Mineralization Of Dental Pulp Stem Cells Research Articles

Wnt/β-catenin plays a dual function in calcium hydroxide induced proliferation, migration, osteogenic differentiation and mineralization in vitro human dental pulp stem cells.

Calcium hydroxide is the gold standard material for pulp capping and has been widely used in clinical dentistry. Calcium hydroxide promotes proliferation, migration and osteogenic differentiation of dental pulp stem cells (DPSCs). However, the underlying mechanism is not clear. Our study investigated the role of Wnt/β-catenin pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation and mineralization of human DPSCs. Protein and gene expression was detected by western blot (WB), immunofluorescence staining and quantitative real-time PCR (qPCR). Cell viability was analysed using the Cell Counting Kit-8 (CCK-8) assay. Wound-healing assay was used to analyse cell migration. The expression of alkaline phosphatase (ALP) was detected using ALP staining. Mineralization was analysed by alizarin red staining. Calcium hydroxide increased the protein expression of phosphorylated-GSK3β/GSK3β, β-catenin and the gene expression of LEF-1. Inhibition of Wnt/β-catenin abolished calcium hydroxide-induced proliferation and migration of DPSCs in 24 h. However, incubation with calcium hydroxide for 7 days and 14 days reduced Wnt/β-catenin signalling. Inhibition of Wnt/β-catenin promoted calcium hydroxide-induced osteogenic differentiation and mineralization in DPSCs. Wnt/β-catenin pathway plays a dual role in calcium hydroxide-regulated DPSC behaviour. Incubation with calcium hydroxide promoted rapid proliferation and migration of DPSCs, while prolonged incubation negatively regulated osteogenic differentiation and mineralization.

The Antimicrobial, Antioxidative, and Anti-Inflammatory Effects of Polycaprolactone/Gelatin Scaffolds Containing Chrysin for Regenerative Endodontic Purposes.

The appropriate endodontic material should eliminate the infection and inflammation to provide a situation for regeneration and healing of pulp tissue besides biomineralization. Chrysin is one of the active ingredients of plant flavonoids, which has significant anti-inflammatory and antimicrobial properties. In the present study, this natural substance was evaluated for antioxidant, anti-inflammatory, and mineralization properties on dental pulp stem cells (DPSCs). SEM, FTIR, and TGA tests were used to determine the successful synthesize of chrysin-loaded scaffolds. The antimicrobial effects of the synthesized scaffold against Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis were assessed by the agar diffusion test and live/dead assay. The proliferation of DPSCs on these scaffolds was determined by the MTT assay, DAPI staining, and DNA extraction. Moreover, the antioxidant and anti-inflammation activity of chrysin-loaded scaffolds on inflamed DPSCs was evaluated. Alkaline phosphatase activity and Alizarin Red S Stain tests were done to evaluate the mineralization of DPSCs seeded on these scaffolds. The chrysin-loaded scaffolds reported antimicrobial effects against evaluated bacterial strains. The proliferation of DPSCs seeded on these scaffolds was increased significantly (p < 0.05). The TNFα and DCF levels in inflamed DPSCs showed a significant decrease in the presence of chrysin-loaded scaffolds (p < 0.05). The ALP activity and formation of mineralized nodules of DPSCs on these scaffolds were significantly increased compared with the control group (p < 0.05). These results indicated that chrysin as an ancient therapeutic agent can accelerate the healing and regeneration of damaged pulp tissue, and this active ingredient can be a potential natural substance for regenerative endodontic procedures.

Lipoteichoic acid stimulates the proliferation, migration and cytokine production of adult dental pulp stem cells without affecting osteogenic differentiation.

To model in vitro the contact between adult dental pulp stem cells (DPSCs) and lipoteichoic acid (LTA), a cell wall component expressed at the surface of most Gram-positive bacteria. Human DPSCs obtained from impacted third molars were cultured and exposed to various concentrations of S.aureus LTA (0.1, 1.0 and 10µgmL-1 ). The effects of LTA on DPSCs proliferation and apoptosis were investigated by MTT assay and flow cytometry. Mineralization of DPSCs was evaluated by alizarin red staining assay. Migration was investigated by microphotographs of wound-healing and Transwell migration assays. Reverse transcription polymerase chain reaction was used to examine the effects of LTA on p65 NF-κB translocation and TLR1, TLR2 or TLR6 regulation. Enzyme-linked immunosorbent assay was used to investigate LTA-stimulated DPSCs cytokine production. One-way or two-way ANOVA and Tukey post hoc multiple comparison were used for statistical analysis. DPSCs expressed TLR1, TLR2 and TLR6 involved in the recognition of various forms of LTA or lipoproteins. Exposure to LTA did not up- or down-regulate the mRNAs of TLR1, TLR2 or TLR6 whilst LPS acted as a potent inducer of them [TLR1 (P≤0.05), TLR2 (P≤0.001) and TLR6 (P≤0.001)]. Translocation of p65 NF-κB to the nucleus was detected in LTA-stimulated cells, but to a lesser extent than LPS-stimulated DPSCs (P≤0.001). The viability of cells exposed to LTA was greater than unstimulated cells, which was attributed to an increased proliferation and not to less cell death [LTA 1μgmL-1 (P≤0.001) and 10μgmL-1 (P≤0.01)]. For specific doses of LTA (1.0µgmL-1 ), adhesion of DPSCs to collagen matrix was disturbed (P≤0.05) and cells enhanced their horizontal mobility (P≤0.001). LTA-stimulated DPSCs released IL-6 and IL-8 in a dose-dependent manner (P≤0.0001). At all concentrations investigated, LTA did not influence osteogenic/odontoblastic differentiation. Human DPSCs were able to sense the wall components of Gram-positive bacteria likely through TLR2 signalling. Consequently, cells modestly proliferated, increased their migratory behaviour and contributed significantly to the local inflammatory response through cytokine release.

SFRP2 enhances dental pulp stem cell-mediated dentin regeneration in rabbit jaw.

Dental tissue-derived mesenchymal stem cell (MSC)-mediated tooth regeneration may be a useful therapeutic tool for repairing tooth loss. However, the low success rate of tooth regeneration restricts its clinical application. Identifying key factors for enhancing dentinogenesis in MSCs is crucial for promoting tooth regeneration. Human dental pulp stem cells (DPSCs) were transfected with retrovirus to obtain SFRP2-over-expressing DPSCs. Alkaline phosphatase (ALP) activity assay, Alizarin red staining, quantitative analysis of calcium, and dentinogenesis-related genes were detected. Additionally, transplantation in a rabbit tooth extraction model was used to explore the role of SFRP2 in dentin regeneration. We found SFRP2 over-expression greatly enhanced ALP activity, and mineralization in DPSCs. Real-time RT-PCR revealed SFRP2 over-expression promoted the expressions of OSX, RUNX2, DSPP, DMP1, and BSP. Moreover, Micro CT analysis showed high-density calcification occurred to a much higher extent in SFRP2 over-expressing group compared to control group in vivo. Additionally, HE staining, immmunohistochemistry staining, and scanning electron microscopy results showed much more dentin-like tissue formed in SFRP2 over-expressing group compared to control group. Our findings revealed SFRP2 is an important regulator that enhances the dentinogenesis of DPSCs and dentin regeneration in the jaw, which may have clinical applications.

Comparison of Osteogenic Potential of Phenytoin with Dexamethasone in Cultured Dental Pulp Stem Cells.

One of the adverse effects of phenytoin (diphenylhydantoin, DPH) is enlargement of facial features. Although there are some reports on anabolic action of phenytoin on bone cells, the osteogenic potential of DPH on mesenchymal stem cells has not been studied. The purpose of this study was to evaluate the osteogenic potential of DPH on dental pulp stem cells (DPSCs). Human DPSCs were isolated and characterized by flow cytometry; presence of CD29 and CD44 and absence of CD34 and CD45 were performed to confirm the mesenchymal stem cells. Isolated DPSCs were differentiated either in conventional osteogenic medium with Dexamethasone or medium containing different concentration of phenytoin (12.5, 25, 100, and 200 µM). The osteogenic differentiation evaluated by performing western blot test for Runt-related transcription factor 2 (RUNX2), osteopontin and alkaline phosphatase (ALP) also alizarin red S staining to measure the mineralization of cells. Our results showed morphological changes and mineralization of DPSCs by using DPH were comparable with dexamethasone. Moreover, western blot results of DPH group showed significant increase of ALP, RUNX2 and osteopontin (OSP) in comparison with control. The data of present study showed the osteogenic activity of phenytoin, considering as an alternative of dexamethasone for inducing osteogenic differentiation of dental pulp stem cells.

Ageing affects the proliferation and mineralization of rat dental pulp stem cells under inflammatory conditions.

To comparatively evaluate changes inthe proliferation and mineralization abilities of dental pulp stem cells (DPSCs) from juvenile and adult rats in a lipopolysaccharide (LPS)-induced inflammatory microenvironment to provide a theoretical basis for the age-related differences observed in DPSCs during repair of inflammatory injuries. DPSCs were isolated from juvenile (JDPSCs) and adult rats (ADPSCs), and senescence-associated β-galactosidase staining was used to compare senescence between JDPSCs and ADPSCs. Effects of LPS on JDPSCs and ADPSCs proliferation were investigated by cell counting kit-8 assays and flow cytometry. Alizarin red staining, quantitative reverse transcription polymerase chain reaction and Western blot assay were used to examine the effects of LPS on mineralization-related genes and proteins in JDPSCs and ADPSCs. Immunohistochemistry was used to compare interleukin-1β (IL-1β) and osteocalcin (OCN) expression in the pulpitis model. Unpaired Student's t-tests and one-way anova were used for statistical analysis. DPSCs were isolated from juvenile and adult rat dental pulp tissues. At low concentrations (0.1-1μgmL-1 ), LPS significantly promoted the proliferation of JDPSCs (P<0.01) and ADPSCs (P<0.01 or P<0.05), with the effect being stronger in JDPSCs than in ADPSCs. In addition, mineralized nodules and the expression of mineralization-related genes (OCN, DSPP, ALP, BSP) increased significantly after stimulation with LPS (0.5μgmL-1 ) in JDPSCs and ADPSCs (P<0.01 or P<0.05), and JDPSCs displayed a more obvious increase than ADPSCs. Western blots revealed OCN and ALP expression levels in JDPSCs treated with LPS were significantly upregulated (P<0.05); meanwhile, ALP expression in ADPSCs increased slightly but significantly (P<0.05), and OCN expression was not affected. Finally, IL-1β expression was significantly higher (P<0.05) and OCN expression was significantly lower (P<0.05) in the inflamed dental pulp of adult rats than in juvenile rats. A certain degree of inflammatory stimulation promoted the proliferation and mineralization of DPSCs; however, this effect declined with age. The DPSCs of adult donors in an inflammatory microenvironment have a weaker repair ability than that of juvenile donors, who are better candidates for tissues damage repair.

Combination of mineral trioxide aggregate and propolis promotes odontoblastic differentiation of human dental pulp stem cells through ERK signaling pathway

The aim of this study is to investigate combined effects of mineral trioxide aggregate (MTA) and propolis on odontoblastic differentiation of human dental pulp stem cells (DPSCs) and to find a signaling pathway involved. Combination of MTA and propolis significantly up-regulated the expression of DSPP and DMP1, and facilitated a mineral nodule formation (p < 0.05). Treatments with MTA, propolis or combined increased the phosphorylation of extracellular signal-regulated kinases (ERK), one of mitogen-activated protein kinases signaling cascades during odontogenic differentiation of DPSCs (p < 0.05), and U0126, an inhibitor of ERK, decreased calcium deposits (p < 0.05). Combination of MTA and propolis promotes odontogenic differentiation and mineralization of DPSCs through ERK pathway.

The Forkhead Box C1, a Novel Negative Regulator of Osteogenesis, Plays a Crucial Role in Odontogenic Differentiation of Dental Pulp Stem Cells.

The forkhead box C1 (Foxc1) protein, a member of the forkhead/winged helix transcription factor family, is required in stem cell developmental processes. Recently, multiple studies have indicated the crucial role of Foxc1 in mesenchymal stem cell differentiation, but the precise effects and mechanisms on dental pulp stem cells (DPSCs) remain unclear. In this study, we evaluate the role of Foxc1 on the odontogenic differentiation and proliferation of DPSCs. Our results show that Foxc1 decreases time dependently in odontogenic differentiation of DPSCs. Meanwhile, overexpression of Foxc1 could significantly inhibit the mineralization of DPSCs and the expression of odontogenic-related genes, such as runt-related transcription factor 2 (Runx2), dentin sialophosphoprote (DSPP), and dentin matrix acidic phosphoprotein 1 (DMP-1). Foxc1 overexpression does not significantly alter the proliferation of DPSCs. In addition, Foxc1 reduces the expression of p-Smad1/5, an important modulator of bone morphogenetic protein (BMP)/Smad signaling pathway, inhibiting BMP/Smad signaling pathway. In conclusion, our data demonstrated that Foxc1 inhibits odontogenic differentiation of DPSCs and odontogenic-related gene expression through the BMP/Smad signaling pathway which may be useful for the dental regeneration and repair.

Static magnetic field regulates proliferation, migration, differentiation and YAP/TAZ activation of human dental pulp stem cells

The dental pulp stem cells (DPSCs) are a population of mesenchymal stem cells, which have multilineage potential and high proliferation. DPSCs are regarded as a promising tool for tissue regeneration of dentine, dental pulp, bone, cartilage, and muscle. Recently, magnetic materials have become commonly applied in dental clinics. Static magnetic field has been reported to regulate the proliferation, migration, or differentiation of stem cells. However, whether static magnetic fields affect DPSCs is still unknown. In our study, we investigated the effect of static magnetic field on the proliferation, migration, and differentiation of DPSCs. The results indicated that static magnetic field rearranged the cytoskeleton of DPSCs. A static magnetic field of 1mT increased DPSC proliferation, as well as the gene expression of several growth factors such as FGF-2, TGF-β, and VEGF. Moreover, the static magnetic field promoted the migration of DPSCs by regulating MMP-1 and MMP-2 gene expression. Static magnetic field of 1mT also induced osteo/odontogenesis and mineralization in DPSCs. Otherwise, the static magnetic field recruited YAP/TAZ to the nucleus, inhibited the phosphorylation of YAP/TAZ, and upregulated the two YAP/TAZ-regulated genes, CTGF and ANKRD1. Cytoskeleton inhibitor, cytochalasin D, obviously inhibited the nuclear localization of YAP/TAZ. When YAP/TAZ were knocked-down, the static magnetic field-induced mineralization of DPSCs was diminished. Our findings provide an insight into the effect of static magnetic field on DPSCs and provide the foundation for the future tissue regeneration.

Bone morphogenetic protein 7 promotes odontogenic differentiation of dental pulp stem cells in vitro

Aimsin vitro effects of bone morphogenetic protein 7 (BMP-7) on proliferation and differentiation of dental pulp stem cells (DPSCs) have not been investigated, nor has an appropriate dose been established. Main methodsHuman DPSCs obtained from healthy volunteers were cultured with BMP-7 at 25, 50, and 100 ng/ml. Cell viability was measured by Cell Counting Kit-8 assay. Expression profiles of selected odontogenic differentiation-related markers in DPSCs were evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunocytochemistry, and western blot analysis. Mineralization of DPSCs was evaluated by alizarin red staining. The Smad5 signaling pathway was examined by qRT-PCR and western blot analysis. Key findingsDiminished cell viability was found in DPSCs induced with 25, 50, and 100 ng/ml of BMP-7 for 7 days, showing a dose-response effect (P-trend = 0.03). DSPP, OCN, DMP-1, and RUNX2 were upregulated by BMP-7 induction after 7 and 14 days, especially at 50 and 100 ng/ml (P < 0.05). Immunocytochemical staining revealed strong expression of DSPP, DMP-1 and ALP in DPSCs induced by BMP-7, whereas null or weak expression in untreated cells. Western blot analysis confirmed over-expression of DSPP in cells induced by BMP-7. Alizarin red staining confirmed formation of mineralized nodules 4 weeks after BMP-7 induction. BMP-7 treated cells showed dose-dependently increased expression of BMPR1A, Smad5, and p-Smad5. SignificanceOur data indicated that BMP-7 at 50 ng/ml and 100 ng/ml was capable to induce DPSCs toward odontogenic differentiation through the Smad5 signaling pathway and not dramatically halt cell proliferation in vitro.

NRP1 Accelerates Odontoblast Differentiation of Dental Pulp Stem Cells Through Classical Wnt/β-Catenin Signaling.

Neuropilin-1 (NRP1) is one of the members of neuropilin family. It can combine with disparate ligands involved in regulating cell proliferation, apoptosis, and differentiation. The binding of NRP1 to Sema3A stimulates osteoblast differentiation through the classical Wnt/β-catenin pathway. However, the functions of NRP1 in dental pulp stem cells (DPSCs) are not clear. The aim of our study was to investigate how NRP1 controlled odontoblast differentiation in DPSCs and clarified the underlying mechanisms. NRP1 expression was increased in time-dependent manner along with cell odontoblast differentiation. Overexpression of NRP1 upregulated dentin matrix protein-1, dentin sialophosphoprotein, alkaline phosphatase protein level, and mineralization in DPSCs, while knockdown of NRP1 induced the opposite effects. SiNRP1 similar to DKK1 availably blocked classical Wnt/β-catenin signaling and odontoblast differentiation. In summary, NRP1, as a promoter of odontoblast differentiation, regulates DPSCs via the classical Wnt/β-catenin pathway.

TNF-α was involved in calcium hydroxide-promoted osteogenic differentiation of human DPSCs through NF-κB/p38MAPK/Wnt pathway.

It has been reported that calcium hydroxide can induce proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying molecular mechanisms are still unclear. In this study, we sought to explore the role of calcium hydroxide in the cell proliferation and directional differentiation of DPSCs and to study the regulatory effect of NF-κB, p38MAPK, and Wnt signaling on differentiation of DPSCs. CCK8 cell assay, Wound Healing Assay, and Alkaline Phosphatase Staining Assay were respectively used to determine the proliferation rate, migration and ALP expression of DPSCs. Alizarin Red Staining Assay was used to observe the mineralization of DPSCs. RT-PCR analysis and Western Blot Analysis displayed the expression of related fators at mRNA and protein level, respectively. In the present study, we found that NF-κB, p38MAPK, and Wnt signaling could abolish calcium hydroxide-induced proliferation of DPSCs. The inhibition of NF-κB, p38MAPK, and Wnt signaling suppressed the migration, ALP expression, and mineralization of DPSCs. NF-κB, p38MAPK, and Wnt signaling involved in directional differentiation of DPSCs. Moverover, calcium hydroxide could activate NF-κB, p38MAPK, and Wnt pathway by regulating TNF-α. Our study showed that NF-κB, p38MAPK, and Wnt signaling pathway were involved in calcium hydroxide-induced proliferation, migration, mineralization, and osteogenic differentiation in DPSCs. Calcium hydroxide affected NF-κB, p38MAPK, and Wnt pathway by regulating TNF-α.

Deferoxamine-Induced Migration and Odontoblast Differentiation via ROS-Dependent Autophagy in Dental Pulp Stem Cells

Background/Aims: As a vital degradation and recycling system, autophagy plays an essential role in regulating the differentiation of stem cells. We previously showed that iron chelator deferoxamine (DFO) could promote the repair ability of dental pulp stem cells (DPSCs). Here, we investigated the effect of DFO in autophagy and the role of autophagy in regulating the migration and odontoblast differentiation of DPSCs. Methods: Transmission electron microscopy, immunofluorescence staining and western blotting were performed to evaluate the autophagic activity of DPSCs. Transmigration assay, alkaline phosphatase staining/activity, alizarin red S staining and quantitative PCR were performed to examine the migration and odontoblast differentiation of DPSCs. Reactive oxygen species (ROS) levels and the effects of ROS scavenger in autophagy induction were also detected. Autophagy inhibitors (3-MA and bafilomycin A1) and lentiviral vectors carrying ATG5 shRNA sequences were used for autophagy inhibition. Results: Early exposure to DFO promoted the mineralization of DPSCs and increased autophagic activity. Autophagy inhibition suppressed DFO-induced DPSC migration and odontoblast differentiation. Furthermore, DFO treatment could induce autophagy partly through hypoxia-inducible factor 1α/B cell lymphoma 2/adenovirus E1B 19K-interacting protein 3 (HIF-1α/BNIP3) pathway in a ROS-dependent manner. Conclusion: DFO increased DPSC migration and differentiation, which might be modulated through ROS-induced autophagy.

MEF2 Transcription Factor Regulates Osteogenic Differentiation of Dental Pulp Stem Cells

The myocyte enhancer factor-2 (MEF2) is a member of the MADS-box family. It controls the expression of genes that are critical for biological processes such as proliferation, cell death, and differentiation. Some studies have shown that MEF2 expression is enhanced in osteogenic progenitor cells established from bone marrow stromal cells with other types of mesenchymal progenitor cells. However, the effect of MEF2 on dental pulp stem cells (DPSCs) is unclear. In this study, we investigate the effect of MEF2 on regulating osteogenic differentiation and proliferation of DPSCs. We find that MEF2 is stably expressed in DPSCs, and the expression is increased time-dependently along with cell osteogenic differentiation. MEF2 expression also increases the alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) activity, and enhances mineralization in DPSCs. SB202190, inhibitor of p38, blocks the p38/MEF2 pathway and osteogenic differentiation. In addition, MEF2 overexpression inhibits DPSC proliferation. In summary, our data indicate that MEF2 not only regulates DPSCs as an inhibitor of cell proliferation but is also a promoter of osteogenic differentiation through the p38/MEF2 signaling pathway.

Calcium Hydroxide–induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells

IntroductionCalcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide–induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. MethodsHuman DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. ResultsCalcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal–regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide–induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal–regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. ConclusionsOur study showed that the MAP kinase pathway was involved in calcium hydroxide–induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs.

The Role of ORAI1 in the Odontogenic Differentiation of Human Dental Pulp Stem Cells

Pulp capping, or placing dental materials directly onto the vital pulp tissues of affected teeth, is a dental procedure that aims to regenerate reparative dentin. Several pulp capping materials are clinically being used, and calcium ion (Ca2+) released from these materials is known to mediate reparative dentin formation. ORAI1 is an essential pore subunit of store-operated Ca2+ entry (SOCE), which is a major Ca2+ influx pathway in most nonexcitable cells. Here, we evaluated the role of ORAI1 in mediating the odontogenic differentiation and mineralization of dental pulp stem cells (DPSCs). During the odontogenic differentiation of DPSCs, the expression of ORAI1 increased in a time-dependent manner. DPSCs knocked down with ORAI1 shRNA (DPSC/ORAI1sh) or overexpressed with dominant negative mutant ORAI1E106Q (DPSC/E106Q) exhibited the inhibition of Ca2+ influx and suppression of odontogenic differentiation and mineralization as demonstrated by alkaline phosphatase (ALP) activity/staining as well as alizarin red S staining when compared with DPSCs of their respective control groups (DPSC/CTLsh and DPSC/CTL). The gene expression for odontogenic differentiation markers such as osteocalcin, bone sialoprotein, and dentin matrix protein 1 (DMP1) was also suppressed. When DPSC/CTL or DPSC/E106Q cells were subcutaneously transplanted into nude mice, DPSC/CTL cells induced mineralized tissue formation with significant increases in ALP and DMP1 staining in vivo, whereas DPSC/E106Q cells did not. Collectively, our data showed that ORAI1 plays critical roles in the odontogenic differentiation and mineralization of DPSCs by regulating Ca2+ influx and that ORAI1 may be a therapeutic target to enhance reparative dentin formation.

Proteomic analysis of mesenchymal stem cells from normal and deep carious dental pulp.

Dental pulp stem cells (DPSCs), precursor cells of odontoblasts, are ideal seed cells for tooth tissue engineering and regeneration. Our previous study has demonstrated that stem cells exist in dental pulp with deep caries and are called carious dental pulp stem cells (CDPSCs). The results indicated that CDPSCs had a higher proliferative and stronger osteogenic differentiation potential than DPSCs. However, the molecular mechanisms responsible for the biological differences between DPSCs and CDPSCs are poorly understood. The aim of this study was to define the molecular features of DPSCs and CDPSCs by comparing the proteomic profiles using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Our results revealed that there were 18 protein spots differentially expressed between DPSCs and CDPSCs in a narrow pH range of 4 to 7. These differently expressed proteins are mostly involved in the regulation of cell proliferation, differentiation, cell cytoskeleton and motility. In addition, our results suggested that CDPSCs had a higher expression of antioxidative proteins that might protect CDPSCs from oxidative stress. This study explores some potential proteins responsible for the biological differences between DPSCs and CDPSCs and expands our understanding on the molecular mechanisms of mineralization of DPSCs in the formation of the dentin-pulp complex.

In vitro differentiation and mineralization of dental pulp stem cells on enamel-like fluorapatite surfaces.

Our previous studies have shown good biocompatibility of fluorapatite (FA) crystal surfaces in providing a favorable environment for functional cell-matrix interactions of human dental pulp stem cells (DPSCs) and also in supporting their long-term growth. The aim of the current study was to further investigate whether this enamel-like surface can support the differentiation and mineralization of DPSCs, and, therefore, act as a potential model for studying the enamel/dentin interface and, perhaps, dentine/pulp regeneration in tooth tissue engineering. The human pathway-focused osteogenesis polymerase chain reaction (PCR) array demonstrated that the expression of osteogenesis-related genes of human DPSCs was increased on FA surfaces compared with that on etched stainless steel (SSE). Consistent with the PCR array, FA promoted mineralization compared with the SSE surface with or without the addition of a mineralization promoting supplement (MS). This was confirmed by alkaline phosphatase (ALP) staining, Alizarin red staining, and tetracycline staining for mineral formation. In conclusion, FA crystal surfaces, especially ordered (OR) FA surfaces, which mimicked the physical architecture of enamel, provided a favorable extracellular matrix microenvironment for the cells. This resulted in the differentiation of human DPSCs and mineralized tissue formation, and, thus, demonstrated that it may be a promising biomimetic model for dentin-pulp tissue engineering.

Retracted: Pro‐inflammatory cytokines induce odontogenic differentiation of dental pulp‐derived stem cells

Postnatal dental pulp stem cells (DPSCs) represent a unique precursor population in the dental pulp, which have multipotential and harbor great potential for tissue engineering purposes. However, for therapy applications, transplanted cells are often exposed to unfavorable conditions such as cytokines released from necrotic or inflammatory cells in injured tissues. It is not clear how stem cells exposed to these conditions changes in their characteristics. In this study, the effects of pro-inflammatory cytokines, such as IL-1 and TNF, on DPSCs were investigated. Cells were treated with IL-1, TNF, or both for 3, 7, and 12 days. The cultures were evaluated for cell proliferation, ALP activity, and real-time PCR. We found that a short treatment (3 days) of pro-inflammatory cytokines induced the odontogenic differentiation of DPSCs. Furthermore, post 3 days treatment with pro-inflammatory cytokines, the cell-scaffold complexes were implanted subcutaneously in mice for 8 weeks. Histological analysis demonstrated that the cultures gave obviously mineralized tissue formation, especially for both IL-1 and TNF applied. These data suggest that IL-1 and TNF produced in the early inflammatory reaction may induce the mineralization of DPSCs.

Effects of interleukin-1β on mineralization potential of dental pulp stem cells

To investigate the effects of pro-inflammatory cytokine interleukin-1β (IL-1β) on mineralization potential of dental pulp stem cells (DPSC). Rat DPSC were cultured in vitro and randomly divided into three groups, IL-1β (10 µg/L), osteogenic inductive medium and non-osteogenic inductive medium. After 3, 7, and 12 days of treatment, the cultures were evaluated for cell proliferation and calcium deposit. Real-time polymerase chain reaction was used to detect the gene expression levels of osteocalcin (OC), bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP-1). In vivo test, after 3 day's treatment with IL-1β, the cell-scaffold complexes were implanted subcutaneously in mice for 8 weeks. Histological analysis was performed to evaluate hard tissue formation. In vitro test, after 3-day's treatment, IL-1β improved cell proliferation to 137.22 DNA µg/L and cell viability becomes (97.12 ± 7.18)% of control. The gene expression levels of OC, BSP, DSPP and DMP-1 are (378.19 ± 16.22)%, (427.12 ± 18.22)%, (247.19 ± 10.11)% and (198.29 ± 10.23)% respectively. The results of IL-1β's group was notable increased compared with non-osteogenic induction medium and the statistical differences are significant. IL-1β induced the odontogenic differentiation of DPSC. However, these effects tended to continuously decrease with treatment time. Histological analysis demonstrated that in the group treated with IL-1β hard tissue was markedly formed in vivo. IL-1β may induce the mineralization of DPSC and play an important role in host defenses and tissue repair.