solutions containing SV. The following groups were established: chitosan-calcium-aluminate scaffolds (CHAlCa - Control), chitosan calcium-aluminate with 0.5 µM SV (CHAlCa-SV0.5), and chitosan calcium-aluminate with 1.0 µM SV (CHAlCa-SV1.0). The morphology and composition of the scaffolds were evaluated by SEM and EDS, respectively. After 14 days of HDPCs culture on scaffolds, cell viability, adhesion and spread, mineralized matrix deposition as well as gene expression of odontogenic markers were assessed. Calcium aluminate particles were incorporated into the chitosan matrix, which exhibited regular pores homogeneously distributed throughout its structure. The selected SV dosages were biocompatible with HDPCs. Chitosan-calcium-aluminate scaffolds with 1 µM SV induced the odontoblastic phenotype in the HDPCs, which showed enhanced mineralized matrix deposition and up-regulated ALP, Col1A1, and DMP-1 expression. Therefore, one can conclude that the incorporation of calcium aluminate and simvastatin in chitosan scaffolds had a synergistic effect on HDPCs, favoring odontogenic cell differentiation and mineralized matrix deposition.

A novel bio-active adhesive monomer induces odontoblast differentiation: a comparative study.

To evaluate the in vitro effect of the novel adhesive monomer CMET, a calcium salt of 4-methacryloxyethyl trimellitate (4-MET), on the proliferation, mineralization and differentiation of odontoblast-like cells, comparing with 4-MET, calcium hydroxide (CH) and mineral trioxide aggregate (MTA). Rat odontoblast-like MDPC-23 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% foetal bovine serum. The powder of four tested materials (CMET, 4-MET, CH and MTA) was first dissolved in distilled water (dH2O) and then was diluted by DMEM to yield final concentrations. Solvent (dH2O) was used as a control. Cell viability was assessed using CCK-8 assay. Real-time RT-PCR was used to quantify the mRNA expression of odontogenic markers, cytokines and integrins. Mineralization inducing capacity was evaluated by alkaline phosphatase (ALPase) activity and alizarin red S staining. Statistical analyses were performed using one-way anova and post hoc Tukey's HSD test, with the significance level at 1%. Cell viability was significantly greater in the CMET- (83 to 828mmolL-1), CH- and MTA-treated (low concentrations) groups than that in the control group (P<0.01). Higher concentrations of each material decreased the viable cells to different extents (P<0.01). CMET treatment augmented the expression of several integrin subunits and exhibited the highest mRNA expression levels of odontogenic markers among all groups (P<0.01). CH and MTA treatment caused significantly greater upregulation of pro-inflammatory cytokines expression than the other groups (P<0.01). The calcific deposition of MDPC-23 cells was dose-dependently accelerated by the addition of CMET (P<0.01); the enhancement of mineralization was also found in the fresh prepared CH and MTA treatments. Besides, CMET showed consistency in mineralization induction after 8weeks storage. Exposure to SB202190, a specific p38 mitogen-activated protein kinases inhibitor, significantly decreased the ALPase activity as well as the mineral deposition which was enhanced by CMET treatment (P<0.01). The novel bio-active monomer had the lowest cytotoxicity among all groups and it induced the proliferation, mineralization and differentiation of odontoblast-like cells under appropriate concentrations. This adhesive monomer possesses excellent biocompatibility and hence exhibits great potential in dentine regeneration.

Electrical Stimulation Decreases Dental Pulp Stem Cell Osteo-/Odontogenic Differentiation.

Dental pulp stem cells (DPSCs) have great potential for use in tissue engineering (TE)-based dental treatments. Electrical stimulation (EStim) has been shown to influence cellular functions that could play an important role in the success of TE treatments. Despite many recent studies focused on DPSCs, few have investigated the effect EStim has on these cells. The aim of this research was to investigate the effects of direct current (DC) EStim on osteo-/odontogenic differentiation of DPSCs. To do so cells were isolated from male Sprague Dawley rats (7–8 weeks old), and phenotype characterization and multilineage differentiation analysis were conducted to verify their “stemness.” Different voltages of DC EStim were administrated 1 h/day for 7 days, and the effect of EStim on DPSC osteo-/odontogenic differentiation was assessed by measuring calcium and collagen deposition, alkaline phosphatase (ALP) activity, and expression of osteo- and odontogenic marker genes at days 7 and 14 of culture. We found that while 10 and 50 mV/mm of EStim had no effect on cell number or metabolic activity, 100 mV/mm caused a significant reduction in cell number, and 150 mV/mm resulted in cell death. Despite increased gene expression of osteo-/odontogenic gene markers, Osteocalcin, RunX2, BSP, and DMP1, at day 7 in EStim treated cells, 50 mV/mm of EStim decreased collagen deposition and ALP activity at both time points, and calcium deposition was found to be lower at day 14. In conclusion, under the conditions tested, EStim appears to impair DPSC osteo-/odontogenic differentiation. Additional studies are needed to further characterize and understand the mechanisms involved in DPSC response to EStim, with an eye toward its potential use in TE-based dental treatments.

Effect of parathyroid hormone-related protein on odontogenic differentiation in human dental pulp cells

BackgroundParathyroid hormone-related protein (PTHrP) plays an important role in many physiological processes, including bone regeneration. The function of PTHrP is similar to PTH. It promotes osteogenic differentiation in MC3T3-E1 cells. The aim of this study was to investigate whether PTHrP might have odontogenic differentiation ability in human dental pulp cells (hDPCs).MethodsThe viability of hDPCs after stimulation with PTHrP was measured. Real-time polymerase chain reaction and Western blot analysis were performed to evaluate the expression levels of odontogenic markers and activation of protein kinase B (PKB/AKT), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). To evaluate mineralized nodule formation, alkaline phosphatase (ALP) staining and alizarin red S staining were performed.ResultsPTHrP promoted odontogenic differentiation as evidenced by the formation of mineralized nodules, the induction of ALP activity, and the upregulation of odontogenic markers (dentin sialophosphoprotein and dentin matrix protein-1). The phosphorylation of AKT, ERK, JNK, and p38 was increased by PTHrP. However, an AKT inhibitor (LY294002), an ERK inhibitor (U0126), a JNK inhibitor (SP600125), and a p38 inhibitor (SB203580) inhibited the increase of mineralization induced by PTHrP.ConclusionThe present study revealed that PTHrP could promote odontogenic differentiation and mineralization through activating the AKT, ERK, JNK, and p38 signaling pathways. These results provide novel insights into the odontogenic action of PTHrP.

The Effect of Mesoporous Bioactive Glass Nanoparticles/Graphene Oxide Composites on the Differentiation and Mineralization of Human Dental Pulp Stem Cells.

The purpose of this study was to investigate the effects of mesoporous bioactive glass nanoparticle (MBN)/graphene oxide (GO) composites on the mineralization ability and differentiation potential of human dental pulp stem cells (hDPSCs). MBN/GO composites were synthesized using the sol-gel method and colloidal processing to enhance the bioactivity and mechanical properties of MBN. Characterization using FESEM, XRD, FTIR, and Raman spectrometry showed that the composites were successfully synthesized. hDPSCs were then cultured directly on the MBN/GO (40:1 and 20:1) composites in vitro. MBN/GO promoted the proliferation and alkaline phosphatase (ALP) activity of hDPSCs. In addition, qRT-PCR showed that MBN/GO regulated the mRNA levels of odontogenic markers (dentin sialophosphoprotein (DSPP), dentine matrix protein 1 (DMP-1), ALP, matrix extracellular phosphoglycoprotein (MEPE), bone morphogenetic protein 2 (BMP-2), and runt-related transcription factor 2 (RUNX-2)). The mRNA levels of DSPP and DMP-1, two odontogenesis-specific markers, were considerably upregulated in hDPSCs in response to growth on the MBN/GO composites. Western blot analysis revealed similar results. Alizarin red S staining was subsequently performed to further investigate MBN/GO-induced mineralization of hDPSCs. It was revealed that MBN/GO composites promote odontogenic differentiation via the Wnt/β-catenin signaling pathway. Collectively, the results of the present study suggest that MBN/GO composites may promote the differentiation of hDPSCs into odontoblast-like cells, and potentially induce dentin formation.

Platelet-rich Fibrin Improves the Osteo-/Odontogenic Differentiation of Stem Cells from Apical Papilla via the Extracellular Signal–regulated Protein Kinase Signaling Pathway

IntroductionThe aim of this study was to investigate the cytobiological effects of platelet-rich fibrin (PRF) on stem cells from the apical papilla (SCAPs) in vitro and to further explore the underlying molecular mechanisms. MethodsSCAPs were isolated from immature third molars. Different concentrations of PRF conditioned medium (one eighth, one quarter, and one half PRF) were prepared. After pretreatment with PRF, the proliferation rate and migration capacity of SCAPs were examined by the Cell Counting Kit-8 assay and wound healing assay, respectively. Alizarin red S staining was performed to examine mineralized nodule formation. Western blot analysis was used to detect the expression of osteo-/odontogenic markers and the extracellular signal–regulated kinase (ERK) pathway in SCAPs. Data were analyzed by one-way analysis of variance. P values <.05 were considered statistically significant. ResultsThe Cell Counting Kit-8 assay showed that one eighth PRF improved the proliferation rate and the migration capacity of SCAPs (P < .005), whereas one quarter PRF and one half PRF showed no significant difference compared with the control group. The expression of osteo-/odontogenic markers and the capacity to form mineralized nodules of SCAPs were promoted by one eighth PRF and one quarter PRF. In addition, PRF activated ERK signaling, and the ERK inhibitor attenuated PRF-induced osteo-/odontogenesis of SCAPs. ConclusionsPRF improved the osteo-/odontogenic differentiation of SCAPs by activating the ERK pathway; meanwhile, PRF improved the proliferation and migration of SCAPs, and one eighth PRF achieved the most obvious promotion effect. The favorable cytobiological effects of PRF on SCAPs might serve as basis for PRF applications in regenerative endodontic treatment.

Effect of histone demethylase KDM5A on the odontogenic differentiation of human dental pulp cells

ABSTRACT Human dental pulp cells (hDPCs) possess the capacity to differentiate into odontoblast-like cells in response to exogenous stimuli. Histone methylation is one of the most robust epigenetic marks and is essential for the regulation of multiple cellular processes. Previous studies have shown that histone methyltransferases (HMTs) and histone demethylases (HDMs) are crucial for the osteogenic differentiation of human bone marrow, adipose tissue, and tooth tissue. However, little is known about the role of histone methylation in hDPC differentiation. Here, the expression levels of HMTs and HDMs were profiled in hDPCs undergoing odontogenic induction. Among several differentially expressed enzymes, HDM KDM5A demonstrated significantly enhanced expression during cytodifferentiation. Furthermore, KDM5A expression increased during early passages and in a time-dependent manner during odontogenic induction. Using a shRNA-expressing lentivirus, KDM5A was knocked down in hDPCs. KDM5A depletion resulted in greater alkaline phosphatase activity and more mineral deposition formation. Meanwhile, the expression levels of the odontogenic markers DMP1, DSPP, OSX, and OCN were increased by KDM5A knockdown. As a histone demethylase specific for tri- and dimethylated histone H3 at lysine 4 (H3K4me3/me2), KDM5A deficiency led to a significant increment in total H3K4me3 levels, whereas no significant difference was found for H3K4 me2. H3K4me3 levels on the promoters of the odontogenic markers increased after KDM5A knockdown in hDPCs. These results demonstrated that KDM5A is present in hDPCs and inhibits the odontogenic differentiation potentiality of hDPCs by removing H3K4me3 from specific gene promoters, suggesting that KDM5A-dependent histone demethylation may play an important role in reparative dentinogenesis.

Coordination of WNT signaling and ciliogenesis during odontogenesis by piezo type mechanosensitive ion channel component 1

Signal transmission from the mechanical forces to the various intracellular activities is a fundamental process during tissue development. Despite their critical role, the mechanism of mechanical forces in the biological process is poorly understood. In this study, we demonstrated that in the response to hydrostatic pressure (HP), the piezo type mechanosensitive ion channel component 1 (PIEZO1) is a primary mechanosensing receptor for odontoblast differentiation through coordination of the WNT expression and ciliogenesis. In stem cells from human exfoliated deciduous teeth (SHED), HP significantly promoted calcium deposition as well as the expression of odontogenic marker genes, PANX3 and DSPP, and WNT related-genes including WNT5b and WNT16, whereas HP inhibited cell proliferation and enhanced primary cilia expression. WNT signaling inhibitor XAV939 and primary cilia inhibitor chloral hydrate blocked the HP-induced calcium deposition. The PIEZO1 activator Yoda1 inhibited cell proliferation but induced ciliogenesis and WNT16 expression. Interestingly, HP and Yoda1 promoted nuclear translocation of RUNX2, whereas siRNA-mediated silencing of PIEZO1 decreased HP-induced nuclear translocation of RUNX2. Taken together, these results suggest that PIEZO1 functions as a mechanotransducer that connects HP signal to the intracellular signalings during odontoblast differentiation.

Role of osteoprotegerin in the regulation of dental epithelial‑mesenchymal signaling during tooth development

Dental epithelial-mesenchymal signaling is crucial for tooth development, but the detailed mechanism is not fully understood. Using microarray analysis, it was revealed that the expression of osteoprotegerin, an important factor regulating bone remodeling, significantly increased after removal of the dental epithelium. Immunohistochemical staining revealed that osteoprotegerin expression within the dental mesenchyme was quite low during the prenatal period, but significantly increased after birth. To investigate the influence of osteoprotegerin upon tooth development, first-molar tooth germs from embryonic day 14.5 (E14.5) Chinese Kunming mice were treated with different concentrations of osteoprotegerin. It was revealed that osteoprotegerin could inhibit the expression of odontogenic markers while promoting the expression of osteogenic markers, thereby disrupting tooth morphogenesis. These findings were further supported by in vitro and in vivo cultures. Finally, quantitative reverse transcription-polymerase chain reaction and immunofluorescence studies revealed that, after osteoprotegerin treatment, the activity of the wingless/integrated (Wnt)/β-catenin pathway increased, indicating that increased osteoprotegerin expression in prenatal tooth development could lead to uncontrolled upregulation of the Wnt/β-catenin pathway.

Effects of mineral trioxide aggregate, calcium hydroxide, biodentine and Emdogain on osteogenesis, Odontogenesis, angiogenesis and cell viability of dental pulp stem cells

BackgroundVital pulp therapy preserves and maintains the integrity and the health of dental pulp tissue that has been injured by trauma, caries or restorative procedures. The enhancement of cells viability and formation of reparative dentine and new blood vessels are vital determinants of the success of direct pulp capping. Therefore, the aims of this study was to evaluate and compare the in vitro osteogenic, odontogenic and angiogenic effects of mineral trioxide aggregate (MTA), calcium hydroxide [Ca(OH)2], Biodentine and Emdogain on dental pulp stem cells (DPSCs) and examine the effects of the tested materials on cell viability.MethodsDPSCs were treated with MTA, Ca(OH)2, Biodentine or Emdogain. Untreated cells were used as control. The cell viability was measured by MTT assay on day 3. Real-Time PCR with SYBR green was used to quantify the gene expression levels of osteogenic markers (alkaline phosphatase and osteopontin), odontogenic marker (dentin sialophosphoprotein) and angiogenic factor (vascular endothelial growth factor) on day 7 and day 14.ResultsAll capping materials showed variable cytotoxicity against DPSCs (77% for Emdogain, 53% for MTA, 26% for Biodentine and 16% for Ca(OH)2 compared to control (P value < 0.0001). Osteopontin (OPN) and dentin sialophosphoprotein (DSPP) gene expression was increased by all four materials. However, alkaline phosphatase (ALP) was upregulated by all materials except Emdogain. Vascular endothelial growth factor (VEGF) expression was upregulated by all four tested materials except Ca(OH)2.ConclusionsOur results suggest MTA, Biodentine and Emdogain exhibit similar attributes and may score better than Ca(OH)2. Emdogain could be a promising alternative to MTA and Biodentine in enhancing pulp repair capacity following dental pulp injury. However, further future research is required to assess the clinical outcomes and compare it with the in vitro findings.

Emulating the early phases of human tooth development in vitro

Functional in vitro models emulating the physiological processes of human organ formation are invaluable for future research and the development of regenerative therapies. Here, a developmentally inspired approach is pursued to reproduce fundamental steps of human tooth organogenesis in vitro using human dental pulp cells. Similar to the in vivo situation of tooth initiating mesenchymal condensation, a 3D self-organizing culture was pursued resulting in an organoid of the size of a human tooth germ with odontogenic marker expression. Furthermore, the model is capable of epithelial invagination into the condensed mesenchyme, mimicking the reciprocal tissue interactions of human tooth development. Comprehensive transcriptome analysis revealed activation of well-studied as well as rather less investigated signaling pathways implicated in human tooth organogenesis, such as the Notch signaling. Early condensation in vitro revealed a shift to the TGFß signal transduction pathway and a decreased RhoA small GTPase activity, connected to the remodeling of the cytoskeleton and actin-mediated mechanotransduction. Therefore, this in vitro model of tooth development provides a valuable model to study basic human developmental mechanisms.

Sclerostin inhibits odontogenic differentiation of human pulp-derived odontoblast-like cells under mechanical stress.

Sclerotic dentin is a natural self-protective barrier beneath non-carious cervical lesions (NCCLs), which are mainly induced by mechanical stress. Sclerostin is a mechanosensory protein and serves as an inhibitor of dentinogenesis. However, its function on mechanotransduction in dentine-pulp complex has not been elucidated yet. In this study, decreased sclerostin expression was detected in odontoblasts beneath NCCL-affected sclerotic dentin. Then human pulp-derived odontoblast-like cells (hOBs) were subjected to mechanical strain (MS) in vitro: the results showed that MS-induced upregulation of odontogenic differentiation markers (dentin sialophosphoprotein, osteopontin, osteocalcin, and runt-related transcription factor 2) in hOBs with downregulated sclerostin expression, and this inductive differentiation was attenuated when sclerostin was overexpressed. Additionally, MS activated ERK1/2 pathway and ERK1/2 inhibition restored MS-induced downregulation of sclerostin. Proteasome inhibitor MG132 could also rescue MS-induced decrease of sclerostin. Furthermore, MS suppressed STAT3 pathway, which could be reversed by sclerostin overexpression. STAT3 inhibition was shown to ameliorate the reduction of odontogenic markers induced by sclerostin overexpression. Taken together, we conclude that MS downregulates sclerostin expression via the ERK1/2 and proteasome signaling pathways to promote odontogenic differentiation of hOBs through the STAT3 signaling pathway. It can therefore be inferred that under mechanical stress, sclerostin inhibition promotes reactive dentin formation by enhancing odontogenic differentiation of odontoblasts, which might be one of potential forming mechanisms of sclerotic dentin beneath NCCLs.

Novel Calcium Phosphate Cement with Metformin-Loaded Chitosan for Odontogenic Differentiation of Human Dental Pulp Cells

Metformin is an old and widely accepted first-line drug for treating type 2 diabetes. Our previous studies demonstrate that metformin can stimulate the osteo/odontogenic differentiation of human-induced pluripotent stem cell-derived mesenchymal stem cells and human dental pulp cells (DPCs). Due to the rapid dilution of metformin from the defect area, the aim of this study was to develop a drug delivery system with controlled release of metformin to promote cell viability and odontogenic differentiation of DPCs favoring dentin regeneration. Calcium phosphate cement (CPC) containing chitosan and metformin as a scaffold was synthesized. DPCs were seeded onto the scaffold, and the viability and proliferation were evaluated at several time points. For osteogenic differentiation analysis, alkaline phosphatase (ALP) activity was tested, cells were stained with Alizarin Red, and the expression of odontogenic markers was evaluated by real-time polymerase chain reaction. DPCs remained viable and attached well to the CPC-chitosan composite scaffold. Moreover, the addition of metformin to the CPC-chitosan composite did not adversely affect cell proliferation, compared to that of CPC control. Our data further revealed that the novel CPC-chitosan-metformin composite enhanced the odontogenic differentiation of DPCs, as evidenced by higher ALP activity, elevated expression of odontoblastic markers, and strong mineral deposition. These results suggest that the new CPC-chitosan-metformin composite is a highly promising scaffold with the potential for tissue engineering applications including dentin regeneration.

HDAC6 regulates dental mesenchymal stem cells and osteoclast differentiation

BackgroundDental and periodontal tissue development is a complicated process involving a finely regulated network of communication among various cell types. Understanding the mechanisms involved in regulating dental mesenchymal stem cells (MSCs) and osteoclast cell differentiation is critical. However, it is still unclear whether histone deacetylase HDAC6 is involved in dental MSCs fate determination and osteoclast differentiation.MethodsWe used shRNA and siRNA knockdown to explore the role of HDAC6 in dental MSCs odontogenic differentiation and osteoclasts maturation.ResultsBased on HDAC6 knockdown dental MSCs, our data suggest that HDAC6 knockdown significantly increases alkaline phosphate activity and mineralized nodules formation. Additionally, mRNA expression of odontogenic marker genes (OSX, OCN, and OPN) was induced by HDAC6 knockdown. By using HDAC6 siRNA, we knocked down HDAC6 in osteoclast precursor RAW 264.7 cells. Our data suggests that HDAC6 knockdown significantly inhibited osteoclasts differentiation. Additionally, mRNA expression of osteoclast marker genes Trap, Mmp9, and Ctsk was decreased by HDAC6 knockdown.ConclusionsOur study demonstrated that HDAC6 plays an important role in regulating dental MSCs and osteoclasts differentiation.