Odontogenic Markers Research Articles

The Enhancing Effects of Amelogenin Exon 5-Encoded Peptide from Enamel Matrix Derivative on Odontoblast-Like KN-3 Cells

Enamel matrix derivative (EMD) is applied for periodontal therapy. We created a synthetic amelogenin peptide (SP) derived from EMD, and have previously investigated the biological function of SP. However, it is unknown whether SP affects odontoblastic differentiation. In this study, we tested the effects of SP in the odontoblast-like cells, KN-3 cells. KN-3 cells were cultured with SP (0 to 1000 ng/mL) and then cultured for 3, 8, 24, or 48 h in order to determine the effects of SP on cell proliferation and detect its optimum concentration. KN-3 cells were treated with SP in odontogenic differentiation medium cultured for 3 or 7 days. Odontogenic markers were measured by the detection of alkaline phosphatase (ALP) activity and dentin sialo phosphoprotein (DSPP) expression, the calcified nodule formation, and calcium deposition. The addition of SP significantly promoted cell proliferation at 100 ng/mL, generating the greatest change in cell proliferation. SP also showed increased odontogenic expression markers and mineralization. These results suggest that SP, derived from EMD, could have potential for application in dental pulp capping.

Effects of silicate-based composite material on the proliferation and mineralization behaviors of human dental pulp cells: An in vitro assessment.

The objective of this study was to investigate the effects of a silicate-based composite material on proliferation and mineralization of human dental pulp cells (hDPCs), which was compared with those of calcium hydroxide (Ca(OH)2, CH) and tricalcium silicate (Ca3SiO5, C3S). HDPCs were cultured with CH, C3S and tricalcium silicate/dicalcium silicate (Ca3SiO5/Ca2SiO4, C3S/C2S) composites extract. The CCK-8 assay showed that the composite material stimulated the proliferation of hDPCs. The odontogenic marker genes and DSPP protein expression were more significantly up-regulated by the C3S/C2S composite material compared with pure CH and C3S. HDPCs cultured with composite material extract exert stronger ALP activity and alizarin red S staining. C3S/C2S composite material was advantageous over pure C3S by showing enhanced ability to stimulate the proliferation and odontogenic differentiation of hDPCs, suggesting that the C3S/C2S composite materials possess desirable biocompatibility and bioactivity, and might be a new type of pulp-capping agent and dentin alternative materials.

Poly(Adenosine Phosphate Ribose) Polymerase 1 Inhibition Enhances Brain-derived Neurotrophic Factor Secretion in Dental Pulp Stem Cell–derived Odontoblastlike Cells

IntroductionThe nuclear enzyme poly(adenosine phosphate ribose) polymerase 1 (PARP-1) has been implicated in the maintenance and differentiation of several stem cells. The role of PARP-1 in dental pulp stem cell (DPSC) differentiation, especially in the context of its ability to modulate nerve regeneration factors, has not been investigated. Regeneration of neuronal components in pulp tissue is important for the assessment of tooth vitality. Brain-derived neurotrophic factor (BDNF) is known to play an integral signaling factor during nerve regeneration. In this study, we identified the role of PARP-1 in the modulation of BDNF in DPSC differentiation into odontoblastlike cells. MethodsHuman DPSCs were prepared from healthy molars and cultured in regular and osteogenic media treated with PARP-1 antagonist and PARP-1 exogeneous protein. Polymerase chain reaction and immunohistochemistry analysis for BDNF and various differentiation markers were performed. ResultsOur polymerase chain reaction results showed that differentiated cells show odontoblastlike properties because they express odontogenic markers such as dentin sialophosphoprotein and dentin matrix protein 1. Both PARP-1 inhibitor and protein did not affect odontogenic differentiation and proliferation because the number of the differentiated cells was unaffected, and the expression of dentin sialophosphoprotein and dentin matrix protein 1 was not significantly changed. There is the possibility that PARP-1 treatment induces DPSCs into the unique cell lineage. Some differentiated cells show a very unique morphology with large irregular cytoplasm and an oval nucleus. Moreover, PARP-1 inhibition significantly increased BDNF secretion in DPSC-derived odontoblastlike cells. This observation was also confirmed by immunohistochemistry. ConclusionsTaken together, our results indicate PARP-1 as a negative regulator in BDNF secretion during odontogenic DPSC differentiation, showing its potential application for translational nerve regeneration strategies to improve dental pulp tissue vitality assessments.

Odontogenic Potential of Parathyroid Hormone–related Protein (107-111) Alone or in Combination with Mineral Trioxide Aggregate in Human Dental Pulp Cells

IntroductionParathyroid hormone–related protein plays an important role in bone remodeling. Its N-terminal domain parathyroid hormone–related protein (107-111) is called osteostatin (OST). OST has demonstrated osteogenic potential when combined with biomaterials such as hydroxyapatite or bioceramics. However, the odontogenic potential of OST has not yet been reported. Therefore, the aim of this study was to determine whether OST has an odontogenic effect or a synergistic effect with mineral trioxide aggregate (MTA) in human dental pulp cells (hDPCs) and to examine the underlying signaling mechanisms involved in OST-mediated odontogenic differentiation. MethodsViability of hDPCs on stimulation with OST or MTA was measured. Real-time polymerase chain reaction and Western blot analyses were performed to evaluate the expression levels of odontogenic markers and the activation of extracellular signal-regulated kinase (ERK). To evaluate mineralized nodule formation, alkaline phosphatase (ALP) staining and alizarin red S staining were performed. Combined effects of OST and MTA were evaluated. ResultsOST promoted odontogenic differentiation, as evidenced by the formation of mineralized nodules, induction of ALP activity, and upregulation of odontogenic markers (dentin sialophosphoprotein, dentin matrix protein-1, and ALP). Phosphorylation of ERK was increased by OST. However, ERK inhibitor (U0126) inhibited the increase in dentin sialophosphoprotein and dentin matrix protein-1 expression and mineralization induced by OST. A combination of MTA and OST upregulated odontogenic differentiation-associated gene expression and calcium nodule mineralization in hDPCs compared with MTA alone. ConclusionsThe present study revealed that OST can promote odontogenic differentiation and mineralization through activating the ERK signaling pathway. A combination of MTA and OST showed a synergistic effect compared with MTA alone in hDPCs.

The effect of chlormadinone acetate on odontogenic differentiation of human dental pulp cells: in vitro study

BackgroundChlormadinone acetate (CMA) is a derivative of progesterone and is used as an oral contraceptive. The aim of this study was to investigate the effects of CMA on odontogenic differentiation and mineralization of human dental pulp cells (hDPCs) and related signaling pathways.MethodsCell viability was determined by the water-soluble tetrazolium (WST)-1 assay. Odontogenic differentiation of hDPCs was evaluated by real-time polymerase chain reaction using odontogenic marker genes, such as alkaline phosphatase (ALP), osteocalcin (OCN), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1). Mineralization of hDPCs was evaluated by ALP staining and alizarin red staining. The extracellular signal-regulated kinase (ERK) pathway was examined by Western blot analysis.ResultsThere was no statistically significant difference in cell viability between the control and CMA-treated groups. Our analysis of odontogenic marker genes indicated that CMA enhanced the expression of those genes. CMA-treated hDPCs showed increased ALP activity and formation of mineralized nodules, compared with control-treated cells. In addition, CMA stimulation resulted in phosphorylation of ERK and resulted in inhibition of downstream molecules by the ERK inhibitor U0126.ConclusionsThese findings suggest that CMA improves odontogenic differentiation and mineralization of hDPCs through the ERK signaling pathway.

Zinc bioglasses regulate mineralization in human dental pulp stem cells

ObjectiveA promising strategy in regenerative endodontics is the combination of human dental pulp stem cells (hDPSCs) with an appropriate biomaterial substrate. The effects of zinc and zinc containing bioactive glasses (ZnBGs) on hDPSCs have been characterized in this study. MethodsZnBGs were designed and produced. Then the odontogenic differentiation and mineralization potential of hDPSCs upon ZnBGs treatment were investigated. ResultsFree Zn ions (0–5ppm) enhanced proliferation and alkaline phosphatase (ALP) activity of hDPSCs. Further, ZnBGs conditioned medium (ZnBG-CM) increased the production and secretion of odontogenic markers: dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1). In addition, we identified that mRNA expression of the osteogenic markers RUNX2, OCN, BSP, BMP-2, MEPE and ON was increased following treatment with ZnBG-CM. Long term treatment with ZnBG-CM increases the formation rate of mineralized nodules (similar to hydroxyapatite, Ca:P=1.6), as confirmed by scanning electron microscopy combined with energy dispersive X-ray spectroscopy (SEM-EDX). Lastly, the administration of ZnBG-CM induces VEGF expression. SignificanceThese findings implicate that ZnBG would be beneficial in regenerative endodontics and could influence the way present Zn containing clinical products are used.

Nephronectin Stimulates the Differentiation of MDPC-23 Cells into an Odontoblast-like Phenotype

IntroductionThe present study investigated the in vitro effects of nephronectin (Npnt) on the proliferation, differentiation, and mineralization of a rat odontoblast-like cell line (MDPC-23 cells). MethodsMDPC-23 cells were cultured on Npnt-coated polystyrene or in the presence of soluble Npnt. Cell proliferation was analyzed using a Cell Counting Kit-8 kit (Dojindo, Kumamoto, Japan). Alkaline phosphatase (ALP) activity was quantified using an ALP activity assay. A reverse-transcription polymerase chain reaction was performed to evaluate the messenger RNA (mRNA) expression level of odontogenic markers and integrin(s). Alizarin red staining was conducted to quantify the calcium deposition. ResultsSoluble Npnt had no adverse effect on the proliferation of MDPC-23 cells, but it exhibited concentration-dependent inhibitory activity toward differentiation. In contrast, coated Npnt promoted cell proliferation dramatically and significantly up-regulated the mRNA expression of odontogenesis-related genes; moreover, mRNA expression of integrin α1, α3, α5, β1, and β5 was found to be augmented. MDPC-23 cells cultured on Npnt-coated polystyrene displayed markedly higher ALP activity as early as day 3 after inoculation. In addition, mineralization was accelerated on Npnt-coated polystyrene. ConclusionsNpnt in its immobilized form enhanced the proliferation of MDPC-23 cells and induced this odontoblastic precursor cell line to differentiate into a mineralizing phenotype.

DLX3 mutation negatively regulates odontogenic differentiation of human dental pulp cells

ObjectivesThe purpose of this study was to investigate the role of a novel mutant DLX3 on the odontogenic differentiation of human dental pulp cells (hDPCs) in tricho-dento-osseous (TDO) syndrome. DesignhDPCs were obtained from the healthy premolars, stably-expressing wild-type DLX3 (WT), novel mutant DLX3 (Mu) and control vector (NC) cells were generated using recombinant lentiviruses. The proliferation rates of WT-hDPCs and Mu-hDPCs were measured by CCK8 assay. Odonto-differentiation of hDPCs was assessed by alkaline phosphatase (ALP) activity assay, and mineralization ability was assessed by Alizarin red staining. Odontogenic markers, including DMP-1, DSPP, Nes, ALP, and DLX5, were analyzed using real-time polymerase chain reaction (qPCR). DMP-1 and DSPP expressions were further confirmed by Western blotting. ResultsCCK8 results showed that the novel mutant DLX3 decreased the proliferation rate of hDPCs compared with wild-type DLX3. qPCR showed that the novel mutant DLX3 weakened odontogenic differentiation by downregulating the expression of odontogenic genes. These results were further confirmed by Western blotting and ALP activity assay. Additionally, Alizarin red staining showed that the novel mutant DLX3 decreased the mineralization of hDPCs compared with wild-type DLX3. ConclusionsNovel de novo mutation of DLX3 significantly decreases the proliferation rate and inhibits the odontogenic differentiation and mineralization of hDPCs, suggesting that this novel mutation of DLX3 can influence the dentinogenesis in TDO syndrome.

Expression of Odontogenic and Osteogenic Markers in DPSCs and SHED: A Review.

The odontogenic and osteogenic potential of dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous tooth (SHED) have been shown clearly by various in vitro and in vivo studies. The findings are promising and demonstrated that dental tissue engineering can give a new hope to the individuals suffering from tooth loss and dental diseases. The evaluation of odontogenic and osteogenic differentiation of DPSCs and SHED is commonly carried out by an illustration of the expression of varied related markers. In this review, few commonly used markers such as alkaline phosphatase (ALP), collagen type 1 (Col I), dentin matrix acid phosphoprotein 1 (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE), osteocalcin (OCN), and osteopontin (OPN). DSPP, DMP1, and MEPE (odontogenic markers), which play an important role during early odontoblastic differentiation and late dentin mineralization, have been highlighted. Osteoblastic proliferation and early/late osteoblastic differentiation can be assessed by estimating the expression of Col I, ALP, OCN and OPN. Despite that, till date, there is no marker which could demonstrate for certain, the differentiation of human DPSCs and SHED towards the odontogenic and osteogenic lineage. This review suggests that SHED are noticeably different from DPSCs and exhibited higher capacity for osteogenic differentiation compared to DPSCs. On the other hand, different expression levels are shown by SHED and DPSCs with regards to the osteoblast markers for osteoblastic differentiation, where, SHED expressed higher levels of ALP, Col I and OCN compared to DPSCs.

Odontogenic differentiation and biomineralization potential of dental pulp stem cells inside Mg-based bioceramic scaffolds under low-level laser treatment

This study aimed to investigate the potential of low-level laser irradiation (LLLI) to promote odontogenic differentiation and biomineralization by dental pulp stem cells (DPSCs) seeded inside bioceramic scaffolds. Mg-based, Zn-doped bioceramic scaffolds, synthesized by the sol-gel technique, were spotted with DPSCs and exposed to LLLI at 660nm with maximum output power of 140mw at fluencies (a) 2 and 4J/cm2 to evaluate cell viability/proliferation by the MTT assay and (b) 4J/cm2 to evaluate cell differentiation, using real-time PCR (expression of odontogenic markers) and a p-nitrophenylphosphate (pNPP)-based assay for alkaline phosphatase (ALP) activity measurement. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis were used for structural/chemical characterization of the regenerated tissues. Exposure of the DPSCs/scaffold complexes to the proposed LLLI scheme was associated with statistically significant increase of odontogenesis-related markers (bone morphogenetic protein 2 (BMP-2): 22.4-fold, dentin sialophosphoprotein (DSPP): 28.4-fold, Osterix: 18.5-fold, and Runt-related transcription factor 2 (Runx2): 3.4-fold). ALP activity was significantly increased at 3 and 7days inside the irradiated compared to that in the non-irradiated SC/DPSC complexes, but gradually decreased until 14days. Newly formed Ca-P tissue was formed on the SC/DPSC complexes after 28days of culture that attained the characteristics of bioapatite. Overall, LLLI treatment proved to be beneficial for odontogenic differentiation and biomineralization of DPSCs inside the bioceramic scaffolds, making this therapeutic modality promising for targeted dentin engineering.

Strontium (Sr) elicits odontogenic differentiation of human dental pulp stem cells (hDPSCs): A therapeutic role for Sr in dentine repair?

Strontium (Sr) forms a significant component of dental restorative materials and although it is widely used in toothpastes, the biological effects of Sr on the dentine-pulp complex have not been investigated. In this first study, we characterise the Sr elicited effects on human dental pulp stem cells (hDPSC) in vitro using exogenously Sr added to culture medium, and bioavailable Sr derived from a novel bioactive glass (BG). The related mechanisms were also investigated. Our results indicate that low dose Sr (between 0.1 and 2.5mM) induces proliferation and alkaline phosphatase (ALP) activity of hDPSCs, but has no effect on colony formation or cell migration. Sr at specific concentrations (1 and 2.5mM) stimulated collagen formation and mineralisation of the hDPSC generated matrix. In addition, qRT-PCR, Western blotting and immunocytochemistry revealed that Sr regulates gene expression and the protein secretion of the odontogenic markers: dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP-1) and protein localisation (DSPP was localised to the Golgi, while no apparent changes occur in DMP-1 distribution which remains in both cytosol and the nucleus). Additionally, the calcium sensing receptor (CaSR) and downstream pathway MAPK/ERK signalling pathway in hDPSCs were activated by Sr. Bioavailable Sr from the BG revealed novel biological insights of regulating metabolic and ALP activities in hDPSCs. Taken together, these results suggest that Sr at specific doses significantly influences proliferation, odontogenic differentiation and mineralisation of hDPSCs in vitro via the CaSR using a pathway with similarities to osteoblast differentiation. These are the first such studies and indicate that Sr treatment of hDPSCs could be a promising therapeutic agent in dental applications. In conclusion, we propose that Sr from a substituted BG could be used more effectively in biomaterials designed for dental applications. Statement of SignificanceDespite the fact that strontium (Sr) is used widely in dental practise, its potential effects on odontoblasts have been ignored. Our study provides the first evidence that Sr (exogenous and that derived from a bioglass (BG)) can stimulate dentinogenesis in human dental pulp stem cells (hDPSCs) by promoting their proliferation, differentiation and mineralisation in vitro. Therefore, while previously unrecognised, Sr BG is likely to be beneficial in atraumatic dentistry practise and maintenance of a competent tooth in conditions such as caries. Repair of defected dentine is still one of the main challenges in dental research and annually untreated caries results in the loss of productivity equivalent to US$ 27 billion. Advances in tissue engineering technology, alongside the use of dental pulp stem cells provide an approach to achieve dentine regeneration. Understanding the actions of Sr will permit a more controlled application of Sr in the clinic. These data are thus likely to be of great interest to the material scientists, biological researchers, clinicians and manufacturers of dental products.

Isolation, Characterization, and Differentiation of Dental Pulp Stem Cells in Ferrets

IntroductionThe ferret canine tooth has been introduced as a suitable model for studying dental pulp regeneration. The aim of this study was to isolate and characterize ferret dental pulp stem cells (fDPSCs) and their differentiation potential. MethodsDental pulp stem cells were isolated from freshly extracted ferret canine teeth. The cells were examined for the expression of stem cell markers STRO-1, CD90, CD105, and CD146. The osteo/odontogenic and adipogenic differentiation potential of fDPSCs was evaluated. Osteogenic and odontogenic marker genes were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) on days 1, 4, and 8 after osteo/odontogenic induction of fDPSCs including dentin sialophosphoprotein (DSPP), dentin matrix protein-1, osteopontin, and alkaline phosphatase. Human dental pulp cells were used as the control. The results were analyzed using 3-way analysis of variance. ResultsfDPSCs were positive for STRO1, CD90, and CD105 and negative for CD146 markers with immunohistochemistry. fDPSCs showed strong osteogenic and weak adipogenic potential. The overall expression of DSPP was not significantly different between fDPSCs and human dental pulp cells. The expression of DSPP in osteo/odontogenic media was significantly higher in fDPSCs on day 4 (P < .01). The overall expression of dentin matrix protein-1, osteopontin, and alkaline phosphatase was significantly higher in fDPSCs (P = .0005). ConclusionsfDPSCs were positive for several markers of dental pulp stem cells resembling human DPSCs and appeared to show a stronger potential to differentiate to osteoblastic rather than odontoblastic lineage.

In vitro osteogenic and odontogenic differentiation of human dental pulp stem cells seeded on carboxymethyl cellulose-hydroxyapatite hybrid hydrogel.

Stem cells from human dental pulp have been considered as an alternative source of adult stem cells in tissue engineering because of their potential to differentiate into multiple cell lineages. Recently, polysaccharide based hydrogels have become especially attractive as matrices for the repair and regeneration of a wide variety of tissues and organs. The incorporation of inorganic minerals as hydroxyapatite nanoparticles can modulate the performance of the scaffolds with potential applications in tissue engineering. The aim of this study was to verify the osteogenic and odontogenic differentiation of dental pulp stem cells (DPSCs) cultured on a carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. Human DPSCs were seeded on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel and on carboxymethyl cellulose hydrogel for 1, 3, 5, 7, 14, and 21 days. Cell viability assay and ultramorphological analysis were carried out to evaluate biocompatibility and cell adhesion. Real Time PCR was carried out to demonstrate the expression of osteogenic and odontogenic markers. Results showed a good adhesion and viability in cells cultured on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel, while a low adhesion and viability was observed in cells cultured on carboxymethyl cellulose hydrogel. Real Time PCR data demonstrated a temporal up-regulation of osteogenic and odontogenic markers in dental pulp stem cells cultured on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. In conclusion, our in vitro data confirms the ability of DPSCs to differentiate toward osteogenic and odontogenic lineages in presence of a carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. Taken together, our results provide evidence that DPSCs and carboxymethyl cellulose—hydroxyapatite hybrid hydrogel could be considered promising candidates for dental pulp complex and periodontal tissue engineering.

Magnetic Nanocomposite Scaffold-Induced Stimulation of Migration and Odontogenesis of Human Dental Pulp Cells through Integrin Signaling Pathways.

Magnetism is an intriguing physical cue that can alter the behaviors of a broad range of cells. Nanocomposite scaffolds that exhibit magnetic properties are thus considered useful 3D matrix for culture of cells and their fate control in repair and regeneration processes. Here we produced magnetic nanocomposite scaffolds made of magnetite nanoparticles (MNPs) and polycaprolactone (PCL), and the effects of the scaffolds on the adhesion, growth, migration and odontogenic differentiation of human dental pulp cells (HDPCs) were investigated. Furthermore, the associated signaling pathways were examined in order to elucidate the molecular mechanisms in the cellular events. The magnetic scaffolds incorporated with MNPs at varying concentrations (up to 10%wt) supported cellular adhesion and multiplication over 2 weeks, showing good viability. The cellular constructs in the nanocomposite scaffolds played significant roles in the stimulation of adhesion, migration and odontogenesis of HDPCs. Cells were shown to adhere to substantially higher number when affected by the magnetic scaffolds. Cell migration tested by in vitro wound closure model was significantly enhanced by the magnetic scaffolds. Furthermore, odontogenic differentiation of HDPCs, as assessed by the alkaline phosphatase activity, mRNA expressions of odontogenic markers (DMP-1, DSPP,osteocalcin, and ostepontin), and alizarin red staining, was significantly stimulated by the magnetic scaffolds. Signal transduction was analyzed by RT-PCR, Western blotting, and confocal microscopy. The magnetic scaffolds upregulated the integrin subunits (α1, α2, β1 and β3) and activated downstream pathways, such as FAK, paxillin, p38, ERK MAPK, and NF-κB. The current study reports for the first time the significant impact of magnetic scaffolds in stimulating HDPC behaviors, including cell migration and odontogenesis, implying the potential usefulness of the magnetic scaffolds for dentin-pulp tissue engineering.

치과용 임플란트 삽입을 위한 이종 이식골에서 보이는 치성상피의 이형성증식

A 57 years old female received xenogenic bone graft for the extraction socket augmentation of right maxillary molars and for the sinus floor elevation six months ago. The bone graft sites were healed uneventfully and showed marked radiopacity in the postoperative X-ray view. Before dental implant insertion the bone biopsy was made using trephine bur and examined pathologically. The graft bones showed minimum new bone deposition with dysplastic epithelium. The epithelium was proliferative on the surface of graft bones forming epithelial strands and nests, similar to the odontogenic epithelium. The immunohistochemical study was performed using different antisera of odontogenic markers, growth factors, oncogenes, etc. The epithelial cells were strongly positive for pan-keratins, EGF, pAKT, and HSP-70, consistently positive for PCNA, p53, EGFR, 14-3-3, and survivin, slightly positive for ameloblastin, but rarely positive for amelogenin. Particularly the matrix of graft bone was slightly positive for EGF. Taken together, it is presumed that the abnormal epithelium on the graft bones was derived from odontogenic epithelial elements, Malassez epithelial rests, distributed at the periodontal tissue of maxillary molars, and that they might undergo dysplastic proliferation affected by the release of growth factors and osteogenic proteins from the graft bones. It is also suggested that the graft bone substitutes inserted for the dental implant possibly have a potential to induce the proliferation of odontogenic epithelial rests leading to the pathogenesis of odontogenic cysts and tumors.

Effect of 5-Aza-2′-deoxycytidine on Odontogenic Differentiation of Human Dental Pulp Cells

IntroductionHuman dental pulp cells (hDPCs) comprise a heterogeneous cell population that possesses the capacity to differentiate into osteoblasts and plays an important role in reparative dentinogenesis. 5-Aza-2′-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, is known to be involved in cell differentiation. However, its role in the differentiation program of hDPCs remains unknown. The purpose of this study was to explore the role of 5-Aza-CdR in the regulation of odontogenic growth and differentiation of hDPCs. MethodshDPCs were treated with 1 μmol/L 5-Aza-CdR for 24 hours before being incubated in odontogenic medium for 2 weeks. To identify the effect of 5-Aza-CdR on proliferation and the odontogenic differentiation potential of hDPCs, the cell growth was measured using the Cell Counting Kit-8 assay (Dojindo, Kumamoto, Japan). The expression levels of the odontogenic markers, dentin sialophosphoprotein (DSPP), dentin matrix protein 1, (DMP1) and transcription factors, runt-related transcription factor 2 (RUNX2), distal-less homeobox 5 (DLX5), osterix (OSX) were analyzed. The activity of alkaline phosphatase was determined, and the formation of mineralized nodules was assessed using alizarin red S staining. ResultsAfter treatment with 5-Aza-CdR, the proliferation capacity of hDPCs was suppressed (n = 3, P < .05). 5-Aza-CdR up-regulated the expression of DSPP, DMP-1, OSX, RUNX2, and DLX5; increased the level of alkaline phosphatase activity; and accelerated the formation of calcified nodules (n = 3, P < .05). ConclusionsDNA methyltransferase inhibitor 5-Aza-CdR significantly inhibits the proliferation and enhances the capability of odontogenic differentiation of hDPCs, suggesting that DNA methylation may play an important role in reparative dentinogenesis.

Genipin, a Cross-linking Agent, Promotes Odontogenic Differentiation of Human Dental Pulp Cells

IntroductionThe aim of this study was to investigate the effects of genipin, a natural collagen cross-linking agent, on odontogenic differentiation of human dental pulp cells (hDPCs) because the mechanical properties of collagen allow it to serve as a scaffold for engineering of pulp-dentin complex. Furthermore, the role of extracellular signal–regulated kinase (ERK) was investigated as a mediator of the differentiation. MethodsThe odontogenic differentiation was analyzed by alkaline phosphatase activity, real time-polymerase chain reaction, Western blotting, and alizarin red S staining. The morphologic features of hDPCs cultured in genipin-treated collagen were evaluated by scanning electron microscopy. For the assessment of mechanical properties of collagen treated with genipin, the surface roughness and compressive strength were measured. ResultsAlkaline phosphatase activity, the expression of odontogenic markers, and mineralized nodule formation increased in the genipin-treated group. Genipin also activated ERK, and treatment with ERK inhibitor blocked the expression of the markers. The cells cultured in genipin-treated collagen spread across the substrate and attached in close proximity to one another. The proliferation and differentiation of hDPCs cultured in genipin-treated collagen were facilitated. The mechanical properties of collagen, such as surface roughness and compressive strength, were increased by treatment with genipin. ConclusionsOur results show that genipin promotes odontogenic differentiation of hDPCs via the ERK signaling pathway. Furthermore, the enhanced mechanical properties of the collagen scaffold induced by genipin may play important roles in cell fate. Consequently, the application of genipin might be a new strategy for dentin-pulp complex regeneration.

Effects of Interferon-γ on odontoblastic differentiation and mineralization of odontoblast-like cells

Introduction: Dentinogenesis is regulated by cytokines and growth factors, and modulated by alterations in the extracellular microenvironment. Dental matrix protein-1 (DMP-1), which is predominantly expressed in odontoblasts, is required during the early and late stages of odontogenesis. In the present study, we examined the involvement of proinflammatory cytokines in the expression of odontogenic markers in the rat odontoblast-like cell line KN-3. Methods: The expression of DMP-1 and p38 mitogen-activated protein kinase (MAPK) in proinflammatory cytokine-treated KN-3 cells was evaluated by immunoblot analysis. Alkaline phosphatase (ALP) activity in proinflammatory cytokine-treated KN-3 cells was measured by ALP staining and a colorimetric assay. Results: DMP-1 protein was downregulated in KN-3 cells treated with interferon-γ (IFN-γ) for 3 days, but not in interleukin-1β (IL-1β)-treated cells. The IFN-γ-induced downregulation of DMP-1 was rescued by treatment with IL-1β for 3 days. Interestingly, ALP activity was also suppressed in IFN-γtreated KN-3 cells and no significant change was induced by IL-1β treatment for 5 days. In addition, IFN-γ treatment for 3 days remarkably upregulated the phosphorylation of p38 MAPK in KN-3 cells. Conclusions: INF-γ treatment downregulated DMP-1 expression and ALP activity in KN-3 cells through prolonged phosphorylated p38 MAPK. IL-1β treatment restored these odontogenic reactions mediated by IFN-γ. Taken together, these findings suggest that IFN-γ and IL-β may be involved in the complex regulation of odontogenesis in the microenvironment of dental pulp. Rec.3/18/2015, Acc.5/24/2015, pp210-217

Iloprost Induces Tertiary Dentin Formation

IntroductionProstacyclin (PGI2), a member of the prostaglandin family, can promote angiogenesis and cell proliferation. MethodsIn this study, the effect of the application of a PGI2 analog (iloprost) on dentin repair was examined in vitro and in vivo. ResultsIloprost significantly stimulated the expression of vascular endothelial growth factor and osteo-/odontogenic marker messenger RNA in human dental pulp cells (HDPCs) under osteoinductive conditions in vitro. In addition, iloprost enhanced HDPC alkaline phosphatase enzymatic activity and mineral deposition. An in vivo study was performed using a rat molar mechanical pulp exposure model. After 30 days, histologic analysis revealed that there was a dramatic tertiary dentin formation in the iloprost-treated group compared with the calcium hydroxide and the untreated control groups. Furthermore, vascular endothelial growth factor protein expression in dental pulp tissue was increased in the iloprost-treated group as determined by immunohistochemical staining. ConclusionsTaken together, the present study, for the first time, shows that iloprost induces the expression of osteo-/odontogenic markers in vitro and promotes angiogenic factor expression and enhances tertiary dentin formation in vivo. This implies the potential clinical usefulness of iloprost in vital pulp therapy.

The Histone Acetyltransferase p300 Regulates the Expression of Pluripotency Factors and Odontogenic Differentiation of Human Dental Pulp Cells

p300 is a well-known histone acetyltransferase (HAT) and coactivator that plays vital roles in many physiological processes. Despite extensive research on the involvement of p300 in the regulation of transcription in numerous cell lines, the roles of this protein in regulating pluripotency genes and odontogenic differentiation in human dental pulp cells (HDPCs) are poorly understood. To address this issue, we investigated the expression of OCT4, NANOG and SOX2 and the proliferation and odontogenic differentiation capacity of HDPCs following p300 overexpression. We found that p300 overexpression did not overtly affect the ability of HDPCs to proliferate. The overexpression of p300 upregulated the promoter activity and the mRNA and protein expression of NANOG and SOX2. The HAT activity of p300 appeared to partially mediate the regulation of these factors; indeed, when a mutant form of p300 lacking the HAT domain was overexpressed, the promoter activity and expression of NANOG and SOX2 decreased relative to p300 overexpression but was greater than in the control. Furthermore, we demonstrated that the mRNA levels of the odontogenic marker genes dentine matrix protein-1 (DMP-1), dentin sialophosphoprotein (DSPP), dentin sialoprotein (DSP), osteopontin (OPN) and osteocalcin (OCN) were significantly decreased in HDPCs overexpressing p300 cultured under normal culture conditions and increased in HDPCs inducted to undergo odontogenic differentiation. This finding was further confirmed by measuring levels of alkaline phosphatase (ALP) activity and assessing the formation of mineralized nodules. The HAT activity of p300 had no significant effect on odontogenic differentiation. p300 was recruited to the promoter regions of OCN and DSPP and might be acting as a coactivator to increase the acetylation of lysine 9 of histone H3 of OCN and DSPP. Collectively, our results show that p300 plays an important role in regulating the expression of key pluripotency genes in HDPCs and modifying odontogenic differentiation.