Heparan sulfate proteoglycans (HSPGs) surround the surface of odontoblasts, and their modification affects their affinity for Wnt ligands. This study proposes applying Matching Transformation System® (MA-T), a novel chlorinated oxidant, to enhance dentinogenesis. MA-T treatment in odontoblasts decreased sulfation of HSPG and upregulated the expression of dentin sialophosphoprotein (Dspp) and Dentin Matrix Protein 1 (Dmp1) via activation of canonical Wnt signaling in vitro. Ex vivo application of MA-T also enhanced dentin matrix formation in developing tooth explants. Reanalysis of a public single-cell RNA-seq dataset revealed significant Wnt activity in the odontoblast population, with enrichment for Wnt10a and Wnt6. Silencing assays showed that Wnt10a and Wnt6 were redundant in inducing Dspp and Dmp1 mRNA expression. These Wnt ligands' expression was upregulated by MA-T treatment, and TCF/LEF binding sites are present in their promoters. Furthermore, the Wnt inhibitors Notum and Dkk1 were enriched in odontoblasts, and their expression was also upregulated by MA-T treatment, together suggesting autonomous maintenance of Wnt signaling in odontoblasts. This study provides evidence that MA-T activates dentinogenesis by modifying HSPG and through subsequent activation of Wnt signaling.

AKT from dental epithelium to papilla promotes odontoblast differentiation

The development of multifunctional materials has been expected in dentistry. This study investigated the effects of a novel universal bond containing a bioactive monomer, calcium 4-methacryloxyethyl trimellitic acid (CMET), on odontoblast differentiation in vitro. Eluates from bioactive universal bond with CMET (BA (+), BA bond), bioactive universal bond without CMET (BA (-)), and Scotchbond Universal Plus adhesive (SC, 3M ESPE, USA) were added to the culture medium of the rat odontoblast-like cell line MDPC-23. Then, cell proliferation, differentiation, and mineralization were examined. Statistical analyses were performed using a one-way ANOVA and Tukey's HSDtest. The cell counting kit-8 assay and alkaline phosphatase (ALP) assay showed that cell proliferation and ALP were significantly higher in the 0.5% BA (+) group than in the other groups. In a real-time reverse-transcription polymerase chain reaction, mRNA expression of the odontogenic markers, dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1), was significantly higher in the 0.5% BA (+) group than in the BA (-) and SC groups. Calcific nodule formation in MDPC-23 cells was accelerated in the BA (+) group in a dose-dependent manner (p < 0.01); however, no such effect was observed in the BA (-) and SC groups. Thus, the BA bond shows excellent potential for dentin regeneration.

Osteolectin Promotes Odontoblastic Differentiation in Human Dental Pulp Cells

An experimental study was conducted to examine whether melatonin influences osteogenic/odontogenic differentiation of human stem cells derived from the apical papilla (hSCAPs). In order to isolate hSCAPs, the undeveloped root of a third molar of a human tooth was used. Melatonin was administered to the experimental groups in an osteogenic medium. No treatment was administered to the control group. The methyl thiazolyl tetrazolium (MTT) assay was performed on days 1, 2, and 3 to assess cell viability (n = 8). A determination of odontogenic/osteogenic differentiation was accomplished using alkaline phosphatase (ALP) activity alizarin red staining (ARS) (n = 6), and the expression of osteogenic genes by real-time polymerase chain reaction (RT-PCR) (n = 3) on days 1, 2, and 7. Evaluation of the data was conducted using SPSS version 18. All experiments were conducted at least three times. The Mann Whitney U test, the ANOVA analysis, Tukey's test, and t-test was implemented to analyze the data(α = 0.05). After 24h, 48h, and 72h, No significant difference was observed between the control group and the melatonin treatment group in terms of viability of hSCAPs. (from 1 up to 10µg/ml) (P > 0.05). The assessment of ARS and ALP activity showed that melatonin treatment enhanced osteogenic differentiation of hSCAPs (P < 0.001). Melatonin treatment caused hSCAPs to show an increase of genes related to osteogenic/odontogenic differentiation. These genes included ALP, dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1), and bone sialoprotein (BSP) (P < 0.001). Melatonin treatment enhanced osteogenic/odontogenic differentiation of hSCAPs with a dose dependent effect on cell viability.

Introduction: Oral carcinogenesis is a multistage process, featuring genetic and molecular alterations leading to rapid cell division, invasion, metastasis, and increased cell survival. Many of these alterations are due to perturbations in the cell signaling networks, which in turn lead to constitutive deregulation of the proteins involved in the regulatory pathways. Our recent reports show that the silencing of dentin sialophosphoprotein (DSPP) and its cognate matrix metalloproteinases 20 (MMP20) alters key tumorigenic hallmarks of oral squamous cell carcinoma (OSCC). Objective: This study, intended to advance our recent findings, focuses on determining the effects of silencing DSPP and its cognate MMP20 on the signaling pathways that control cell proliferation, differentiation, invasion and metastasis. Materials and Methods: DSPP and MMP20 were silenced individually and in combination, using adenovirus-mediated short hairpin RNA (shRNA) in OSCC cell line, OSC2, and the effects of silencing on the following pathways: EFGR; RAS-RAF; MEK; MAPK; ERK; JNK; NF-kB; TGFβ; and GSK3β, were analysed by western blot. Results: DSPP and MMP20 silencing decreased EGFR, KRAS, MEK1/2, MAPK, ERK, MEEK1, JNK, CREBP, p300, NF-kB,TGF β, SMAD7, GSK3 β, and β-catenin expressions. In contrast, the expression of IKKα and SMAD4 were increased in DSPP/MMP20-silenced group, compared with control group. Furthermore, DSPP-silencing alone was more effective than MMP20, or combined DSPP-MM20 silencing, in altering the levels of key proteins of each signaling pathway investigated. Conclusion: Our findings provide the basis for further studies aimed at verifying the effects of these alterations in the profiles of these proteins on the various hallmarks of oral carcinogenesis, and for understanding the molecular role of DSPP and MMP20 in OSCC. This is with a view to evaluating their diagnostic and prognostic utility as well as the values of DSPP/MMP20 as potential targets for design of chemotherapeutic agents for the treatment of OSCC patients.

Regenerative endodontic procedures rely on the delivery of mesenchymal stem cells into the root canal and on the effect of local growth factors from the dentin and blood clot. The aim of this study was to assess the effect of dentin conditioning with ethylenediamine tetraacetic acid (EDTA) and diode lasers with different wavelengths (808 nm and 980 nm) on the expression of odontoblast-like cell markers. Forty dentin cylinders were divided into four groups according to the irrigation protocol: EDTA, EDTA + 808 nm diode laser, EDTA + 980 nm diode laser, and phosphate-buffered saline as the control group. Dental pulp stem cells were seeded into the previously conditioned cylinders and incubated for 14 days. The quantitative real-time polymerase chain reaction was used to evaluate the expression of dentin sialophosphoprotein (DSPP), dentin morphoprotein-1 (DMP-1), and transforming growth factor-beta 1 (TGF-β1). Data analysis was performed using the Kruskal-Wallis test. The activation of EDTA with 980 nm and 808 nm diode lasers resulted in lower DSPP and DMP-1 expression than that for EDTA alone (p < 0.05 and p < 0.01, respectively). The expression of TGF was similar among all groups. The highest level of expression of odontoblast-like differentiation markers was observed with EDTA alone. However, the use of an 808 nm diode laser during EDTA irrigation reduced the expression of odontoblastic differentiation markers.

Biological Function and Potential Applications of Garcinol in Human Dental Pulp Stem Cells

Dentin dysplasia type Ⅱ (DD-Ⅱ) is a subtype of hereditary dentin disorders. The dentin sialophosphoprotein (DSPP) gene has been revealed to be the causative gene, whose mutations could affect the normal tooth development process. The lesions involve both deciduous and permanent dentition, mainly manifested as tooth discoloration, attrition and even the subsequent malocclusion. If not treated in time, it will significantly affect the physical and psychological health of patients. The disease is difficult to be diagnosed in clinic accurately as its low incidence and hidden manifestations. The present article aims to discuss the clinical and radiographic characteristics, diagnosis, treatment of DD-Ⅱ, in order to improve the overall understanding on DD-Ⅱ for clinicians.

The premixed bioceramic sealer iRoot SP that is widely used clinically has been reported to kill bacterial biofilms and promote osteogenic differentiation of human stem cells from the apical papilla (hSCAPs). Although miR-141-3p has been substantiated to be involved in the osteogenic process, the underlying mechanisms remain unclear. The aim of this study was to investigate the role of miR-141-3p in osteogenic differentiation and underlying mechanisms of iRoot SP-treated hSCAPs. hSCAPs were extracted from tissue blocks with enzyme digestion and identified by using immunofluorescence, flow cytometry and alizarin red staining. The mRNA expression level of miR-141-3p in hSCPAs after culture with iRoot SP was examined by quantitative real-time PCR (qRT-PCR) assay. SPAG9 was identified as a downstream target gene of miR-141-3p by dual-luciferase report assay. Alkaline phosphatase (ALP) staining and activity detection, alizarin red staining, calcium concentration assay, qRT-PCR and western blot were used to estimate osteogenic differentiation ability and involved protein expression levels of the osteogenic makers and signalling pathway-related factors in iRoot SP-treated hSCAPs. Data were analysed by one-way anova and post hoc Tukey's test to determine any statistical differences between the experimental groups and the control group. p < .05 was considered statistically significant. Expression of miR-141-3p was reduced in iRoot SP-treated hSCAPs with the increased exposure time up to 7 days, and the western blot and qRT-PCR results revealed that the osteogenic markers osteocalcin (OCN), osterix (OSX), runt-related transcription factor 2 (RUNX2) and dentin sialophosphoprotein (DSPP) were inversely correlated with miR-141-3p. The negative regulatory relationship between miR-141-3p and SPAG9/ mitogen-activated protein kinases (MAPK) signalling axis was validated in this in vitro experiments. The bioceramic sealer iRoot SP promoted osteogenic differentiation of hSCAPs by inhibiting miR-141-3p following down-regulated SPAG9 expression, and activated MAPK pathway. These findings proposed a novel therapeutic impact of bioceramic sealer iRoot SP inducing bone regeneration in refractory periapical periodontitis.

The odontoblastic differentiation of dental pulp stem cells (DPSCs) associated with caries injury happens in an inflammatory context. We recently demonstrated that there is a link between inflammation and dental tissue regeneration, identified via enhanced DPSC-mediated dentinogenesis in vitro. Brain-derived neurotrophic factor (BDNF) is a nerve growth factor-related gene family molecule which functions through tropomyosin receptor kinase B (TrkB). While the roles of BDNF in neural tissue repair and other regeneration processes are well identified, its role in dentinogenesis has not been explored. Furthermore, the role of BDNF receptor-TrkB in inflammation-induced dentinogenesis remains unknown. The role of BDNF/TrkB was examined during a 17-day odontogenic differentiation of DPSCs. Human DPSCs were subjected to odontogenic differentiation in dentinogenic media treated with inflammation inducers (LTA or TNFα), BDNF, and a TrkB agonist (LM22A-4) and/or antagonist (CTX-B). Our data show that BDNF and TrkB receptors affect the early and late stages of the odontogenic differentiation of DPSCs. Immunofluorescent data confirmed the expression of BDNF and TrkB in DPSCs. Our ELISA and qPCR data demonstrate that TrkB agonist treatment increased the expression of dentin matrix protein-1 (DMP-1) during early DPSC odontoblastic differentiation. Coherently, the expression levels of runt-related transcription factor 2 (RUNX-2) and osteocalcin (OCN) were increased. TNFα, which is responsible for a diverse range of inflammation signaling, increased the levels of expression of dentin sialophosphoprotein (DSPP) and DMP1. Furthermore, BDNF significantly potentiated its effect. The application of CTX-B reversed this effect, suggesting TrkB`s critical role in TNFα-mediated dentinogenesis. Our studies provide novel findings on the role of BDNF-TrkB in the inflammation-induced odontoblastic differentiation of DPSCs. This finding will address a novel regulatory pathway and a therapeutic approach in dentin tissue engineering using DPSCs.

Dentin regeneration is the preferred method used to preserve dental pulp vitality after pulp exposure due to caries. Red light-emitting diode irradiation (LEDI), which is based on photobiomodulation (PBM), has been used to promote hard-tissue regeneration. However, the underlying mechanism still needs elucidation. This study aimed to explore the mechanism involved in red LEDI affecting dentin regeneration. Alizarin red S (ARS) staining revealed that red LEDI induced mineralization of human dental pulp cells (HDPCs) in vitro. We further distinguished the cell proliferation (0-6 d), differentiation (6-12 d), and mineralization (12-18 d) of HDPCs in vitro and treated cells either with or without red LEDI in each stage. The results showed that red LEDI treatment in the mineralization stage, but not the proliferation or differentiation stages, increased mineralized nodule formation around HDPCs. Western blot also indicated that red LEDI treatment in the mineralization stage, but not the proliferation or differentiation stages, upregulated the expression of dentin matrix marker proteins (dentin sialophosphoprotein, DSPP; dentin matrix protein 1, DMP1; osteopontin, OPN) and an intracellular secretory vesicle marker protein (lysosomal-associated membrane protein 1, LAMP1). Therefore, the red LEDI might enhance the matrix vesicle secretion of HDPCs. On the molecular level, red LEDI enhanced mineralization by activating the mitogen-activated protein kinase (MAPK) signaling pathways (ERK and P38). ERK and P38 inhibition reduced mineralized nodule formation and the expression of relevant marker proteins. In summary, red LEDI enhanced the mineralization of HDPCs by functioning to produce a positive effect in the mineralization stage in vitro.

ABSTRACT Aim Inflammation is a complex host response to harmful infection or injury, and it seems to play a crucial role in tissue regeneration both positively and negatively. We have previously demonstrated that the activation of the complement C5a pathway affects dentin-pulp regeneration. However, limited information is available to understand the role of the complement C5a system related to inflammation-mediated dentinogenesis. The aim of this study was to determine the role of complement C5a receptor (C5aR) in regulating lipopolysaccharide (LPS)-induced odontogenic differentiation of dental pulp stem cells (DPSCs). Material and Methods Human DPSCs were subjected to LPS-stimulated odontogenic differentiation in dentinogenic media treated with the C5aR agonist and antagonist. A putative downstream pathway of the C5aR was examined using a p38 mitogen-activated protein kinase (p38) inhibitor (SB203580). Results Our data demonstrated that inflammation induced by the LPS treatment potentiated DPSC odontogenic differentiation and that this is C5aR dependent. C5aR signaling controlled the LPS-stimulated dentinogenesis by regulating the expression of odontogenic lineage markers like dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP-1). Moreover, the LPS treatment increased the total p38, and the active form of p38 expression, and treatment with SB203580 abolished the LPS-induced DSPP and DMP-1 increase. Conclusions These data suggest a significant role of C5aR and its putative downstream molecule p38 in the LPS-induced odontogenic DPSCs differentiation. This study highlights the regulatory pathway of complement C5aR/p38 and a possible therapeutic approach for improving the efficiency of dentin regeneration during inflammation.

Objective: Single-cell RNA sequencing (scRNA-seq) was used to analyze the developing mouse molars, in order to construct a spatiotemporal development atlas of pulp cells, and further to reveal the developmental process and regulatory mechanism of tooth development. Methods: Ten mandibular first molars from C57BL/6 mice in postnatal day (PN) 0 and 3 were respectively dissected and digested to obtain single-cell suspensions. scRNA-seq was performed on 10× Genomics platform. PN 7 mouse molar scRNA-seq data were obtained from our previous study. PN 0, 3, and 7 scRNA-seq data were integrated for following analysis. The initial quality control, mapping and single cell expression matrix construction were performed by Cell Ranger. Quality control, standardization, dimensional reduction and cluster analysis were performed by using Seurat. Monocle was used to generate the pseudotime trajectory. Scillus was used to perform gene ontology analysis. In order to detect the spatiotemporal change of different population of pulp cells, the marker genes of each cluster were demonstrated by RNAscope in situ hybridization. Results: There were twenty-six cell clusters within mouse molars, which were identified as eight different cell types, including dental pulp cells, dental follicle cells, epithelial cells, immune cells, endothelial cells, perivascular cells, glial cells and erythrocytes. We further re-clustered and analyzed dental pulp cells. Cluster 0 were mature pulp cells, which located at the upper portion of crown. The main functions of cluster 0 were osteogenesis and extracellular structure organization. Cluster 1 were apical papilla cells, which located at the apical part of roots, whose main functions were extracellular structure organization and organ development. Cluster 2 were cycling cells, which were actively proliferated, resided in the lower portion of the crown. Cluster 3 and 4 were preodontoblasts and odontoblasts, respectively. Their functions were closely related to biomineralization. The proportion of mature pulp cells increased with the development process, while the proportion of cycling cells and odontoblast lineage decreased. According to the expression pattern of marker genes of each cluster, we constructed a cell atlas of dental pulp. Pseudotime trajectory analysis found there were two development trajectories within dental pulp. They both started from SPARC related modular calcium binding 2 (Smoc2)+ dental papilla cells, then went through DNA topoisomerase Ⅱ alpha (Top2a)+ cycling cells, and finally divided into coxsackie virus and adenovirus receptor (Cxadr)+ mature pulp cells or dentin sialophosphoprotein (Dspp)+ odontoblasts two lineages. Conclusions: scRNA-seq could fully discover the intercellular heterogeneity of cells on transcriptome level, which provides a powerful tool to study the process and regulatory mechanism of organ development.

BackgroundTo study the odontogenic potential of dental pulp stem cells (DPSCs) after induction with three different bioactive materials: activa bioactive (base/liner) (AB), TheraCal LC (TC), and mineral trioxide aggregate (MTA), when combined with two different types of scaffolds.MethodsDPSCs were isolated from freshly extracted premolars of young orthodontic patients, cultured, expanded to passage 4 (P), and characterized by flow cytometric analysis. DPSCs were seeded onto two scaffolds in contact with different materials (AB, TC, and MTA). The first scaffold contained polycaprolactone-nano-chitosan and synthetic hydroxyapatite (PCL-NC-HA), whereas the second scaffold contained polycaprolactone-nano-chitosan and synthetic Mg-substituted hydroxyapatite (PCL-NC-Mg-HA). DPSC viability and proliferation were evaluated at various time points. To assess odontoblastic differentiation, gene expression analysis of dentin sialophosphoprotein (DSPP) by quantitative real-time polymerase chain reaction (qRT-PCR) and morphological changes in cells were performed using inverted microscope phase contrast images and scanning electron microscopy. The fold-change in DSPP between subgroups was compared using a one-way ANOVA. Tukey's test was used to compare the fold-change in DSPP between the two subgroups in multiple comparisons, and P was set at p < 0.05.ResultsDSPP expression was significantly higher in the PCL-NC-Mg-HA group than in the PCL-NC-HA group, and scanning electron microscopy revealed a strong attachment of odontoblast-like cells to the scaffold that had a stronger odontogenic differentiation effect on DPSCs than the scaffold that did not contain magnesium. MTA has a significantly higher odontogenic differentiation effect on cultured DPSCs than AB or TC does. The combination of scaffolds and bioactive materials improves DPSCs induction in odontoblast-like cells.ConclusionsThe PCL-NC-Mg-HA scaffold showed better odontogenic differentiation effects on cultured DPSCs. Compared to AB and TC, MTA is the most effective bioactive material for inducing the odontogenic differentiation of cultured DPSCs.

ObjectivesThis experimental study aimed to assess the effect of copper oxide nanoparticles (CuONPs) and light-emitting diode (LED) irradiation on the cell viability and osteogenic/odontogenic differentiation of human SCAPs.MethodsAfter the culture of SCAPs, the effects of different concentrations of CuONPs on cell viability were evaluated by the methyl thiazolyl tetrazolium (MTT) assay after 24 and 48 h, and the optimal concentration was determined (n = 12). SCAPs were then divided into four groups based on the type of treatment: (I) no-treatment control group, (II) exposure to CuONPs, (III) LED irradiation (635 nm, 200 mW/cm2) for 30 s, and (IV) exposure to CuONPs combined with LED irradiation. CuONPs were synthesized by a green technique, which was based on reduction and simultaneous stability of copper ions by using the pomegranate peel extract. After treatments, the expression of osteogenic/odontogenic markers including dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), alkaline phosphatase (ALP), and dentin matrix acidic phosphoprotein 1 (DMP1) was evaluated in all four groups using quantitative real-time polymerase chain reaction (PCR) (n = 16). Also, osteogenic differentiation of SCAPs was evaluated qualitatively by alizarin red staining (ARS) to assess the matrix mineralization (n = 4). SPSS version 18 was used for data evaluation. The Kruskal–Wallis and Mann–Whitney tests were used to compare the groups.ResultsExposure to 1 µg/mL CuONPs resulted in maximum viability of SCAPs. Concentrations of CuONPs over 10 µg/mL significantly decreased the viability of SCAPs. Real-time PCR showed that the expression of DMP1, BSP, ALP, and DSPP in CuONPs + LED and LED groups was significantly higher than that in CuONPs and control groups at both 24 and 48 h (P < 0.05). The density of ARS increased in all experimental groups after 24 h, and in CuONPs + LED and CuONPs groups after 48 h, compared to the control group.ConclusionAddition of CuONPs and LED irradiation of SCAPs in the culture medium significantly enhanced their osteogenic/odontogenic differentiation.

Dentin sialophosphoprotein (DSPP) is primarily expressed by differentiated odontoblasts (dentin-forming cells), and transiently expressed by presecretory ameloblasts (enamel-forming cells). Disease-causing DSPP mutations predominantly fall into two categories: 5’ mutations affecting targeting and trafficking, and 3’ − 1 frameshift mutations converting the repetitive, hydrophilic, acidic C-terminal domain into a hydrophobic one. We characterized the dental phenotypes and investigated the pathological mechanisms of DsppP19L and Dspp−1fs mice that replicate the two categories of human DSPP mutations. In DsppP19L mice, dentin is less mineralized but contains dentinal tubules. Enamel mineral density is reduced. Intracellular accumulation and ER retention of DSPP is observed in odontoblasts and ameloblasts. In Dspp−1fs mice, a thin layer of reparative dentin lacking dentinal tubules is deposited. Odontoblasts show severe pathosis, including intracellular accumulation and ER retention of DSPP, strong ubiquitin and autophagy activity, ER-phagy, and sporadic apoptosis. Ultrastructurally, odontoblasts show extensive autophagic vacuoles, some of which contain fragmented ER. Enamel formation is comparable to wild type. These findings distinguish molecular mechanisms underlying the dental phenotypes of DsppP19L and Dspp−1fs mice and support the recently revised Shields classification of dentinogenesis imperfecta caused by DSPP mutations in humans. The Dspp−1fs mice may be valuable for the study of autophagy and ER-phagy.

Novel dentin sialophosphoprotein gene frameshift mutations affect dentin mineralization

Heterogeneous mutations in dentin sialophosphoprotein (DSPP) gene, which is located on autosome 4, are associated with hereditary dentin developmental disorders. According to the new classification proposed by de La Dure-Molla et al, diseases caused by DSPP gene mutations mainly manifested as abnormal dentin development are collectively referred to as dentinogenesis imperfecta (DI), including dentin dysplasia type Ⅱ (DD-Ⅱ), dentinogenesis imperfecta type Ⅱ (DGI-Ⅱ) and dentinogenesis imperfecta type Ⅲ (DGI-Ⅲ) in Shields classification. And dentin dysplasia type Ⅰ (DD-Ⅰ) in Shields classification is redesignated as radicular dentin dysplasia. In this paper, progress in the classification, clinical characteristics and genetic mechanisms of DI are reviewed. This paper also provides clinical management and treatment strategies for patients suffering DI.

The purpose of this study was to investigate whether fibroblast growth factor 4 (FGF4) and FGF9 are active in dentin differentiation. Dentin matrix protein 1 (Dmp1) -2A-Cre transgenic mice, which express the Cre-recombinase in Dmp1-expressing cells, were crossed with CAG-tdTomato mice as reporter mouse. The cell proliferation and tdTomato expressions were observed. The mesenchymal cell separated from neonatal molar tooth germ were cultured with or without FGF4, FGF9, and with or without their inhibitors ferulic acid and infigratinib (BGJ398) for 21days. Their phenotypes were evaluated by cell count, flow cytometry, and real-time PCR. Immunohistochemistry for FGFR1, 2, and 3 expression and the expression of DMP1 were performed. FGF4 treatment of mesenchymal cells obtained promoted the expression of all odontoblast markers. FGF9 failed to enhance dentin sialophosphoprotein (Dspp) expression levels. Runt-related transcription factor 2 (Runx2) was upregulated until day 14 but was downregulated on day 21. Compared to Dmp1-negative cells, Dmp1-positive cells expressed higher levels of all odontoblast markers, except for Runx2. Simultaneous treatment with FGF4 and FGF9 had a synergistic effect on odontoblast differentiation, suggesting that they may play a role in odontoblast maturation.