Dental Papilla Cells Research Articles

Odontoblasts: Specialized hard-tissue-forming cells in the dentin-pulp complex.

Odontoblasts are specialized cells that produce dentin and exhibit unique morphological characteristics; i.e., they extend cytoplasmic processes into dentinal tubules. While osteoblasts, which are typical hard-tissue-forming cells, are generated from mesenchymal stem cells during normal and pathological bone metabolism, the induction of odontoblasts only occurs once during tooth development, and odontoblasts survive throughout the lives of healthy teeth. During the differentiation of odontoblasts, signaling molecules from the inner enamel epithelium are considered necessary for the differentiation of odontoblast precursors, i.e., peripheral dental papilla cells. If odontoblasts are destroyed by severe external stimuli, such as deep caries, the differentiation of dental pulp stem cells into odontoblast-like cells is induced. Various bioactive molecules, such as non-collagenous proteins, might be involved in this process, although the precise mechanisms responsible for odontoblast differentiation have not been fully elucidated. Recently, our knowledge about the other functional activities of odontoblasts (apart from dentin formation) has increased. For example, it has been suggested that odontoblasts might act as nociceptive receptors, and surveillance cells that detect the invasion of exogenous pathogens. The regeneration of the dentin-pulp complex has recently gained much attention as a promising future treatment modality that could increase the longevity of pulpless teeth. Finally, congenital dentin anomalies, which are concerned with the disturbance of odontoblast functions, are summarized.

The Novel NF‐κB Inhibitor, MTI‐II Peptide Anti‐Inflammatory Drug, Suppresses Inflammatory Responses in Odontoblast‐Like Cells

Regulation of inflammation is important for pulp wound healing, including protective responses by odontoblast-like cells. However, methods for directly regulating pulp inflammation have not yet been described. The inflammatory response is mediated by a transcription factor, nuclear factor-κB (NF-κB), which activates inflammatory cytokines including tumor necrosis factor (TNF)-α. Macromolecular translocation inhibitor II (MTI-II) was previously demonstrated as an enhancer of the transcriptional activity of glucocorticoid-bound glucocorticoid receptor. Recently, a MTI-II peptide anti-inflammatory drug (MPAID) was bioengineered from the structure of MTI-II as an inhibitor of NF-κB transactivation. Here, we examined the effects of MTI-II and MPAID on the inflammatory responses of odontoblast-like cells. TNF-α inhibited alkaline phosphatase (ALP) activity, a marker of odontoblast/osteogenic differentiation, and induced NF-κB transcriptional activity in KN-3 cells, which are odontoblast-like cells derived from dental papilla cells of rat incisors, without affecting their viability. Exogenous expression of MTI-II suppressed the NF-κB transcriptional activity induced by TNF-α or overexpression of p65 (a main subunit of NF-κB) in the cells, whereas it failed to inhibit degradation of IκBα and nuclear translocation of p65 after TNF-α treatment, suggesting that MTI-II inhibits NF-κB transcriptional activity by modulating the duration of DNA binding by p65. MPAID also inhibited TNF-α-induced NF-κB transcriptional activity, the mRNA expression of IL-6 and IL-8, and IL-6 production. Furthermore, pretreatment of the cells with MPAID restored the inhibitory effect of TNF-α on ALP activity. These results suggest that MPAID may be able to regulate the inflammatory response and maintain a protective response of dental pulp. J. Cell. Biochem. 117: 2552-2558, 2016. © 2016 Wiley Periodicals, Inc.

Spatial and temporal expression of histone demethylase, Kdm2a, during murine molar development

The histone demethylase, lysine (K)-specific demethylase 2A (Kdm2a), is highly conserved and expressed ubiquitously. Kdm2a can regulate cell proliferation and osteo/dentinogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) derived from dental tissue. We used quantitative real-time RT-PCR analysis and immunohistochemistry to detect Kdm2a expression during development of the murine molar at embryonic days E12, E14, E16 and E17 and postnatal days P3 and P14. Immunohistochemistry results showed no positive staining of Kdm2a at E12. At E14, Kdm2a was expressed weakly in the inner enamel epithelium, stellate reticulum cells and dental sac. At E16, Kdm2a was expressed mainly in the inner and outer enamel epithelium, stratum intermedium and dental sac, but weaker staining was found in cervical loop and dental papilla cells adjacent to the basement membrane. At E17, the strongest Kdm2a staining was detected in the ameloblasts and stronger Kdm2a staining also was detected in the stratum intermedium, outer enamel epithelium and dental papilla cells compared to the expression at E16. Postnatally, we found that Kdm2a was localized in secretory and mature ameloblasts and odontoblasts, and dentin was unstained. Real-time RT-PCR showed that Kdm2a mRNA levels in murine germ cells increased from E12 to E14 and from E14 to E16; no significant change occurred at E16, E17 or P3, then the levels decreased at P14 compared to P3. Kdm2a expression may be closely related to cell proliferation, to ameloblast and odontoblast differentiation and to the secretion of extracellular enamel and dentin during murine tooth development.

Inhibition of Ape1 Redox Activity Promotes Odonto/osteogenic Differentiation of Dental Papilla Cells.

Dentinogenesis is the formation of dentin, a substance that forms the majority of teeth, and this process is performed by odontoblasts. Dental papilla cells (DPCs), as the progenitor cells of odontoblasts, undergo the odontogenic differentiation regulated by multiple cytokines and paracrine signal molecules. Ape1 is a perfect paradigm of the function complexity of a biological macromolecule with two major functional regions for DNA repair and redox regulation, respectively. To date, it remains unclear whether Ape1 can regulate the dentinogenesis in DPCs. In the present study, we firstly examed the spatio-temporal expression of Ape1 during tooth germ developmental process, and found the Ape1 expression was initially high and then gradually reduced along with the tooth development. Secondly, the osteo/odontogenic differentiation capacity of DPCs was up-regulated when treated with either Ape1-shRNA or E3330 (a specific inhibitor of the Ape1 redox function), respectively. Moreover, we found that the canonical Wnt signaling pathway was activated in this process, and E3330 reinforced-osteo/odontogenic differentiation capacity was suppressed by Dickkopf1 (DKK1), a potent antagonist of canonical Wnt signaling pathway. Taken together, we for the first time showed that inhibition of Ape1 redox regulation could promote the osteo/odontogenic differentiation capacity of DPCs via canonical Wnt signaling pathway.

Effect of dentine phosphophoryn-derived RGD peptides on odontoblast-like cells.

To investigate the effect of RGD peptides derived from dentine phosphophoryn (DPP) on odontoblast-like cell in terms of differentiation and mineralization. Mouse dental papilla cell line (MDPC-23), a rat odontoblast-like cell line, was used. Briefly, RGD peptides (RGD-1: SESDNNSSSRGDASYNSDES, RGD-2: ANSESDNNSSSRGDA, RGD-3: SRGDASYNSDESKD) were immobilized onto tissue culture polystyrene dishes (TCPS) assisted by carbodiimide chemistry. Surface characterization including carboxyl group quantification and amino acid analysis was carried out to ensure the existence of peptides on plates. Cells were inoculated to those peptides-modified and control dishes. Next, cell morphology was observed under phase contrast microscopy; cell numbers were counted manually using a hemocytometer. Furthermore, differentiation was examined by alkaline phosphatase (ALP) activity quantification, conventional and real-time RT-PCR. Finally, calcific deposition was observed by alizarin red staining and quantified using the cetylpyridinium chloride extraction method. Differences between the experimental groups and the control group were analysed statistically using one-way anova and Tukey's multiple comparison tests. Peptides were immobilized onto TCPS successfully as evidenced by carboxyl group density and amino acid analysis. Cell morphology remained unchanged between peptides-immobilized groups and control, but adhered cell numbers were higher on those peptides-immobilized dishes (significant differences existed between RGD-1-0.5 with control, RGD-2-0.1 with control, and RGD-3-0.5 with control, respectively). RGD-3-0.5 exhibited the highest ALP activity on day 7 (P<0.05) and promoted a twofold greater DMP-1 mRNA expression compared to the control on day 10 (P<0.05). RGD peptides grafted dishes accelerated the mineralization of cells, amongst the experimental groups tested, RGD-3 groups (comprising RGD-3-0.1 and RGD-3-0.5) had significantly higher amounts of calcific deposition as compared to the control (P<0.05). RGD peptides originated from DPP especially RGD-3 promoted MDPC-23 differentiation and mineralization.

SP1 promotes the odontoblastic differentiation of dental papilla cells.

Odontoblasts are a type of terminally differentiated and matrix-secreting cells that are responsible for dentinogenesis. The process of odontoblast differentiation is regulated by a variety of transcription factors. The transcription factor SP1 is known to play an essential regulatory role in cell proliferation and differentiation. The purpose of this study was to investigate the role of SP1 in odontoblastic differentiation. Immunohistochemistry verified that SP1 was specifically expressed in polarizing and secretory odontoblasts invivo. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence revealed that the expression of SP1 was significantly upregulated during odontoblastic differentiation of mDPC6T cells, a dental papilla cell line. Overexpression of SP1 significantly increased the expression of odontoblast-related genes, including DSPP, DMP1 and ALP, and promoted the formation of mineralized nodules. Meanwhile, knockdown of SP1 decreased the expression of these odontoblast-related genes and suppressed the formation of mineralized nodules. Our results demonstrate that SP1 promotes the odontoblastic differentiation and mineralization of dental papilla cells.

Comparison of the Odontogenic Differentiation Potential of Dental Follicle, Dental Papilla, and Cranial Neural Crest Cells

IntroductionDuring tooth development, cells originating from the neural crest serve as precursors to the cells in the dental follicle and dental papilla. Therefore, the current study aimed to understand the associations of cranial neural crest cells (CNCCs), dental follicle cells (DFCs), and dental papilla cells (DPCs) by performing a parallel comparison to evaluate their odontogenic differentiation capacities. MethodsIn this study, we harvested the 3 cells from C57/green fluorescent protein–positive mice or embryos and compared the cell morphology, surface antigens, microstructures, and gene and protein expression. Under the odontogenic microenvironments provided by treated dentin matrix, the odontogenic differentiations of the 3 cells were further compared in vitro and in vivo. ResultsThe gene levels of DFCs in neurofilament, tubulin, and nestin were close to the DPCs, and in alkaline phosphatase, osteopontin, dentin matrix protein 1, and dentin sialophosphoprotein were the lowest in the 3 cells. However, Western blot results showed that DFCs possessed more similar protein profiles to CNCCs than DPCs, including collagen 1, transforming growth factor beta 1, osteopontin, neurofilament, and dentin matrix protein 1. Meanwhile, DFCs as 1 source of dental stem cells possessed high potency in odontogenic differentiation in vitro. Moreover, similar dentinlike tissues were observed in all 3 groups in vivo. ConclusionsCNCCs, DFCs, and DPCs possessed different biological characteristics in odontogenic differentiation.

Anatomical abnormalities in multiple permanent teeth.

Anatomical abnormalities in multiple permanent teeth.

Canonical Wnt signaling acts synergistically on BMP9-induced osteo/odontoblastic differentiation of stem cells of dental apical papilla (SCAPs)

Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. Here, we investigate if BMP9 and Wnt/β-catenin act synergistically on odontogenic differentiation. Using the immortalized SCAPs (iSCAPs) isolated from mouse apical papilla tissue, we demonstrate that Wnt3A effectively induces early osteogenic marker alkaline phosphatase (ALP) in iSCAPs, which is reduced by β-catenin knockdown. While Wnt3A and BMP9 enhance each other's ability to induce ALP activity in iSCAPs, silencing β-catenin significantly diminishes BMP9-induced osteo/odontogenic differentiation. Furthermore, silencing β-catenin reduces BMP9-induced expression of osteocalcin and osteopontin and in vitro matrix mineralization of iSCAPs. In vivo stem cell implantation assay reveals that while BMP9-transduced iSCAPs induce robust ectopic bone formation, iSCAPs stimulated with both BMP9 and Wnt3A exhibit more mature and highly mineralized trabecular bone formation. However, knockdown of β-catenin in iSCAPs significantly diminishes BMP9 or BMP9/Wnt3A-induced ectopic bone formation in vivo. Thus, our results strongly suggest that β-catenin may play an important role in BMP9-induced osteo/ondontogenic signaling and that BMP9 and Wnt3A may act synergistically to induce osteo/odontoblastic differentiation of iSCAPs. It's conceivable that BMP9 and/or Wnt3A may be explored as efficacious biofactors for odontogenic regeneration and tooth engineering.

Effect of melatonin on human dental papilla cells.

Melatonin regulates a variety of biological processes, which are the control of circadian rhythms, regulation of seasonal reproductive function and body temperature, free radical scavenging and so on. Our previous studies have shown that various cells exist in human and mouse tooth germs that express the melatonin 1a receptor (Mel1aR). However, little is known about the effects of melatonin on tooth development and growth. The present study was performed to examine the possibility that melatonin might exert its influence on tooth development. DP-805 cells, a human dental papilla cell line, were shown to express Mel1aR. Expression levels of mRNA for Mel1aR in DP-805 cells increased until 3 days after reaching confluence and decreased thereafter. Real-time reverse transcription-polymerase chain reaction showed that melatonin increased the expression of mRNAs for osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotin (DSPP). Melatonin also enhanced the mineralized matrix formation in DP-805 cell cultures in a dose-dependent manner. These results strongly suggest that melatonin may play a physiological role in tooth development/growth by regulating the cellular function of odontogenic cells in tooth germs.

The Effects of Osterix on the Proliferation and Odontoblastic Differentiation of Human Dental Papilla Cells

IntroductionDental papilla cells (DPCs) are precursors of odontoblasts and have the potential to differentiate into odontoblasts. Osteoblasts and odontoblasts have many common characteristics. Osterix (Osx) is essential for osteoblast differentiation. However, no information is available for the effects of Osx on the odontoblastic differentiation of DPCs. The purpose of this study was to investigate the effects of Osx on the proliferation and odontoblastic differentiation of DPCs. MethodsAn immortalized human dental papilla cell (hDPC) line was used. Osx was stably overexpressed or knocked down in hDPCs with infection of lentiviral particles to determine its biological effects on hDPCs. The proliferation of cells was measured by the 5-ethynyl-2′-deoxyuridine incorporation assay and direct cell counting. Expressions of dentin sialophosphoprotein, nestin, dentin matrix protein 1, and alkaline phosphatase were detected by real-time polymerase chain reaction to determine the odontoblastic differentiation of cells. The mineralization ability of cells was evaluated by von Kossa staining and alkaline phosphatase activity assay. ResultsOverexpression of Osx retarded the proliferation of hDPCs, whereas knockdown of Osx increased the cell proliferation. Overexpression of Osx promoted the odontoblastic differentiation of hDPCs by up-regulating odontoblastic differentiation genes and increased the mineralization ability of hDPCs. Knockdown of Osx down-regulated odontoblastic differentiation genes and decreased the mineralization ability of hDPCs. ConclusionsOsx might function as a potential regulator for the proliferation and odontoblastic differentiation of hDPCs.

Expression of Nfic during root formation in first mandibular molar of rat.

The transcription factor Nfic is a key regulator during tooth development. Nfic deficient mice exhibit short and abnormal molar roots and severely deformed incisors. Dental epithelial cells, known as Hertwig's epithelial root sheath (HERS), participate in root formation. However, whether Nfic is involved in HERS-mesenchyme interaction remains unclear. In this study, the detail temporal and spatial expression pattern of Nfic during rat molar development was examined using immunohistochemistry and in situ hybridization. Nfic was detected in ameloblasts, dental follicle cells (DFCs) and dental papilla cells (DPCs), especially the DPCs close to dentin, from postnatal day 5 to day 16. Nfic expression in DPCs, DFCs and HERS cells was also examined by western blot and RT-PCR. Nfic was detected in DPCs and DFCs, but not in HERS cells. Co-culture experiment further indicated that Nfic mRNA expression in DPCs was elevated by the presence of HERS cells. Our results revealed that Nfic could be a marker gene for root odontoblasts differentiation initiation and its expression might be regulated through epithelial-mesenchymal interaction.

Expression of Oct-4, SOX-2, and MYC in dental papilla cells and dental follicle cells during in-vivo tooth development and in-vitro co-culture.

During tooth development, the special structure of dental follicle and dental papilla enables dental papilla cells (DPCs) and dental follicle cells (DFCs) to make contact with each other. Octamer-binding transcription factor 4 (Oct-4), sex determining region Y box-2 (SOX-2), and cellular homologue of avian myelocytomatosis virus oncogene (MYC) (OSM) are associated with reprogramming and pluripotency. However, whether the expression of OSM could be activated through cell-cell communication is not known. In this study, the distribution of OSM in rat tooth germ was investigated by immunohistochemical staining. An in-vitro co-culture system of DPCs and DFCs was established. Cell proliferation, cell apoptosis, cell cycle stages, and expression of OSM were investigated by Cell Counting Kit 8 (CCK8) analysis, flow cytometry, real-time PCR, and immunohistochemical staining. We found that Oct-4 and SOX-2 were strongly expressed in tooth germ on days 7 and 9 after birth, whereas MYC was expressed only on day 9. Cell proliferation and apoptosis were inhibited, the cell cycle was arrested in the G0/G1 phase, and the propidium iodide (PI) value was downregulated. Expression of Oct-4 and SOX-2 was significantly elevated in both cell types after 3 d of co-culture, whereas expression of MYC was not significantly elevated until day 5. These results indicate that the optimized microenvironment with cell-cell communication enhanced the expression of reprogramming markers associated with reprogramming capacity in DPCs and DFCs, both in vivo and in vitro.

Periostin is a negative regulator of mineralization in the dental pulp tissue

The dental pulp tissue is encased in hard tissue and surrounded by hard tissue-forming cells, but remains in a non-mineralized state itself, suggesting the presence of regulatory mechanisms precluding pulp mineralization. This study aimed to reveal the regulatory function of periostin (Postn), which is essential for osteoblast differentiation, for odontoblast differentiation/mineralization. We evaluated the effects of Postn overexpression and RNAi-mediated suppression in mouse dental papilla cells (MDPs) on the expression of odontoblastic markers and Notch signaling molecules, and on the formation of mineralized nodules. Localization of Postn in the dental pulp tissue of normal and cavity-prepared molars was observed immunohistologically. Enforced overexpression of Postn in MDPs induced down-regulation of odontoblastic markers and in vitro mineralization. Conversely, silencing of Postn mRNA in MDPs induced up-regulation of odontoblastic markers and ALP activity. Up- and down-regulation of Postn caused increased and decreased expression, respectively, of Notch signaling molecules. Postn expression was minimal in normal dental pulp, but was rapidly and globally increased in the whole pulp tissue of molar teeth at 1day after cavity preparation, decreasing thereafter. These results indicate that Postn may be a negative regulator of odontoblast differentiation/mineralization, and that may exert its actions via Notch signals.

Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells

ObjectiveThree-dimensional (3D) spheroid culture is a method for creating 3D aggregations of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into odontoblasts. MethodsWe cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture. Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signalling-related proteins were detected immunohistochemically. ResultsOdonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs (p<0.05). Histologically, 3D spheroid colonies consisted of two compartments: a cell-dense peripheral zone and cell-sparse core zone. Proliferating cells with high ALP activity and DMP1 expression were found mainly in the peripheral zone that also showed strong expression of vinculin and integrin signalling-related proteins. In contrast, apoptotic and hypoxic cells were detected in the core zone. Conclusion3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling.

Wnt11 expression in rat dental pulp and promotional effects of Wnt signaling on odontoblast differentiation

Congenital anomalies of wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt) are frequently accompanied with tooth and dentin abnormality. The aim of this study was to investigate the effects of Wnt signaling on odontoblast differentiation of mouse dental papilla cells (MDPs). Mouse dental papilla cells were cultured in α-modified minimum essential medium containing 10% fetal bovine serum and antibiotics. Odontoblast differentiation was induced by bone morphogenic protein 2 (BMP2), and the expression of odontoblast-specific markers and Wnt-related signaling molecules was analyzed by real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Odontoblast differentiation was evaluated by dentin sialophosphoprotein (Dspp) and dentin matrix protein (DMP) 1 expression. Localization of β-catenin in MDPs was detected by immunocytochemistry using an anti-β-catenin antibody. Dspp expression in MDPs was upregulated in the presence of BMP2. Wnt5a, Wnt11, Lef1 and Tcf4 expression was upregulated in BMP2-treated MDPs. Wnt11 expression was detected in rat dental pulp in vivo, and particularly strong expression of Wnt11 was detected in odontoblasts. Enhanced Dspp and DMP1 expression and alkaline phosphatase activity induced by BMP2 were completely negated by the Wnt antagonist: IWR-1-endo treatment. Nuclear translocation of β-catenin observed in BMP2-treated MDPs was also negated by IWR-1-endo treatment. These results indicate that Wnt signaling upregulates odontoblast marker expression in MDPs, suggesting a promoting effect of Wnt signaling on odontoblast differentiation.

Role of the epithelial cell rests of Malassez in the development, maintenance and regeneration of periodontal ligament tissues

Periodontitis is a highly prevalent inflammatory disease that results in damage to the tooth-supporting tissues, potentially leading to tooth loss. Periodontal tissue regeneration is a complex process that involves the collaboration of two hard tissues (cementum and alveolar bone) and two soft tissues (gingiva and periodontal ligament). To date, no periodontal-regenerative procedures provide predictable clinical outcomes. To understand the rational basis of regenerative procedures, a better understanding of the events associated with the formation of periodontal components will help to establish reliable strategies for clinical practice. An important aspect of this is the role of the Hertwig's epithelial root sheath in periodontal development and that of its descendants, the epithelial cell rests of Malassez, in the maintenance of the periodontium. An important structure during tooth root development, the Hertwig's epithelial root sheath is not only a barrier between the dental follicle and dental papilla cells but is also involved in determining the shape, size and number of roots and in the development of dentin and cementum, and may act as a source of mesenchymal progenitor cells for cementoblasts. In adulthood, the epithelial cell rests of Malassez are the only odontogenic epithelial population in the periodontal ligament. Although there is no general agreement on the functions of the epithelial cell rests of Malassez, accumulating evidence suggests that the putative roles of the epithelial cell rests of Malassez in adult periodontal ligament include maintaining periodontal ligament homeostasis to prevent ankylosis and maintain periodontal ligament space, to prevent root resorption, to serve as a target during periodontal ligament innervation and to contribute to cementum repair. Recently, ovine epithelial cell rests of Malassez cells have been shown to harbor clonogenic epithelial stem-cell populations that demonstrate similar properties to mesenchymal stromal/stem cells, both functionally and phenotypically. Therefore, the epithelial cell rests of Malassez, rather than being 'cell rests', as indicated by their name, are an important source of stem cells that might play a pivotal role in periodontal regeneration.

C-Jun N-Terminal Kinase (JNK) Mediates Wnt5a-Induced Cell Motility Dependent or Independent of RhoA Pathway in Human Dental Papilla Cells

Wnt5a plays an essential role in tissue development by regulating cell migration, though the molecular mechanisms are still not fully understood. Our study investigated the pathways involved in Wnt5a-dependent cell motility during the formation of dentin and pulp. Over-expression of Wnt5a promoted cell adhesion and formation of focal adhesion complexes (FACs) in human dental papilla cells (hDPCs), while inhibiting cell migration. Instead of activating the canonical Wnt signal pathway in hDPCs, Wnt5a stimulation induced activation of the JNK signal in a RhoA-dependent or independent manner. Inhibiting JNK abrogated Wnt5a-induced FACs formation but not cytoskeletal rearrangement. Both dominant negative RhoA (RhoA T19N) and constitutively active RhoA mutants (RhoA Q63L) blocked the Wnt5a-dependent changes in hDPCs adhesion, migration and cytoskeletal rearrangement here too, with the exception of the formation of FACs. Taken together, our study suggested that RhoA and JNK signaling have roles in mediating Wnt5a-dependent adhesion and migration in hDPCs, and the Wnt5a/JNK pathway acts both dependently and independently of the RhoA pathway.

KLF4 promoted odontoblastic differentiation of mouse dental papilla cells via regulation of DMP1

Odontoblasts, which derive from dental papilla, are a type of terminally differentiated matrix-secreting cells. Previous studies have identified various transcription factors involved in the differentiation process of odontoblasts. We have recently found that Krüppel-like factor 4 (Klf4) was expressed in the polarizing and elongating odontoblasts, but the function of Klf4 in the differentiation of odontoblasts is still unclear. We hypothesized Klf4 promoted the differentiation of odontoblasts by up-regulating some odontoblast-related genes. In this study, we found that the expression of Klf4 increased significantly during the odontoblastic differentiation of primary mouse dental papilla cells and the mouse dental papilla cell line-mDPC6T. Overexpression of Klf4 significantly up-regulated odontoblast-related genes, such as Dmp1, Dspp, and Alp, and promoted the accumulation of mineral nodules. Knock-down of Klf4 down-regulated expression of Dmp1, Dspp, and Alp, and inhibited mineral deposition. We applied in silico analysis and identified one target gene of Klf4-Dmp1. Based on further analysis of ChIP data, EMSA and dual luciferase activity assays, we confirmed that Klf4 was able to specifically bind to the Dmp1 promoter and transactivate its expression. Furthermore, forced expression of Dmp1 in the Klf4 knock-down mDPC6T cell line significantly recovered its odontoblastic differentiation ability. Our data confirmed our hypothesis that Klf4 promotes the differentiation of odontoblasts via the up-regulation of Dmp1.

Effect of mineral trioxide aggregate on proliferation and differentiation of rat dental papilla cells

To investigate the effects of mineral trioxide aggregate(MTA) on the proliferation and differentiation of rat dental papilla cells(RDPC). RDPC were cultured by tissue block method and identified.RDPC of the third passage were cultured with material extract fluid containing different mass concentrations of MTA (0.002, 0.02,0.2, 2 and 20 g/L) for 3 d, those cultured with routine culture fluid served as control group. The proliferation-related parameters were measured by methyl thiazolyl tetrazolium(MTT) assay. RDPC were cultured with material extract fluids containing 0.002 g/L MTA, those cultured with routine culture fluid served as control group, the activity of alkaline phosphatase(ALP) at 1, 3,5, 7 d and the level of collagen I at 1, 3,5 d were detected. MTT results showed that the A value of RDPC of group 20 g/L (0.092 ± 0.011) was less than that of the control group (0.249 ± 0.006) at 3 d(P < 0.01), the A value of RDPC of group 0.02 g/L (0.267 ± 0.005) and 0.002 g/L (0.276 ± 0.006) were more than that of the control group (0.249 ± 0.006) at 3 d(P < 0.01). ALP detection proved that ALP activity of MTA at 3 d (0.217 ± 0.008), 5 d (0.253 ± 0.005) , 7 d (0.279 ± 0.004) were more than that of the control group at 3 d (0.166 ± 0.006) ,5 d (0.221 ± 0.006), 7 d (0.242 ± 0.004) (P < 0.01). Collagen I detection showed that the level of collagen I of MTA at 3 d[(78.46 ± 2.72) µg/L], 5 d[(90.73 ± 3.08) µg/L] were more than that of the control group at 3 d[ (66.75 ± 3.08) µg/L], [5 d (74.27 ± 3.50) µg/L] (P < 0.01). MTA of high concentrations can significantly inhibit cell growth, and of low concentrations can promote cells proliferation and differentiation.