Odontogenic Differentiation Of Dental Pulp Stem Cells Research Articles

Enhancing Dental Pulp Stem Cell Proliferation and Odontogenic Differentiation with Protein Phosphatase 1-Disrupting Peptide: An In Vitro Study.

The reparative and regenerative capabilities of dental pulp stem cells (DPSCs) are crucial for responding to pulp injuries, with protein phosphatase 1 (PP1) playing a significant role in regulating cellular functions pertinent to tissue healing. Accordingly, this study aimed to explore the effects of a novel cell-penetrating peptide Modified Sperm Stop 1-MSS1, that disrupts PP1, on the proliferation and odontogenic differentiation of DPSCs. Employing MSS1 as a bioportide, DPSCs were cultured and characterized for metabolic activity, cell proliferation, and cell morphology alongside the odontogenic differentiation through gene expression and alkaline phosphatase (ALP) activity analysis. MSS1 exposure induced early DPSC proliferation, upregulated genes related to odontogenic differentiation, and increased ALP activity. Markers associated with early differentiation events were induced at early culture time points and those associated with matrix mineralization were upregulated at mid-culture stages. This investigation is the first to document the potential of a PP1-disrupting bioportide in modulating DPSC functionality, suggesting a promising avenue for enhancing dental tissue regeneration and repair.

Inflammatory microenvironment of moderate pulpitis enhances the osteo-/odontogenic potential of dental pulp stem cells by autophagy.

This study investigated the effects of the inflammatory microenvironment of moderate pulpitis on biological properties of human dental pulp stem cells (DPSCs) and further explored the mechanism involved in osteo-/odontogenic induction of the inflammatory microenvironment. Healthy DPSCs (hDPSCs) and inflammatory DPSCs (iDPSCs) were isolated from human-impacted third molars free of caries and clinically diagnosed with moderate pulpitis, respectively. Healthy DPSCs were treated with lipopolysaccharides (LPS) to mimic iDPSCs invitro. The surface markers expressed on hDPSCs and iDPSCs were detected by flow cytometry. A CCK-8 assay was performed to determine cell proliferation. Flow cytometric analysis was used to evaluate cell apoptosis. The osteo-/odontogenic differentiation of DPSCs was evaluated by western blot, alkaline phosphatase staining, and Alizarin Red S staining. The functions of the genes of differentially expressed mRNAs of hDPSCs and iDPSCs were analysed using gene set enrichment analysis. Transmission electron microscopy and western blot were used to evaluate the autophagy changes of LPS-treated DPSCs. Compared with hDPSCs, iDPSCs showed no significant difference in proliferative capacity but had stronger osteo-/odontogenic potential. In addition, the mRNAs differentially expressed between iDPSCs and hDPSCs were considerably enriched in autophagosome formation and assembly-related molecules. Invitro mechanism studies further found that low concentrations of LPS could upregulate DPSC autophagy-related protein expression and autophagosome formation and promote its odontogenic/osteogenic differentiation, whereas the inhibition of DPSC autophagy led to the weakening of the odontogenic/osteogenic differentiation induced by LPS. This explorative study showed that DPSCs isolated from teeth with moderate pulpitis possessed higher osteo-/odontogenic differentiation capacity, and the mechanism involved was related to the inflammatory microenvironment-mediated autophagy of DPSCs. This helps to better understand the repair potential of inflamed dental pulp and provides the biological basis for pulp preservation and hard tissue formation in minimally invasive endodontics.

Effect of PCL/nHAEA nanocomposite to osteo/odontogenic differentiation of dental pulp stem cells

PurposeThe green synthesis of nanoparticles has recently opened up a new route in material production. The aim of this study was to evaluate the effect of nanohydroxyapatite (nHA) synthesized from Elaeagnus angustifolia (EA) extract in polycaprolactone (PCL) nanofibers (PCL/nHAEA) to odontogenic differentiation of dental pulp stem cells (DPSCs) and their potential applications for dentin tissue engineering.MethodsGreen synthesis of nHA via EA extract (nHAEA) was done by the sol–gel technique. Then electrospun nanocomposites containing of PCL blended with nHA (P/nHA) and nHAEA (P/nHAEA) were fabricated, and the characterization was evaluated via X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and the contact angle. The morphology of nanofibers and the cell adhesion capacity of DPSCs on nanofibers were evaluated using SEM. Cytocompatibility was assessed by MTT. Osteo/odontogenic differentiation ability of the nanocomposites were assessed using alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and quantitative real-time polymerase chain reaction (qPCR) technique.ResultsViability and adhesion capacity of DPSCs were higher on P/nHAEA nanofibers than PCL and P/nHA nanofibers. ARS assay, ALP activity, and qPCR analysis findings confirmed that the nHAEA blended nanofibrous scaffolds substantially increased osteo/odontogenic differentiation of DPSCs.ConclusionPCL/nHAEA nanocomposites had a noticeable effect on the odontogenic differentiation of DPSCs and may help to improve cell-based dentin regeneration therapies in the future.

NBCe1 Regulates Odontogenic Differentiation of Human Dental Pulp Stem Cells via NF-κB.

Dental pulp stem cells (DPSCs) play an important role in the repair of tooth injuries. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a Na＋-coupled HCO3- transporter encoded by the solute carrier 4A4 (SLC4A4) gene and plays a crucial role in maintaining the pH of DPSCs. Our previous research confirmed that NBCe1 is highly expressed in odontoblasts during the development of the tooth germ. Therefore, in this study, we aimed to investigate the effect of NBCe1 on odontogenic differentiation of DPSCs and further clarify the underlying mechanisms. DPSCs were isolated and identified, and the selective NBCe1 inhibitor S0859 was used to treat DPSCs. We used a cell counting Kit-8 assay to detect cell proliferative ability, and intracellular pH was assessed using confocal microscopy. Odontogenic differentiation of DPSCs was analyzed using real-time PCR and Alizarin Red S staining, and the NF-κB pathway was assessed using western blotting. Our results indicated that 10 μM S0859 was the optimal concentration for DPSC induction. Intracellular pH was decreased upon treatment with S0859. The mRNA expressions of DSPP, DMP1, RUNX2, OCN, and OPN were upregulated in the NBCe1 inhibited group compared to the controls. Moreover, NBCe1 inhibition significantly activated the NF-κB pathway, and a NF-κB inhibitor reduced the effect of NBCe1 on DPSC differentiation. NBCe1 inhibition significantly promotes odontogenic differentiation of DPSCs, and this process may be regulated by activating the NF-κB signaling pathway.

The role of ZBTB16 in odontogenic differentiation of dental pulp stem cells

ObjectiveOdontogenic differentiation of dental pulp stem cells (DPSCs) is highly controlled by the activation of several transcription factors. The zinc finger and BTB domain-containing 16 (ZBTB16) gene encodes a BTB/POZ domain and zinc finger containing transcription factors and is involved in several biological processes, but little is known about its role in odontogenic differentiation. The main goal of the current study was to determine the role of ZBTB16 in odontogenic differentiation of DPSCs. DesignZBTB16, runt-related transcription factor 2 (RUNX2), and osterix (OSX) were silenced via small-hairpin RNA (shRNA) lentivirus. The odontoblastic differentiation of DPSCs was detected by alkaline phosphatase (ALP) staining, activity measurement, and alizarin red S staining in vitro. The gene and protein expression levels were assessed by RT-qPCR and western blotting. Further, an ectopic implantation experiment was performed to explore the role of ZBTB16 in mineralization regulation in vivo followed by histological examination. ResultsThe silencing of ZBTB16 attenuated ALP activity and mineralized nodules formation by DPSCs. In addition, knockdown of ZBTB16 impaired the expression of markers involved in odontogenic differentiation, including dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, and collagen 1 in vitro and vivo. Silencing the OSX gene suppressed ZBTB16 expression and, in turn, OSX expression decreased after ZBTB16 knockdown. However, shRUNX2 did not suppress ZBTB16 expression and shZBTB16 did not affect RUNX2 expression. ConclusionsZBTB16 may play an important role in modulating the odontoblastic differentiation of DPSCs and act as a regulator of OSX in a possible feed-back cycle independent of RUNX2.

Epigenetic regulation of dental pulp stem cells and its potential in regenerative endodontics.

Regenerative endodontics (RE) therapy means physiologically replacing damaged pulp tissue and regaining functional dentin–pulp complex. Current clinical RE procedures recruit endogenous stem cells from the apical papilla, periodontal tissue, bone marrow and peripheral blood, with or without application of scaffolds and growth factors in the root canal space, resulting in cementum-like and bone-like tissue formation. Without the involvement of dental pulp stem cells (DPSCs), it is unlikely that functional pulp regeneration can be achieved, even though acceptable repair can be acquired. DPSCs, due to their specific odontogenic potential, high proliferation, neurovascular property, and easy accessibility, are considered as the most eligible cell source for dentin–pulp regeneration. The regenerative potential of DPSCs has been demonstrated by recent clinical progress. DPSC transplantation following pulpectomy has successfully reconstructed neurovascularized pulp that simulates the physiological structure of natural pulp. The self-renewal, proliferation, and odontogenic differentiation of DPSCs are under the control of a cascade of transcription factors. Over recent decades, epigenetic modulations implicating histone modifications, DNA methylation, and noncoding (nc)RNAs have manifested as a new layer of gene regulation. These modulations exhibit a profound effect on the cellular activities of DPSCs. In this review, we offer an overview about epigenetic regulation of the fate of DPSCs; in particular, on the proliferation, odontogenic differentiation, angiogenesis, and neurogenesis. We emphasize recent discoveries of epigenetic molecules that can alter DPSC status and promote pulp regeneration through manipulation over epigenetic profiles.

C5a complement receptor modulates odontogenic dental pulp stem cell differentiation under hypoxia

ABSTRACT Aim Alterations in the microenvironment change the phenotypes of dental pulp stem cells (DPSCs). The role of complement component C5a in the differentiation of DPSCs is unknown, especially under oxygen-deprived conditions. The aim of this study was to determine the effect of C5a on the odontogenic differentiation of DPSCs under normoxia and hypoxia. Material and Methods Human DPSCs were subjected to odontogenic differentiation in osteogenic media and treated with the C5a receptor antagonist-W54011 under normal and hypoxic conditions (2% oxygen). Immunochemistry, western blot, and PCR analysis for the various odontogenic differentiation genes/proteins were performed. Results Our results demonstrated that C5a plays a positive role in the odontogenic differentiation of DPSCs. C5a receptor inhibition resulted in a significant decrease in odontogenic differentiation genes, such as DMP1, ON, RUNX2, DSPP compared with the control. This observation was further supported by the Western blot data for DSPP and DMP1 and immunohistochemical analysis. The hypoxic condition reversed this effect. Conclusions Our results demonstrate that C5a regulates the odontogenic DPSC differentiation under normoxia. Under hypoxia, C5a exerts a reversed function for DPSC differentiation. Taken together, we identified that C5a and oxygen levels are key initial signals during pulp inflammation to control the odontogenic differentiation of DPSCs, thereby, providing a mechanism for potential therapeutic interventions for dentin repair and vital tooth preservation.

Demineralized Dentin Matrix Particle-Based Bio-Ink for Patient-Specific Shaped 3D Dental Tissue Regeneration.

Demineralized dentin matrix (DDM)-based materials have been actively developed and are well-known for their excellent performance in dental tissue regeneration. However, DDM-based bio-ink suitable for fabrication of engineered dental tissues that are patient-specific in terms of shape and size, has not yet been developed. In this study, we developed a DDM particle-based bio-ink (DDMp bio-ink) with enhanced three-dimensional (3D) printability. The bio-ink was prepared by mixing DDM particles and a fibrinogen–gelatin mixture homogeneously. The effects of DDMp concentration on the 3D printability of the bio-ink and dental cell compatibility were investigated. As the DDMp concentration increased, the viscosity and shear thinning behavior of the bio-ink improved gradually, which led to the improvement of the ink’s 3D printability. The higher the DDMp content, the better were the printing resolution and stacking ability of the 3D printing. The printable minimum line width of 10% w/v DDMp bio-ink was approximately 252 μm, whereas the fibrinogen–gelatin mixture was approximately 363 μm. The ink’s cytocompatibility test with dental pulp stem cells (DPSCs) exhibited greater than 95% cell viability. In addition, as the DDMp concentration increased, odontogenic differentiation of DPSCs was significantly enhanced. Finally, we demonstrated that cellular constructs with 3D patient-specific shapes and clinically relevant sizes could be fabricated through co-printing of polycaprolactone and DPSC-laden DDMp bio-ink.

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

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

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

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

Antibiotics Disturb Dentin Formation and Differentiation of Dental Pulp Stem Cells: The Role of Microbiota in Cellular Turnover of Mouse Incisor.

Dentin formation was dependent on osteo-/odontogenic differentiation of dental pulp stem cells (DPSCs). It was observed in previous studies that antibiotic treatment in a clinical and animal model resulted in impaired mineralization of dental tissues. We previously reported that microbiota maintained the function of bone marrow mesenchymal stem cells, while whether microbiota dysbiosis caused by antibiotic treatment contributed to DPSCs dysfunction and impaired dentin formation is still not known. In this study, we aimed to clarify the role of microbiota or its metabolic products on dental mineralization and the function of DPSCs. Mice were treated with antibiotics to disrupt microbiota; then, the growth rate and histological characteristics of incisors as well as the biological characteristics of DPSCs in vitro were compared with specific pathogen-free (SPF) mice. In antibiotic-treated mice (AbT), we found a diminished quantity of microbiota and reduced growth rate of mechanical injured incisor, as well as decreased colony-forming rate and impaired ability of osteo-/odontogenic differentiation of DPSCs, in comparison to SPF mice. Colonization of AbT mice with SPF mice replanted the microbiota by cohousing (conventionalized (ConvD)) and normalized the growth rate of injured incisors and colony-forming and osteo-/odontogenic differentiation ability of DPSCs. Giving short-chain fatty acids (SCFAs) by oral gavage after antibiotic treatment also rescued the growth rate of incisors and the differentiation ability of DPSCs and enhanced proliferation ability of DPSCs. Collectively, gut microbiota could make contribution to maintain continuous growth of injured rodent incisor and differentiation capacity of DPSCs; SCFAs might play a crucial role in this process.

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

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

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.

A non-invasive monitoring of USPIO labeled silk fibroin/hydroxyapatite scaffold loaded DPSCs for dental pulp regeneration.

The aim of this study was to prepare a promising biomaterial for dental pulp tissue repair and regeneration. The ultrasmall superparamagnetic iron oxide (USPIO)-labeled hydroxyapatite (HA)/silk fibroin (SF) scaffold loaded dental pulp stem cells (DPSCs) was designed. The odontogenic differentiation of DPSCs was investigated, and USPIO was used as magnetic resonance imaging (MRI) contrast agent. The results indicated that scaffolds freeze-dried from SF solution with 0.025 mg/mL USPIO and 5 mg/mL HA showed stable physical properties, accurate MRI images, and low cytotoxicity in vitro. The composite scaffolds were characterized and implanted to the subcutaneous space under the nude mice with tooth fragment. After 2 W of implantation, the relaxation rate (R2) values increased because of incorporation of USPIO, then decreased with the degradation of scaffolds. The tissue regeneration and scaffolds degradation behaviors were monitored by MRI. The expression of dentin sialophosphoprotein (DSPP) and dentin matrix acidic phosphoprotein 1 (DMP1) indicated that DPSCs differentiated into odontoblast-like cells. Revascularization and mineralization evaluated by HE-stained images and alizarin red staining images showed good formation. The USPIO/HA/SF composite scaffold loaded DPSCs promoted the repair and regeneration of dental pulp tissue and provided the ability of imaging noninvasively.

Rosuvastatin Regulates Odontoblast Differentiation by Suppressing NF-κB Activation in an Inflammatory Environment.

Rosuvastatin is a synthetic statin of 3-hydroxy-methyl-3-glutamyl coenzyme A reductase inhibitor. It has pleiotropic characteristics including hepatic selectivity, minimal metabolism, inhibition of inflammation, and induction of osteoblast differentiation. In this study, dental pulp stem cells (DPSCs) were treated with lipopolysaccharide alone or with rosuvastatin. Then, we examined the accelerative effects of rosuvastatin on odontoblast differentiation and mineralized nodule formation by real-time polymerase chain reaction (RT-PCR), western blot, alizarin red S staining, and alkaline phosphatase staining. The extent of anti-inflammation was determined by RT-PCR and analysis of the expression of tumor necrosis factor α, interleukin 1β (IL-1β), and IL-6. Furthermore, the activation of nuclear factor kappa B (NF-κB) was determined by western blot. This study demonstrates that rosuvastatin may speed up odontoblast differentiation and rescue inflammatory reaction by suppressing the NF-κB signaling pathway. It is believed that our findings provide novel perceptions on odontogenic differentiation of DPSCs.

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.

Wedelolactone Enhances Odontoblast Differentiation by Promoting Wnt/β-Catenin Signaling Pathway and Suppressing NF-κB Signaling Pathway

Wedelolactone is a multitarget natural plant compound with many pharmacological activities, including anti-inflammatory, anticancer, and antiosteoporosis. In this study, dental pulp stem cells (DPSCs) were treated with or without wedelolactone. We found that wedelolactone stimulated odontoblast differentiation and mineralization. At the molecular level, wedelolactone directly promoted the nuclear accumulation of β-catenin, and thereafter stimulated the expression of odontoblast-related marker genes containing dentin matrix protein-1 (DMP1), dentin sialophosphoprotein (DSPP), and runt-related transcription factor 2 (Runx2). Furthermore, wedelolactone upregulated the expression of IκBα and inhibited phosphonation and nuclear migration of p65. As a result, wedelolactone remarkably induced odontoblast differentiation through semaphorin 3A (Sema3A)/neuropilin-1 (NRP1) pathway-mediated β-catenin activation and nuclear factor kappa B (NF-κB) pathway inhibition. Our findings provide novel perceptions on odontogenic differentiation of DPSCs.

In Vitro differentiation potential of Isolated dental pulp stem cells

Objective: The aim of the present study is to isolate and characterize human dental pulp stem cells (DPSCs).Surface markers expression utilizing surface markers for flow cytometry. The odontogenic differentiation of DPSCs was assessed.Material and methods: dental pulp tissuses were collected from impacted third molars. samples were treated with collagenase before centrifugation to allow release of the cells. Cells were cultured in complete culture medium for 12-14 days. After reaching confluence, the isolated cells were characterized by flow cytometry using CD105 and CD45. The cells were induced for odontogenic differentiation by placing the cells in odontogenic induction media for 14 days. Odontogenic differentiation was evaluated by Alizarin Red stain and by the expressions of dentine sialo phospho protein (DSPP), dentin matrix protein-1 (DMP-1) and alkaline phosphatase (ALP) activity, which were assessed by RT-PCR.Results: The results showed that successful isolation of stem cells from human dental pulp was achieved. Cells were successfully sub-cultured and expanded up to the third passage. A flow cytometric analysis was done after stem cells isolation. Isolated DPSCs were identified by positive expression of CD105 and negative expression of hematopoietic marker C. The results showed that differentiatiationed odontoblastic like cells from DPSCs induced by odontogenic medium were stained by Alizarin Red at days 7 and 14. RT-PCR results indicated that all cultured cells efficiently differentiate into dentin forming cells and expressed specific DSPP, DMP1 and ALP genes at days 7 and 14.Conclusion: DPSCs can be a possible source of stable differentiated odontoblastic cells. Their ability of inducing hard tissue formation offer an alternative method to save teeth with compromised structural integrity.

Semaphorin 4D Enhances Angiogenic Potential and Suppresses Osteo-/Odontogenic Differentiation of Human Dental Pulp Stem Cells

IntroductionTo investigate the roles of semaphorin 4D (Sema4D)/plexin-B1 signaling on the angiogenic potential and osteo-/odontogenic differentiation of human dental pulp stem cells (DPSCs) and to uncover the corresponding molecular mechanisms. MethodsDPSCs were treated with Sema4D (10 μg/mL) for different time durations. Osteo-/odontogenic differentiation was assessed by quantifying alkaline phosphatase activity, mineralized nodule formation, and osteo-/odontogenic gene (ALP, Col1A1, BSP, RUNX2, and DSPP) and protein (Col1A1 and DSPP) expression. Involvement of the Sema4D/plexin-B1 signaling pathway was analyzed by Western blot analysis. Additionally, angiogenic gene and protein expression was assessed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. In vitro endothelial tube formation assay on Matrigel (BD Biosciences, San Jose, CA) was performed to evaluate the angiogenic inductive potential of the Sema4D-treated DPSCs conditioned medium. Results were analyzed using 1-way analysis of variance and the Student t test. ResultsSema4D significantly inhibited ALP activity and mineralized nodule formation of DPSCs. Furthermore, Sema4D-treated DPSCs displayed marked down-regulation in the expression of osteo-/odontogenic genes (ALP, Col1A1, BSP, RUNX2, and DSPP) as well as proteins (Col1A1 and DSPP). Elevated levels of plexin-B1 and downstream RhoA protein expression together with phosphorylated plexin-B1 confirmed the involvement of Sema4D/plexin-B1 signaling. Protein expression of ErbB2 was up-regulated, and Met was slightly down-regulated. Furthermore, Sema4D-treated DPSCs exhibited enhanced expression of vascular endothelial growth factor at both the messenger RNA and protein level. Accordingly, the conditioned medium of Sema4D-treated DPSCs promoted the formation of vessel-like structures as shown by the Matrigel assay. ConclusionsSema4D markedly enhances the angiogenic potential but suppresses osteo-/odontogenic differentiation of DPSCs. Sema4D/plexin-B signaling was activated via the RhoA-mediated pathway.

JAB1 accelerates odontogenic differentiation of dental pulp stem cells.

Jun activation domain-binding protein 1 (JAB1) is a multifunctional protein that participates in the control of cell proliferation and the stability of multiple proteins. JAB1 regulates several key proteins, and thereby produces varied effects on cell cycle progression, genome stability and cell survival. Some studies have shown that the loss of JAB1 in osteochondral progenitor cells severely impairs embryonic limb development in mice. However, the biological significance of JAB1 activity in the odontogenic differentiation of dental pulp stem cells (DPSCs) remains unclear. This study aimed to determine the role of JAB1, a key player in tooth development, in reparative dentin formation, especially odontogenic differentiation. We found that increased expression of JAB1 promoted odontogenic differentiation of DPSCs via Wnt/β-catenin signaling. The role of JAB1 in the odontogenic differentiation of DPSCs was further confirmed by knocking down JAB1. Our findings provide novel insights on odontogenic differentiation of DPSCs.