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Dental pulp stem cells (DPSCs) show potential for treating neurodegenerative and traumatic diseases due to their neural crest origin. Melatonin (MT), an endogenous neurohormone with well-documented anti-inflammatory and antioxidant properties, has shown promising results with MSCs in terms of engraftment, proliferation, and neuronal differentiation in animal SCI models. However, the effects of melatonin preconditioning on human dental pulp stem cells (hDPSCs) for SCI treatment remain unclear. This study investigates the impact of melatonin preconditioning on hDPSCs engraftment, neural differentiation, and neurological function in rats with SCI. Forty-two male Sprague–Dawley rats were divided into six groups: Control, Sham, Model, Vehicle, Lesion Treatment A (SCI + hDPSCs), and Lesion Treatment B (SCI + MT-hDPSCs). After obtaining hDPSCs, stem cells were evaluated using flow cytometry. Cell viability was assessed using the MTT assay. SCI was induced in the Model, Vehicle, Lesion Treatment A, and Lesion Treatment B groups. The Lesion Treatment A and B groups received hDPSCs and hDPSCs pretreated with melatonin, respectively, 1 week after SCI, while the Vehicle group received only an intravenous injection of DMEM to simulate treatment. The other groups were used for behavioral testing. Immunohistochemistry (IHC) was employed to assess hDPSCs engraftment and differentiation at the SCI site. Motor function across the six groups was evaluated using the Basso, Beattie, and Bresnahan (BBB) score. Histological studies and cell counts confirmed hDPSCs implantation at the injury site, with a significantly higher presence in the MT-hDPSCs compared to hDPSCs (p < 0.01). IHC revealed that hDPSCs and MT-hDPSCs differentiated into neurons and astrocytes, with greater differentiation observed in the MT-hDPSCs compared to the hDPSCs (p < 0.01 and p < 0.05, respectively). Functional improvement was noted in both SCI + hDPSCs and SCI + MT-hDPSCs groups compared to SCI and Vehicle groups from Week 4 onward (p < 0.001). Significant differences were also observed between the SCI + hDPSCs and SCI + MT-hDPSCs groups starting from Week 7 (p < 0.01). Preconditioning hDPSCs with melatonin enhances engraftment, neuronal differentiation, and greater performance improvement compared to hDPSCs alone in the SCI animal model. [ABSTRACT FROM AUTHOR]

AbstractIntroductionMaterials and methodsResultsConclusionDental pulp stem cells (DPSCs) have the potential to differentiate into various types of tissues including tooth, adipose, cartilage, muscle, nerve, and also possess regenerative properties. Harmine, a beta-carboline alkaloid, has been shown to have antitumor activities and promote bone formation through the differentiation of osteoblasts. The aim of this study was to investigate the effect of harmine on the differentiation of DPSCs into odontoblast cells.DPSCs were obtained from Iran’s National Genetic Reserve Center and cultured under standard stem cell culture conditions. The cells were differentiated in culture medium with and without harmine, and cell viability was evaluated using MTT assay at different harmine concentrations. Moreover, differentiation of cells was measured using Alizarin Red staining, and the expression of Runx2, DSPP, and DMP1 genes was evaluated using western blotting and real-time PCR.Harmine increased the survival rate of DPSCs in a time-­dependent manner, but higher doses (above 80 μM) had a toxic effect. On day 14, Alizarin Red staining showed increased differentiation of odontoblasts in the harmine-treated groups compared to the untreated groups. Furthermore, harmine increased the expression of Runx2, DSPP, and DMP1 genes and proteins.These findings suggest that harmine has a significant impact on the differentiation and proliferation of odontoblasts in DPSCs, likely due to its various properties and role in healing various diseases. Therefore, harmine could serve as a potential therapeutic agent for promoting dental tissue regeneration using DPSCs. [ABSTRACT FROM AUTHOR]

Seed cells and scaffold materials are essential components of tissue engineering. In this study, we investigated the key pathway of the zirconia/dental pulp stem cell composite scaffold in regulating macrophage polarization by transcriptome sequencing. We established N-rGO/ZrO2 composite scaffold and confirmed its structure using various analytical techniques, including SEM, TEM, FTIR, Raman spectra, XPS, and XRD. DPSCs were seeded onto N-rGO/ZrO2 composite scaffold material, and their proliferation, adhesion, and osteogenic differentiation were evaluated by CCK-8, immunofluorescence staining, ALP staining, and alizarin red staining. We then co-cultured DPSCs combined with N-rGO/ZrO2 as composite material with THP-1 cells in a transwell system to investigate the effect of the composite on macrophage polarization. The levels of pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes were assessed by RT-qPCR and western blot. Through bulk RNA sequencing, we detected the transcriptional characteristics of macrophages under the regulation of the composite materials, and identified the differential genes using the DEseq2 package. We also analyzed the cellular and molecular functions of differentially expressed genes (DEGs) in THP-1 cells with DPSCs combined with N-rGO/ZrO2 treatment using GO enrichment analysis and KEGG pathway enrichment analysis. Our results showed that N-rGO/ZrO2 composite scaffold promoted the proliferation, adhesion, and osteogenic differentiation of DPSCs. Moreover, N-rGO/ZrO2 composite scaffold combined with DPSCs regulated macrophage migration, polarization, and glycolysis. Mechanistically, the combination of N-rGO/ZrO2 composite materials and DPSCs regulated macrophage polarization by activating the TNF signaling pathway. This finding provides a new approach to the clinical preservation of maxillofacial bone defect repair. [ABSTRACT FROM AUTHOR]

Background: This study investigates the impact of environmental pollution on the quality and viability of dental stem cells (DSCs) from impacted third molars. By comparing DSCs from patients in industrial areas with high air pollution and those from non-industrial regions, the research assesses the adverse effects of heavy metals on stem cell proliferation. Methods: Impacted lower third molars were collected from 28 patients—10 from industrial and 18 from non-industrial areas. Patients were divided into two age groups: 18–27 years and 28–38 years old. Dental pulp was extracted under sterile conditions, and DSCs were isolated and cultured. Heavy metal concentrations in dental tissues were measured using atomic absorption/emission spectrometry. Results: The study found significantly higher concentrations of copper and lead in the dental tissues of patients in industrial areas. Cell viability was lower in samples from these areas, with a statistically significant difference in average doubling time and the number of cells obtained after the first passage. There was no significant impact of gender on heavy metal content, except for higher iron levels in men. Conclusions: Exposure to industrial pollutants negatively affects the viability and proliferation of DSCs, but there are no differences in differentiation in the osteogenic medium regarding cell mineralization. These studies highlight the importance of environmental factors for oral health, suggesting that residents of polluted areas may face greater difficulties in dental and regenerative treatments. Further research is needed to develop strategies to mitigate the effects and improve clinical outcomes for affected populations. [ABSTRACT FROM AUTHOR]

Human dental pulp stem cells (DPSCs) have become an important component for bone tissue engineering and regenerative medicine due to their ability to differentiate into osteoblast precursors. Two miRNA chip datasets (GSE138180 and E-MTAB-3077) of DPSCs osteogenic differentiation were analyzed respectively to find the expression of miR-483-3p significantly increased in the differentiated groups. We further confirmed that miR-483-3p continued to overexpress during osteogenic differentiation of DPSCs, especially reaching its peak on the 7th day. Moreover, miR-483-3p could significantly promote the expression of osteogenic markers including RUNX2 and OSX, and activate MAPK signaling pathway by inducing phosphorylation of ERK, p38, and JNK. In addition, as a significant gene within the MAPK signaling pathway, ARRB2 was identified as the target gene of miR-483-3p by bioinformatic prediction and experimental verification. In conclusion, we identified miR-483-3p could promote osteogenic differentiation of DPSCs via the MAPK signaling pathway by targeting ARRB2. [ABSTRACT FROM AUTHOR]

Abstract Dental pulp stem cells (DPSCs) show potential for treating neurodegenerative and traumatic diseases due to their neural crest origin. Melatonin (MT), an endogenous neurohormone with well-documented anti-inflammatory and antioxidant properties, has shown promising results with MSCs in terms of engraftment, proliferation, and neuronal differentiation in animal SCI models. However, the effects of melatonin preconditioning on human dental pulp stem cells (hDPSCs) for SCI treatment remain unclear. This study investigates the impact of melatonin preconditioning on hDPSCs engraftment, neural differentiation, and neurological function in rats with SCI. Forty-two male Sprague–Dawley rats were divided into six groups: Control, Sham, Model, Vehicle, Lesion Treatment A (SCI + hDPSCs), and Lesion Treatment B (SCI + MT-hDPSCs). After obtaining hDPSCs, stem cells were evaluated using flow cytometry. Cell viability was assessed using the MTT assay. SCI was induced in the Model, Vehicle, Lesion Treatment A, and Lesion Treatment B groups. The Lesion Treatment A and B groups received hDPSCs and hDPSCs pretreated with melatonin, respectively, 1 week after SCI, while the Vehicle group received only an intravenous injection of DMEM to simulate treatment. The other groups were used for behavioral testing. Immunohistochemistry (IHC) was employed to assess hDPSCs engraftment and differentiation at the SCI site. Motor function across the six groups was evaluated using the Basso, Beattie, and Bresnahan (BBB) score. Histological studies and cell counts confirmed hDPSCs implantation at the injury site, with a significantly higher presence in the MT-hDPSCs compared to hDPSCs (p

Mesenchymal stem-cell-derived extracellular vesicles (MSC-EVs) have been increasingly investigated for cancer therapy and drug delivery, and they offer an advanced cell-free therapeutic option. However, their overall effects and efficacy depend on various factors, including the MSC source and cargo content. In this study, we isolated EVs from the conditioned medium of human immature dental pulp stem cells (hIDPSC-EVs) and investigated their effects on two papillary thyroid cancer (PTC) cell lines (BCPAP and TPC1). We observed efficient uptake of hIDPSC-EVs by both PTC cell lines, with a notable impact on gene regulation, particularly in the Wnt signaling pathway in BCPAP cells. However, no significant effects on cell proliferation were observed. Conversely, hIDPSC-EVs significantly reduced the invasive capacity of both PTC cell lines after 120 h of treatment. These in vitro findings suggest the therapeutic potential of hIDPSC-EVs in cancer management and emphasize the need for further research to develop novel and effective treatment strategies. Furthermore, the successful internalization of hIDPSC-EVs by PTC cell lines underscores their potential use as nanocarriers for anti-cancer agents. [ABSTRACT FROM AUTHOR]

Alginate/gelatin (Alg-Gel) hydrogels have been used experimentally, associated with mesenchymal stromal/stem cells (MSCs), to guide bone tissue formation. One of the main challenges for clinical application is optimizing Alg-Gel stiffness to guide osteogenesis. In this study, we investigated how Alg-Gel stiffness could modulate the dental pulp stem cell (DPSC) attachment, morphology, proliferation, and osteogenic differentiation, identifying the optimal conditions to uncouple osteogenesis from the other cell behaviors. An array of Alg-Gel hydrogels was prepared by casting different percentages of alginate and gelatin cross-linked with 2% CaCl2. We have selected two hydrogels: one with a stiffness of 11 ± 1 kPa, referred to as "low-stiffness hydrogel", formed by 2% alginate and 8% gelatin, and the other with a stiffness of 55 ± 3 kPa, referred to as "high-stiffness hydrogel", formed by 8% alginate and 12% gelatin. Hydrogel analyses showed that the average swelling rates were 20 ± 3% for the low-stiffness hydrogels and 35 ± 2% for the high-stiffness hydrogels. The degradation percentage was 47 ± 5% and 18 ± 2% for the low- and high-stiffness hydrogels, respectively. Both hydrogel types showed homogeneous surface shape and protein (Alg-Gel) interaction with CaCl2 as assessed by physicochemical characterization. Cell culture showed good adhesion of the DPSCs to the hydrogels and proliferation. Furthermore, better osteogenic activity, determined by ALP activity and ARS staining, was obtained with high-stiffness hydrogels (8% alginate and 12% gelatin). In summary, this study confirms the possibility of characterizing and optimizing the stiffness of Alg-Gel gel to guide osteogenesis in vitro without altering the other cellular properties of DPSCs. [ABSTRACT FROM AUTHOR]

Background: This study investigated the contact between adult dental pulp stem cells (DPSCs) and carbamide peroxide (CP), a bleaching agent that is a popular choice for at-home whitening products, using an in vitro model. Objectives: The aim of this study was to evaluate the impact of exposure to different concentrations and timings of a commonly used peroxide-based home tooth-whitening product on DPSCs. Materials and methods: Human DPSCs obtained from impacted third molars were cultured and exposed to various concentrations of carbamide peroxide (0.1%, 0.5%, and 1%). The effects of CP on DPSC proliferation and apoptosis were investigated by MTT assay and flow cytometry. Migration was investigated by micrographs of wound healing. An enzyme-linked immunosorbent assay (IL-6 and IL-8) was used to investigate the CP-stimulated cytokine production of DPSCs. Each experiment was performed three times with independent batches of DPSCs. Statistical analysis of the collected data was performed using one-way and two-way ANOVAs with the significance threshold set at p < 0.05. Tukey's post hoc multiple comparison test was used to identify differences between groups. Results: Cell viability and adherence were lower in the CP-exposed cells compared to the non-stimulated cells, probably due to increased cell death (** p ≤ 0.01, **** p ≤ 0.0001). CP-stimulated DPSCs exhibited a dose-dependent release of IL-6 and IL-8 (**** p ≤ 0.0001). CP did not affect wound healing at any concentration tested. Conclusions: Human DPSCs were able to sense CP. Consequently, CP contributed significantly to cell apoptosis and local inflammatory responses through cytokine release. [ABSTRACT FROM AUTHOR]

Cell outer membranes contain glycosphingolipids and protein receptors, which are integrated into glycoprotein domains, known as lipid rafts, which are involved in a variety of cellular processes, including receptor-mediated signal transduction and cellular differentiation process. In this study, we analyzed the lipidic composition of human Dental Pulp-Derived Stem Cells (DPSCs), and the role of lipid rafts during the multilineage differentiation process. The relative quantification of lipid metabolites in the organic fraction of DPSCs, performed by Nuclear Magnetic Resonance (NMR) spectroscopy, showed that mono-unsaturated fatty acids (MUFAs) were the most representative species in the total pool of acyl chains, compared to polyunsatured fatty acids (PUFAs). In addition, the stimulation of DPSCs with different culture media induces a multilineage differentiation process, determining changes in the gangliosides pattern. To understand the functional role of lipid rafts during multilineage differentiation, DPSCs were pretreated with a typical lipid raft affecting agent (MβCD). Subsequently, DPSCs were inducted to differentiate into osteoblast, chondroblast and adipoblast cells with specific media. We observed that raft-affecting agent MβCD prevented AKT activation and the expression of lineage-specific mRNA such as OSX, PPARγ2, and SOX9 during multilineage differentiation. Moreover, this compound significantly prevented the tri-lineage differentiation induced by specific stimuli, indicating that lipid raft integrity is essential for DPSCs differentiation. These results suggest that lipid rafts alteration may affect the signaling pathway activated, preventing multilineage differentiation.

IntroductionSmoking is known to alter the regenerative and immunomodulatory properties of many types of mesenchymal stem cells (MSCs). This study investigates the impact of cigarette smoke exposure on the regenerative potential of dental pulp stem cells (DPSCs).MethodsDPSCs were treated with various doses of cigarette smoke condensate (CSC) or nicotine. Cell proliferation and survival were evaluated by a water-soluble tetrazolium salt (WST-1) and a survival assay. DPSC migration, cytokine expression, mutagenesis, and the signaling pathway were also measured during CSC and nicotine treatment.ResultsLow concentrations of CSC and nicotine did not impair cell proliferation, but higher concentrations reduced cell proliferation. CSC and nicotine could impede DPSC survival and migration in a dose-dependent manner. In addition, the cytokine secretion expression profile was altered with CSC or nicotine treatments. In particular, secretion of IL-6, TNF-α, and IL-10 significantly increased, while TGF-β1 levels showed different patterns after exposure to CSC or nicotine, as shown by ELISA and quantitative PCR. Nicotine treatment increased AKT (also known as protein kinase B) and extracellular signal-regulated kinase (ERK) phosphorylation. Finally, CSC induced higher levels of mutagenicity than nicotine, as shown by the Ames test.ConclusionsThese findings suggest that cigarette smoke exposure alters the regenerative abilities of DPSCs in various ways. Future studies are warranted to further characterize the underlying molecular mechanisms of smoking-mediated damage to DPSCs, which will guide the personalized stem cell treatment plan for smoking patients.

Introduction: Nonsurgical endodontic therapies have evolved from classic endodontic therapies to regenerative endodontic treatments (RETs) in recent years. In context of the cytotoxic activity of the most commonly used endodontic irrigant, NaOCl, newer endodontic irrigating solutions should be tested for its effective use in RETs. The aim of this trial was to examine and assess the cytotoxic response of 3.8 % SDF and BioAKT irrigating solutions on the viability of DPSCs. Methods: The viability of DPSCs cultivated in 5.25 % NaOCl, 3.8 % SDF & BioAKT at dilutions of 1:100,1:20 &1:10 were evaluated through MTT assay after 10 min, 60 min and 24 h incubation, detection of apoptosis and ALP activity after 7,14 & 21-days incubation. A two-way analysis of variance (ANOVA) with post hoc Turkey HSD was performed to determine significant differences between the specimens tested. Results: When compared to the control at all time periods, all test specimens at varied dilutions (1:100, 1:20, and 1:10) caused no cytotoxic effects. The maximum number of live cells and ALP activity was observed with DPSCs cultivated in BioAKT followed by 3.8 % SDF and 5.25 % NaOCl at all time intervals. Conclusion: Different doses of 3.8 % SDF and BioAKT solution revealed encouraging outcomes when compared to 5.25 % NaOCl in terms of viability, proliferation and long-term ALP functioning potential when cultivated in DPSCs.

Abstract Background Revascularization and restoration of normal pulp-dentin complex are important for tissue-engineered pulp regeneration. Recently, a unique periodontal tip-like endothelial cells subtype (POTCs) specialized to dentinogenesis was identified. We have confirmed that TPPU, a soluble epoxide hydrolase (sEH) inhibitor targeting epoxyeicosatrienoic acids (EETs) metabolism, promotes bone growth and regeneration by angiogenesis and osteogenesis coupling. We hypothesized that TPPU could also promote revascularization and induce POTCs to contribute to pulp-dentin complex regeneration. Here, we in vitro and in vivo characterized the potential effect of TPPU on the coupling of angiogenesis and odontogenesis and investigated the relevant mechanism, providing new ideas for pulp-dentin regeneration by targeting sEH. Methods In vitro effects of TPPU on the proliferation, migration, and angiogenesis of dental pulp stem cells (DPSCs), human umbilical vein endothelial cells (HUVECs) and cocultured DPSCs and HUVECs were detected using cell counting kit 8 (CCK8) assay, wound healing, transwell, tube formation and RT-qPCR. In vivo, Matrigel plug assay was performed to outline the roles of TPPU in revascularization and survival of grafts. Then we characterized the VEGFR2 + POTCs around odontoblast layer in the molar of pups from C57BL/6 female mice gavaged with TPPU. Finally, the root segments with DPSCs mixed with Matrigel were implanted subcutaneously in BALB/c nude mice treated with TPPU and the root grafts were isolated for histological staining. Results In vitro, TPPU significantly promoted the migration and tube formation capability of cocultured DPSCs and HUVECs. ALP and ARS staining and RT-qPCR showed that TPPU promoted the osteogenic and odontogenic differentiation of cultured cells, treatment with an anti-TGF-β blocking antibody abrogated this effect. Knockdown of HIF-1α in HUVECs significantly reversed the effect of TPPU on the expression of angiogenesis, osteogenesis and odontogenesis-related genes in cocultured cells. Matrigel plug assay showed that TPPU increased VEGF/VEGFR2-expressed cells in transplanted grafts. TPPU contributed to angiogenic-odontogenic coupling featured by increased VEGFR2 + POTCs and odontoblast maturation during early dentinogenesis in molar of newborn pups from C57BL/6 female mice gavaged with TPPU. TPPU induced more dental pulp-like tissue with more vessels and collagen fibers in transplanted root segment. Conclusions TPPU promotes revascularization of dental pulp regeneration by enhancing migration and angiogenesis of HUVECs, and improves odontogenic differentiation of DPSCs by TGF-β. TPPU boosts the angiogenic–odontogenic coupling by enhancing VEGFR2 + POTCs meditated odontoblast maturation partly via upregulating HIF-1α, which contributes to increasing pulp-dentin complex for tissue-engineered pulp regeneration.

Purpose: Recently, increasing attention has been paid to the function of long non-coding RNAs (lncRNAs) in osteogenic differentiation (OD) of dental pulp stem cells (DPSCs). LINC01133 was reported to have a close relationship with tumorigenesis for multiple cancers, but no study has yet explored the role of LINC01133 in modulating OD of DPSCs. Materials and Methods: Alizarin red S (ARS) staining and alkaline phosphatase (ALP) staining were perfomed to assess the OD potential of DPSCs. Osteogenic markers including runt-related transcription factor 2 (RUNX2), osterix (OSX) and ALP expression levels in DPSCs were monitored by qRT-PCR and Western blot before and after cell transfection. Luciferase reporter gene assay detected the relationship between LINC01133 and miR-199b-5p. Results: The expression of LINC01133 was low, while miR-199b-5p was increasingly expressed during OD of DPSCs. Overexpression of LINC01133 in DPSCs resulted in decreased expression of RUNX2, OSX, ALP, DSPP and DMP1, whose expression was reversed in DPSCs after transfections of miR-199b-5p overexpression. Co-transfection of pcDNA3.1-LINC01133 and miR-199b-5p mimic led to elevated expression of RUNX2, OSX, ALP, DSPP and DMP1 compared with pcDNA3.1-LINC01133 transfection alone. LINC01133 served as a sponge of miR-199b-5p. AKT3 was verified as a downstream effector of miR-199b-5p in DPSCs. Conclusion: LINC01133 inhibits the OD of DPSCs by upregulating AKT3 via sponging miR-199b-5p, which may act as a potential diagnostic biomarker for dentin regeneration in the dental pulp. [ABSTRACT FROM AUTHOR]

Background: Revascularization and restoration of normal pulp-dentin complex are important for tissue-engineered pulp regeneration. Recently, a unique periodontal tip-like endothelial cells subtype (POTCs) specialized to dentinogenesis was identified. We have confirmed that TPPU, a soluble epoxide hydrolase (sEH) inhibitor targeting epoxyeicosatrienoic acids (EETs) metabolism, promotes bone growth and regeneration by angiogenesis and osteogenesis coupling. We hypothesized that TPPU could also promote revascularization and induce POTCs to contribute to pulp-dentin complex regeneration. Here, we in vitro and in vivo characterized the potential effect of TPPU on the coupling of angiogenesis and odontogenesis and investigated the relevant mechanism, providing new ideas for pulp-dentin regeneration by targeting sEH. Methods: In vitro effects of TPPU on the proliferation, migration, and angiogenesis of dental pulp stem cells (DPSCs), human umbilical vein endothelial cells (HUVECs) and cocultured DPSCs and HUVECs were detected using cell counting kit 8 (CCK8) assay, wound healing, transwell, tube formation and RT-qPCR. In vivo, Matrigel plug assay was performed to outline the roles of TPPU in revascularization and survival of grafts. Then we characterized the VEGFR2 + POTCs around odontoblast layer in the molar of pups from C57BL/6 female mice gavaged with TPPU. Finally, the root segments with DPSCs mixed with Matrigel were implanted subcutaneously in BALB/c nude mice treated with TPPU and the root grafts were isolated for histological staining. Results: In vitro, TPPU significantly promoted the migration and tube formation capability of cocultured DPSCs and HUVECs. ALP and ARS staining and RT-qPCR showed that TPPU promoted the osteogenic and odontogenic differentiation of cultured cells, treatment with an anti-TGF-β blocking antibody abrogated this effect. Knockdown of HIF-1α in HUVECs significantly reversed the effect of TPPU on the expression of angiogenesis, osteogenesis and odontogenesis-related genes in cocultured cells. Matrigel plug assay showed that TPPU increased VEGF/VEGFR2-expressed cells in transplanted grafts. TPPU contributed to angiogenic-odontogenic coupling featured by increased VEGFR2 + POTCs and odontoblast maturation during early dentinogenesis in molar of newborn pups from C57BL/6 female mice gavaged with TPPU. TPPU induced more dental pulp-like tissue with more vessels and collagen fibers in transplanted root segment. Conclusions: TPPU promotes revascularization of dental pulp regeneration by enhancing migration and angiogenesis of HUVECs, and improves odontogenic differentiation of DPSCs by TGF-β. TPPU boosts the angiogenic–odontogenic coupling by enhancing VEGFR2 + POTCs meditated odontoblast maturation partly via upregulating HIF-1α, which contributes to increasing pulp-dentin complex for tissue-engineered pulp regeneration. [ABSTRACT FROM AUTHOR]

Nonsurgical endodontic therapies have evolved from classic endodontic therapies to regenerative endodontic treatments (RETs) in recent years. In context of the cytotoxic activity of the most commonly used endodontic irrigant, NaOCl, newer endodontic irrigating solutions should be tested for its effective use in RETs. The aim of this trial was to examine and assess the cytotoxic response of 3.8 % SDF and BioAKT irrigating solutions on the viability of DPSCs. The viability of DPSCs cultivated in 5.25 % NaOCl, 3.8 % SDF & BioAKT at dilutions of 1:100,1:20 &1:10 were evaluated through MTT assay after 10 min, 60 min and 24 h incubation, detection of apoptosis and ALP activity after 7,14 & 21-days incubation. A two-way analysis of variance (ANOVA) with post hoc Turkey HSD was performed to determine significant differences between the specimens tested. When compared to the control at all time periods, all test specimens at varied dilutions (1:100, 1:20, and 1:10) caused no cytotoxic effects. The maximum number of live cells and ALP activity was observed with DPSCs cultivated in BioAKT followed by 3.8 % SDF and 5.25 % NaOCl at all time intervals. Different doses of 3.8 % SDF and BioAKT solution revealed encouraging outcomes when compared to 5.25 % NaOCl in terms of viability, proliferation and long-term ALP functioning potential when cultivated in DPSCs. [Display omitted] [ABSTRACT FROM AUTHOR]

Polylactic Acid (PLA) and Acrylonitrile–Butadiene–Styrene (ABS) are commonly used polymers in 3D printing for biomedical applications. Dental Pulp Stem Cells (DPSCs) are an accessible and proliferative source of stem cells with significant differentiation potential. Limited knowledge exists regarding the biocompatibility and genetic safety of ABS and PLA when in contact with DPSCs. This study aimed to investigate the impact of PLA and ABS on the adhesion, proliferation, osteogenic differentiation, genetic stability, proteomics, and immunophenotypic profile of DPSCs. A total of three groups, 1- DPSC-control, 2- DPSC+ABS, and 3- DPSC+PLA, were used in in vitro experiments to evaluate cell morphology, proliferation, differentiation capabilities, genetic stability, proteomics (secretome), and immunophenotypic profiles regarding the interaction between DPSCs and polymers. Both ABS and PLA supported the adhesion and proliferation of DPSCs without exhibiting significant cytotoxic effects and maintaining the capacity for osteogenic differentiation. Genetic stability, proteomics, and immunophenotypic profiles were unaltered in DPSCs post-contact with these polymers, highlighting their biosafety. Our findings suggest that ABS and PLA are biocompatible with DPSCs and demonstrate potential in dental or orthopedic applications; the choice of the polymer will depend on the properties required in treatment. These promising results stimulate further studies to explore the potential therapeutic applications in vivo using prototyped polymers in personalized medicine. [ABSTRACT FROM AUTHOR]

Supernumerary teeth are advantaged sources for high‐quality stem cell preparation from both apical papilla (SCAP‐Ss) and dental pulp (DPSCs). However, the deficiency of the systematic and detailed comparison of the biological and transcriptomic characteristics of the aforementioned stem cells largely hinders their application in regenerative medicine. Herein, we collected supernumerary teeth for SCAP‐S and DPSC isolation and identification by utilizing multiple biological tests (e.g., growth curve, cell cycle and apoptosis, adipogenic and osteogenic differentiation, and quantitative real‐time polymerase chain reaction). Furthermore, we took advantage of transcriptome sequencing and multifaceted bioinformatic analyses to dissect the similarities and diversities between them. In this study, we found that SCAP‐Ss and DPSCs showed indistinctive signatures in morphology and immunophenotypes, whereas with diversity in cell vitality and multi‐lineage differentiation as well as gene expression profiling and differentially expressed genes‐associated gene ontology and signaling pathways. Collectively, our data indicated the diversity of the multifaceted signatures of human supernumerary teeth‐derived stem cells both at the cellular and molecular levels, which also supplied new references for SCAP‐Ss serving as splendid alternative stem cell sources for regenerative medicine purposes. [ABSTRACT FROM AUTHOR]

As previously described by several authors, dental pulp stem cells (DPSCs), when adequately stimulated, may acquire a neuronal-like phenotype acting as a favorable source of stem cells in the generation of nerves. Besides, it is known that hypoxia conditioning is capable of stimulating cell differentiation as well as survival and self-renewal, and that multiple growth factors, including Epidermal Growth factor (EGF) and basic fibroblast growth factor (bFGF), are often involved in the induction of the neuronal differentiation of progenitor cells. In this work, we investigated the role of hypoxia in the commitment of DPSCs into a neuronal phenotype. These cells were conditioned with hypoxia (O2 1%) for 5 and 16 days; subsequently, we analyzed the proliferation rate and morphology, and tested the cells for neural and stem markers. Moreover, we verified the possible autocrine/paracrine role of DPSCs in the induction of neural differentiation by comparing the secretome profile of the hypoxic and normoxic conditioned media (CM). Our results showed that the hypoxia-mediated DPSC differentiation was time dependent. Moreover, conditioned media (CM derived from DPSCs stimulated by hypoxia were able, in turn, to induce the neural differentiation of SH-SY5Y neuroblastoma cells and undifferentiated DPSCs. In conclusion, under the herein-mentioned conditions, hypoxia seems to favor the differentiation of DPSCs into neuron-like cells. In this way, we confirm the potential clinical utility of differentiated neuronal DPSCs, and we also suggest the even greater potential of CM-derived-hypoxic DPSCs that could more readily be used in regenerative therapies.

Alginate/gelatin (Alg-Gel) hydrogels have been used experimentally, associated with mesenchymal stromal/stem cells (MSCs), to guide bone tissue formation. One of the main challenges for clinical application is optimizing Alg-Gel stiffness to guide osteogenesis. In this study, we investigated how Alg-Gel stiffness could modulate the dental pulp stem cell (DPSC) attachment, morphology, proliferation, and osteogenic differentiation, identifying the optimal conditions to uncouple osteogenesis from the other cell behaviors. An array of Alg-Gel hydrogels was prepared by casting different percentages of alginate and gelatin cross-linked with 2% CaCl2. We have selected two hydrogels: one with a stiffness of 11 ± 1 kPa, referred to as “low-stiffness hydrogel”, formed by 2% alginate and 8% gelatin, and the other with a stiffness of 55 ± 3 kPa, referred to as “high-stiffness hydrogel”, formed by 8% alginate and 12% gelatin. Hydrogel analyses showed that the average swelling rates were 20 ± 3% for the low-stiffness hydrogels and 35 ± 2% for the high-stiffness hydrogels. The degradation percentage was 47 ± 5% and 18 ± 2% for the low- and high-stiffness hydrogels, respectively. Both hydrogel types showed homogeneous surface shape and protein (Alg-Gel) interaction with CaCl2 as assessed by physicochemical characterization. Cell culture showed good adhesion of the DPSCs to the hydrogels and proliferation. Furthermore, better osteogenic activity, determined by ALP activity and ARS staining, was obtained with high-stiffness hydrogels (8% alginate and 12% gelatin). In summary, this study confirms the possibility of characterizing and optimizing the stiffness of Alg-Gel gel to guide osteogenesis in vitro without altering the other cellular properties of DPSCs.

Introduction Smoking is known to alter the regenerative and immunomodulatory properties of many types of mesenchymal stem cells (MSCs). This study investigates the impact of cigarette smoke exposure on the regenerative potential of dental pulp stem cells (DPSCs). Methods DPSCs were treated with various doses of cigarette smoke condensate (CSC) or nicotine. Cell proliferation and survival were evaluated by a watersoluble tetrazolium salt (WST-1) and a survival assay. DPSC migration, cytokine expression, mutagenesis, and the signaling pathway were also measured during CSC and nicotine treatment. Results Low concentrations of CSC and nicotine did not impair cell proliferation, but higher concentrations reduced cell proliferation. CSC and nicotine could impede DPSC survival and migration in a dose-dependent manner. In addition, the cytokine secretion expression profile was altered with CSC or nicotine treatments. In particular, secretion of IL-6, TNF-α, and IL-10 significantly increased, while TGF-β1 levels showed different patterns after exposure to CSC or nicotine, as shown by ELISA and quantitative PCR. Nicotine treatment increased AKT (also known as protein kinase B) and extracellular signal-regulated kinase (ERK) phosphorylation. Finally, CSC induced higher levels of mutagenicity than nicotine, as shown by the Ames test. Conclusions These findings suggest that cigarette smoke exposure alters the regenerative abilities of DPSCs in various ways. Future studies are warranted to further characterize the underlying molecular mechanisms of smoking-mediated damage to DPSCs, which will guide the personalized stem cell treatment plan for smoking patients.

Abstract Background Dental pulp stem cells (DPSCs) play a crucial role in dentin-pulp complex regeneration. Further understanding of the mechanism by which DPSCs remain in a quiescent state could contribute to improvements in the dentin-pulp complex and dentinogenesis. Methods TSC1 conditional knockout (DMP1-Cre+; TSC1f/f, hereafter CKO) mice were generated to increase the activity of mechanistic target of rapamycin complex 1 (mTORC1). H&E staining, immunofluorescence and micro-CT analysis were performed with these CKO mice and littermate controls. In vitro, exosomes were collected from the supernatants of MDPC23 cells with different levels of mTORC1 activity and then characterized by transmission electron microscopy and nanoparticle tracking analysis. DPSCs were cocultured with MDPC23 cells and MDPC23 cell-derived exosomes. Alizarin Red S staining, ALP staining, qRT‒PCR, western blotting analysis and micro-RNA sequencing were performed. Results Our study showed that mTORC1 activation in odontoblasts resulted in thicker dentin and higher dentin volume/tooth volume of molars, and it increased the expression levels of the exosome markers CD63 and Alix. In vitro, when DPSCs were cocultured with MDPC23 cells, odontoblastic differentiation was inhibited. However, the inhibition of odontoblastic differentiation was reversed when DPSCs were cocultured with MDPC23 cells with mTORC1 overactivation. To further study the effects of mTORC1 on exosome release from odontoblasts, MDPC23 cells were treated with rapamycin or shRNA-TSC1 to inactivate or activate mTORC1, respectively. The results revealed that exosome release from odontoblasts was negatively correlated with mTORC1 activity. Moreover, exosomes derived from MDPC23 cells with active or inactive mTORC1 inhibited the odontoblastic differentiation of DPSCs at the same concentration. miRNA sequencing analysis of exosomes that were derived from shTSC1-transfected MDPC23 cells, rapamycin-treated MDPC23 cells or nontreated MDPC23 cells revealed that the majority of the miRNAs were similar among these groups. In addition, exosomes derived from odontoblasts inhibited the odontoblastic differentiation of DPSCs, and the inhibitory effect was positively correlated with exosome concentration. Conclusion mTORC1 regulates exosome release from odontoblasts to inhibit the odontoblastic differentiation of DPSCs, but it does not alter exosomal contents. These findings might provide a new understanding of dental pulp complex regeneration.

Background: This study investigated the contact between adult dental pulp stem cells (DPSCs) and carbamide peroxide (CP), a bleaching agent that is a popular choice for at-home whitening products, using an in vitro model. Objectives: The aim of this study was to evaluate the impact of exposure to different concentrations and timings of a commonly used peroxide-based home tooth-whitening product on DPSCs. Materials and methods: Human DPSCs obtained from impacted third molars were cultured and exposed to various concentrations of carbamide peroxide (0.1%, 0.5%, and 1%). The effects of CP on DPSC proliferation and apoptosis were investigated by MTT assay and flow cytometry. Migration was investigated by micrographs of wound healing. An enzyme-linked immunosorbent assay (IL-6 and IL-8) was used to investigate the CP-stimulated cytokine production of DPSCs. Each experiment was performed three times with independent batches of DPSCs. Statistical analysis of the collected data was performed using one-way and two-way ANOVAs with the significance threshold set at p < 0.05. Tukey’s post hoc multiple comparison test was used to identify differences between groups. Results: Cell viability and adherence were lower in the CP-exposed cells compared to the non-stimulated cells, probably due to increased cell death (** p ≤ 0.01, **** p ≤ 0.0001). CP-stimulated DPSCs exhibited a dose-dependent release of IL-6 and IL-8 (**** p ≤ 0.0001). CP did not affect wound healing at any concentration tested. Conclusions: Human DPSCs were able to sense CP. Consequently, CP contributed significantly to cell apoptosis and local inflammatory responses through cytokine release.

Cell outer membranes contain glycosphingolipids and protein receptors, which are integrated into glycoprotein domains, known as lipid rafts, which are involved in a variety of cellular processes, including receptor-mediated signal transduction and cellular differentiation process. In this study, we analyzed the lipidic composition of human Dental Pulp-Derived Stem Cells (DPSCs), and the role of lipid rafts during the multilineage differentiation process. The relative quantification of lipid metabolites in the organic fraction of DPSCs, performed by Nuclear Magnetic Resonance (NMR) spectroscopy, showed that mono-unsaturated fatty acids (MUFAs) were the most representative species in the total pool of acyl chains, compared to polyunsatured fatty acids (PUFAs). In addition, the stimulation of DPSCs with different culture media induces a multilineage differentiation process, determining changes in the gangliosides pattern. To understand the functional role of lipid rafts during multilineage differentiation, DPSCs were pretreated with a typical lipid raft affecting agent (MβCD). Subsequently, DPSCs were inducted to differentiate into osteoblast, chondroblast and adipoblast cells with specific media. We observed that raft-affecting agent MβCD prevented AKT activation and the expression of lineage-specific mRNA such as OSX, PPARγ2, and SOX9 during multilineage differentiation. Moreover, this compound significantly prevented the tri-lineage differentiation induced by specific stimuli, indicating that lipid raft integrity is essential for DPSCs differentiation. These results suggest that lipid rafts alteration may affect the signaling pathway activated, preventing multilineage differentiation.

How to repair dentin-pulp injury effectively has always been a clinical problem, and the comparative study of repair process between different injuries is unknown. Dental pulp stem cells (DPSCs) often are selected as seed cells for the study of dentin-pulp injury repair due to excellent advantages in odontogenesis and pulp differentiation. Although many previous researches have indicated that the Wnt protein and Wnt/β-catenin signaling pathway were crucial for dental growth, development, and injury repair, the specific mechanism remained unknown. In this study, different dentine-pulp injury models of adult mice were established successfully by abrasion and cutting methods. The gross morphology and micro-CT were used to observe the repair of injured mice incisor in different groups. We found that the repair time of each group was different. The repair time of the cutting group was longer than the abrasion group and the qRT-PCR detection showed that the expression of DSPP in the cutting group was higher than that in the abrasion group, but there was no significant difference in proliferation among the groups. In vivo and cell experiments showed that activation of Wnt/β-catenin signaling pathway can promote the proliferation and odontoblast differentiation of DPSCs. In addition, by using RNAscope staining, we observed that Wnt10a was mainly expressed in the proliferative region and partially expressed in the odontoblast region. The Western blotting results showed that in the early stage of repair, the expression of Wnt10a increased with the extension of days after injury in both abrasion and cutting group and the increase of Wnt10a was tested obviously on the 5th day after injury. But on the 7th day after injury, the expression of Wnt10a was still obvious in the cutting group, while the expression of Wnt10a was significantly reduced in the abrasion group, which was close to the control group. It is suggested that Wnt10a acts as a repair-related protein and has an important role in tooth injury repair. Wnt10a was activated by R-spondin and LiCl, and Wnt10a-siRNA DPSCs were constructed to inhibit Wnt10a. The results showed that Wnt10a/β-catenin signaling pathway promoted the proliferation and odontoblast differentiation of DPSCs. It plays a crucial role in the repair process of different injuries. This study enriched the mechanisms of Wnt10a /β-catenin signaling pathways in different types of dentin-pulp injury repair, which could provide experimental evidences for the target gene screening and also give some new ideas for the subsequent research on the molecular mechanisms of tooth regeneration. [ABSTRACT FROM AUTHOR]

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

Background: Dental pulp stem cells (DPSCs) play a crucial role in dentin-pulp complex regeneration. Further understanding of the mechanism by which DPSCs remain in a quiescent state could contribute to improvements in the dentin-pulp complex and dentinogenesis. Methods: TSC1 conditional knockout (DMP1-Cre+; TSC1f/f, hereafter CKO) mice were generated to increase the activity of mechanistic target of rapamycin complex 1 (mTORC1). H&E staining, immunofluorescence and micro-CT analysis were performed with these CKO mice and littermate controls. In vitro, exosomes were collected from the supernatants of MDPC23 cells with different levels of mTORC1 activity and then characterized by transmission electron microscopy and nanoparticle tracking analysis. DPSCs were cocultured with MDPC23 cells and MDPC23 cell-derived exosomes. Alizarin Red S staining, ALP staining, qRT‒PCR, western blotting analysis and micro-RNA sequencing were performed. Results: Our study showed that mTORC1 activation in odontoblasts resulted in thicker dentin and higher dentin volume/tooth volume of molars, and it increased the expression levels of the exosome markers CD63 and Alix. In vitro, when DPSCs were cocultured with MDPC23 cells, odontoblastic differentiation was inhibited. However, the inhibition of odontoblastic differentiation was reversed when DPSCs were cocultured with MDPC23 cells with mTORC1 overactivation. To further study the effects of mTORC1 on exosome release from odontoblasts, MDPC23 cells were treated with rapamycin or shRNA-TSC1 to inactivate or activate mTORC1, respectively. The results revealed that exosome release from odontoblasts was negatively correlated with mTORC1 activity. Moreover, exosomes derived from MDPC23 cells with active or inactive mTORC1 inhibited the odontoblastic differentiation of DPSCs at the same concentration. miRNA sequencing analysis of exosomes that were derived from shTSC1-transfected MDPC23 cells, rapamycin-treated MDPC23 cells or nontreated MDPC23 cells revealed that the majority of the miRNAs were similar among these groups. In addition, exosomes derived from odontoblasts inhibited the odontoblastic differentiation of DPSCs, and the inhibitory effect was positively correlated with exosome concentration. Conclusion: mTORC1 regulates exosome release from odontoblasts to inhibit the odontoblastic differentiation of DPSCs, but it does not alter exosomal contents. These findings might provide a new understanding of dental pulp complex regeneration. [ABSTRACT FROM AUTHOR]

Dental Pulp Stem Cells (DPSCs) offer great potential in regenerative medicine. Odontoblasts are naturally occurring cells that are responsible for constructing dentin for restorative dental purposes. Yet, their potential for use in the restoration of non-dental tissues is also being explored. Because of their similarities to BMSCs, DPSCs may be useful in musculoskeletal regenerative medicine, and they may be easily separated from teeth that have been pulled. Information on stem cells in dental pulp, transplantation, and immunomodulation was culled from the databases Scopus, PubMed, Web of Science, Science Direct, and Google Scholar. Differentiated Pluripotent Stem Cells (DPSCs) are not derived from bone marrow like BMSCs are. These phenotypic and genetic distinctions from BMSCs are being used in neurological and other settings. Pulp tissue regeneration and the therapeutic potential of DPSCs were discussed in this article. Prior to discussing their extensive uses in regenerative medicine, this article provides a brief background on the discovery and function of DPSCs. [ABSTRACT FROM AUTHOR]

Despite decades of research, no therapies are available to halt or slow down the course of neuro-degenerative disorders. Most of the drugs developed to fight neurodegeneration are aimed to alleviate symptoms, but none has proven adequate in altering the course of the pathologies. Cell therapy has emerged as an intriguing alternative to the classical pharmacological approach. Cell therapy consists of the transplantation of stem cells that can be obtained from various embryonal and adult tissues. Whereas the former holds notable ethical issue, adult somatic stem cells can be obtained without major concerns. However, most adult stem cells, such as those derived from the bone marrow, are committed toward the mesodermal lineage, and hence need to be reprogrammed to induce the differentiation into the neurons. The discovery of neural crest stem cells in the dental pulp, both in adults' molar and in baby teeth (dental pulp stem cells and stem cells from human exfoliated deciduous teeth, respectively) prompted researchers to investigate their utility as therapy in nervous system disorders. In this review, we recapitulate the advancements on the application of these stem cells in preclinical models of neurodegenerative diseases, highlighting differences and analogies in their maintenance, differentiation, and potential clinical application. [ABSTRACT FROM AUTHOR]

Abstract Background Pulpitis often are characterized as sustained inflammation and impaired pulp self-repair. Circular RNAs (circRNAs) have been reported to be involved in the development of inflammation, but their influence in pulpitis is still unidentified, which was examined in our research. Methods In this study, TNF-α (20 ng/mL) was used to treat DPSCs, then MTS identified cell proliferation. The circRNAs profile in DPSCs with or without TNF-α treatment was evaluated using RNA sequencing and subsequently by bioinformatics analysis. After that, the circular structure was assessed using agarose gel electrophoresis, followed by Sanger sequencing. And the circRNAs expression was ratified using quantitative real-time polymerase chain reaction in cell and tissues samples. Additionally, the plausible mechanism of circRNAs was envisaged, and the circRNA-miRNA-mRNA linkage was plotted using Cytoscape. Results The treatment of TNF-α inhibited cell proliferation capabilities in DPSCs, which also made 1195 circRNA expressions undergo significant alterations. Among these changes, 11 circRNAs associated with inflammation were chosen for circular structure verification, and only seven circRNAs (hsa_circ_0001658, hsa_circ_0001978, hsa_circ_0003910, hsa_circ_0004314, hsa_circ_0004417, hsa_circ_0035915, and hsa_circ_0002545) had circular structure. Additionally, five circRNAs expressions (hsa_circ_0001978, hsa_circ_0003910, hsa_circ_0004314, hsa_circ_0004417, and hsa_circ_0035915) had significantly altered between with or without TNF-α treated DPSCs. Furthermore, hsa_circ_0001978 and hsa_circ_0004417 were increased in patients suffering from pulpitis. Furthermore, their ceRNA linkage and Kyoto Encyclopedia of Genes and Genomes analysis suggested that these two circRNAs may participate in the inflammation development of pulpitis via mitogen-activated protein kinase and the Wnt signaling pathway. Conclusion This study revealed that the circRNAs profile was altered in TNF-α treated DPSCs. Also, hsa_circ_0001978 and hsa_circ_0004417 may be involved in the inflammation progress of pulpitis. These outcomes provided the latest information for additional research on pulpitis.

Background: Pulpitis often are characterized as sustained inflammation and impaired pulp self-repair. Circular RNAs (circRNAs) have been reported to be involved in the development of inflammation, but their influence in pulpitis is still unidentified, which was examined in our research. Methods: In this study, TNF-α (20 ng/mL) was used to treat DPSCs, then MTS identified cell proliferation. The circRNAs profile in DPSCs with or without TNF-α treatment was evaluated using RNA sequencing and subsequently by bioinformatics analysis. After that, the circular structure was assessed using agarose gel electrophoresis, followed by Sanger sequencing. And the circRNAs expression was ratified using quantitative real-time polymerase chain reaction in cell and tissues samples. Additionally, the plausible mechanism of circRNAs was envisaged, and the circRNA-miRNA-mRNA linkage was plotted using Cytoscape. Results: The treatment of TNF-α inhibited cell proliferation capabilities in DPSCs, which also made 1195 circRNA expressions undergo significant alterations. Among these changes, 11 circRNAs associated with inflammation were chosen for circular structure verification, and only seven circRNAs (hsa_circ_0001658, hsa_circ_0001978, hsa_circ_0003910, hsa_circ_0004314, hsa_circ_0004417, hsa_circ_0035915, and hsa_circ_0002545) had circular structure. Additionally, five circRNAs expressions (hsa_circ_0001978, hsa_circ_0003910, hsa_circ_0004314, hsa_circ_0004417, and hsa_circ_0035915) had significantly altered between with or without TNF-α treated DPSCs. Furthermore, hsa_circ_0001978 and hsa_circ_0004417 were increased in patients suffering from pulpitis. Furthermore, their ceRNA linkage and Kyoto Encyclopedia of Genes and Genomes analysis suggested that these two circRNAs may participate in the inflammation development of pulpitis via mitogen-activated protein kinase and the Wnt signaling pathway. Conclusion: This study revealed that the circRNAs profile was altered in TNF-α treated DPSCs. Also, hsa_circ_0001978 and hsa_circ_0004417 may be involved in the inflammation progress of pulpitis. These outcomes provided the latest information for additional research on pulpitis. [ABSTRACT FROM AUTHOR]

This study evaluated exposed dogs’ dental pulp response after direct capping with autologous dental pulp stem cells (DPSCs) and compared the results with MTA (Angelus, Brazil). Sixty teeth from six mongrel dogs were included (n = 60), allocated into four groups; negative control (n = 6), positive control (n = 6), MTA (n = 24), and DPSCs (n = 24). DPSCs were isolated and cultured from extracted first premolars (1premolar/dog). The teeth were prepared for histological and immunohistochemical analyses. Statistical analysis was calculated using ANOVA tests, P ≤ 0.05. MTA induced the formation of an almost complete calcific bridge. DPSCs successfully regenerated the injured pulp and dentin. The comparison between the capping groups and time intervals had a statistically significant effect, as well as, the interaction between the two variables. DPSCs had greater ability in the reparative and regenerative process of dentin and odontoblastic differentiation by having a significantly stronger TGFβ1 expression than MTA.

Background/purpose: SHED and DPSC have stem cell regenerative potential, but comparative research on their cytokine profile is rare. This study aimed to investigate and compare cytokine profiles secreted from stem cells from human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSCs). Materials and methods: SHED-conditioned medium (CM) and DPSC-CM were extracted using seven primary and permanent teeth each. Cytokine membrane array was performed for each CM to quantify and compare the secretomes of 120 cytokines. Enzyme-linked immunosorbent assay, immunocytochemistry, and immunohistochemistry analysis were performed to demonstrate cytokine membrane array analysis. Results: Significant differences were observed in the expression levels of 68 cytokines–27 and 41 cytokines were 1.3-fold more strongly expressed in SHED-CM and DPSC-CM, respectively. Cytokines involved in immunomodulation, odontogenesis and osteogenesis were more strongly expressed in SHED-CM. Cytokines involved in angiogenesis were detected more strongly in DPSCs-CM. SHED and DPSCs have distinctive cytokine profiles and characteristics in terms of their stem cell regenerative potential. Conclusion: These observations suggest that SHED may have a better cytokine profile related to inflammatory, proliferative, osteogenic, and odontogenic potential.

Abstract Background Dental pulp stem cells (DPSCs) are low immunogenic and hold immunomodulatory properties that, along with their well-established multi-potency, might enhance their potential application in autoimmune and inflammatory diseases. The present study focused on the ability of DPSCs to modulate the inflammatory microenvironment through PD1/PD-L1 pathway. Methods Inflammatory microenvironment was created in vitro by the activation of T cells isolated from healthy donors and rheumatoid arthritis (RA) patients with anti-CD3 and anti-CD28 antibodies. Direct and indirect co-cultures between DPSCs and PBMCs were carried out to evaluate the activation of immunomodulatory checkpoints in DPSCs and the inflammatory pattern in PBMCs. Results Our data suggest that the inflammatory stimuli trigger DPSCs immunoregulatory functions that can be exerted by both direct and indirect contact. As demonstrated by using a selective PD-L1 inhibitor, DPSCs were able to activate compensatory pathways targeting to orchestrate the inflammatory process by modulating pro-inflammatory cytokines in pre-activated T lymphocytes. The involvement of PD-L1 mechanism was also observed in autologous inflammatory status (pulpitis) and after direct exposure to pre-activated T cells from RA patients suggesting that immunomodulatory/anti-inflammatory properties are strictly related to their stemness status. Conclusions Our findings point out that the communication with the inflammatory microenvironment is essential in licensing their immunomodulatory properties.

The human dental pulp stem cells (hDPSCs) are one of the readily available sources of multipotent mesenchymal stem cells (MSCs) and can be considered as a type of tool cells for cell‐based therapies. However, the main limitation in the clinical use of these cells is DPSC senescence, which can be induced by lipopolysaccharide (LPS) of oral pathogenic bacteria. Up to now, far little attention has been paid to exploring the molecular mechanisms of senescence in DPSCs. So, the current study aimed to investigate the underlying molecular mechanism of senescence in hDPSCs stimulated with Porphyromonas gingivalis (P. gingivalis) and Escherichia coli (E. coli)‐derived LPSs, by evaluating both mRNA and protein expression of four important senescence‐related genes, including TP53, CDKN1A, CDKN2A and SIRT1. To this purpose, hDPSCs were stimulated with different LPSs for 6, 24 and 48 h and then the gene expression was evaluated using quantitative real‐time polymerase chain reaction (qPCR) and western blotting. Following stimulation with P. gingivalis and E. coli‐derived LPSs, the relative mRNA and protein expression of all genes were significantly up‐regulated in a time‐dependent manner, as compared with unstimulated hDPSCs. Moreover, the hDPSCs stimulated with P. gingivalis LPS for 6 and 24 h had the highest mRNA expression of CDKN1A and SIRT1, respectively (p < 0.0001), whereas the highest mRNA expression of CDKN2A and TP53 was seen in hDPSCs stimulated with E. coli LPS for 48 h (p < 0.0001). In summary, because DPSCs have been reported to have therapeutic potential for several cell‐based therapies, targeting molecular mechanisms aiming at preventing DPSC senescence could be considered a valuable strategy. [ABSTRACT FROM AUTHOR]

Vital pulp treatment (VPT) is crucial for preserving the health and function of the tooth in cases where the pulp tissue remains vital despite exposure. Various materials are introduced for this purpose. However, challenges such as low strength, high solubility, and tooth discoloration persist. Methylmethacrylate-based cement (MC) offers excellent sealing ability, feasibility, and mechanical properties, making it a promising alternative for VPT. Phosphate-based glass (PBG) has the potential to promote hard tissue regeneration by releasing key inducers, phosphorus (P) and calcium (Ca), for reparative odontogenesis. This study investigates PBG-integrated MC (PIMC) by characterizing its properties, assessing human dental pulp stem cell activity related to initial inflammatory adaptation and odontogenic differentiation, and evaluating hard tissue formation using an in vivo dog pulpotomy model. Results indicate that a 5% PBG-integrated MC (5PIMC) maintains the physicochemical properties of MC. Furthermore, 5PIMC demonstrates cytocompatibility, excellent expression of osteo/odontogenic markers, and resistance to inflammatory markers, significantly outperforming MC. Enhanced hard tissue formation is observed in the dental pulp of mongrel dog teeth treated with 5PIMC. These findings suggest that 5PIMC could be an optimal and suitable material for reparative odontogenesis through VPT., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Background: A major challenge in bone tissue regeneration is the use of right combination of stem cells with osteoinductive biomaterials. Hence, the present in vitro study was aimed at evaluating the effect of mineralized teeth matrix (MTM) and demineralized teeth matrix (DTM) on the selected cellular and biological characteristics of human dental pulp stem cells (DPSCs). Methods: Established DPSCs were cultured in conditioned media (CM) of MTM and DTM and analyzed on their morphology, proliferation rate, population doubling time (PDT), viability, migration ability, ploidy and expression of cell surface markers, Further, the effect of MTM and DTM on the biocompatibility and osteogenic differentiation ability of DPSCs was evaluated. Results: The DPSCs exhibited a fibroblast-like morphology with >80% viability. Cells were highly proliferative with an average PDT of 61 ​± ​12 ​h. A greater proliferation of DPSCs in the scratched area was observed when cultured in CM of teeth matrix compared to the cells in basal media. Moreover, no chromosomal abnormalities were induced during the culture of DPSCs. Flow cytometry analysis showed that DPSCs in basal media and CM of MTM and DTM were positive for CD29, CD44, CD73, CD90 (>70%), and negative for CD34 and CD45 (

Abstract Background During the process of deep decay, when decay approaches the pulp, an immune response is triggered inside the pulp, which activates the complement cascade. The effect of complement component 5a (C5a) on the differentiation of dental pulp mesenchymal stem cells (DPSCs) is related to dentin reparation. The aim of the present study was to stimulate DPSCs with different concentrations of C5a and evaluate the differentiation of odontoblasts using dentin sialoprotein (DSP). Methods DPSCs were divided into the following six groups: (i) Control; (ii) DPSCs treated with 50 ng/ml C5a; (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a. Flow cytometry and multilineage differentiation potential were used to identify DPSCs. Mineralization induction, Real-time PCR and Western blot were conducted to evaluate the differentiation of odontoblast in the 6 groups. Result DPSCs can express mesenchymal stem cell markers, including CD105, CD90, CD73 and, a less common marker, mesenchymal stromal cell antigen-1. In addition, DPSCs can differentiate into adipocytes, neurocytes, chondrocytes and odontoblasts. All six groups formed mineralized nodules after 28 days of culture. Reverse transcription-quantitative PCR and western blotting indicated that the high concentration C5a groups expressed higher DSP levels and promoted DPSC differentiation, whereas the low concentration C5a groups displayed an inhibitory effect. Conclusion In this study, the increasing concentration of C5a, which accompanies the immune process in the dental pulp, has demonstrated an enhancing effect on odontoblast differentiation at higher C5a concentrations in vitro.

Alzheimer’s disease (AD) is a destructive neurodegenerative disease with the progressive dysfunction, structural disorders and decreased numbers of neurons in the brain, which leads to long-term memory impairment and cognitive decline. There is a growing consensus that the development of AD has several molecular mechanisms similar to those of other neurodegenerative diseases, including excessive accumulation of misfolded proteins and neurotoxic substances produced by hyperactivated microglia. Nonetheless, there is currently a lack of effective drug candidates to delay or prevent the progression of the disease. Based on the excellent regenerative and reparative capabilities of stem cells, the application of them to repair or replace injured neurons carries enormous promise. Dental pulp stem cells (DPSCs), originated from ectomesenchyme of the cranial neural crest, hold a remarkable potential for neuronal differentiation, and additionally express a variety of neurotrophic factors that contribute to a protective effect on injured neuronal cells. Notably, DPSCs can also express immunoregulatory factors to control neuroinflammation and potentiate the regeneration and recovery of injured neurons. These extraordinary features along with accessibility make DPSCs an attractive source of postnatal stem cells for the regeneration of neurons or protection of existing neural circuitry in the neurodegenerative diseases. The present reviews the latest research advance in the pathophysiology of AD and elaborate the neurodifferentiation and neuroprotective properties of DPSCs as well as their application prospects in AD.

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

Despite decades of research, no therapies are available to halt or slow down the course of neuro-degenerative disorders. Most of the drugs developed to fight neurodegeneration are aimed to alleviate symptoms, but none has proven adequate in altering the course of the pathologies. Cell therapy has emerged as an intriguing alternative to the classical pharmacological approach. Cell therapy consists of the transplantation of stem cells that can be obtained from various embryonal and adult tissues. Whereas the former holds notable ethical issue, adult somatic stem cells can be obtained without major concerns. However, most adult stem cells, such as those derived from the bone marrow, are committed toward the mesodermal lineage, and hence need to be reprogrammed to induce the differentiation into the neurons. The discovery of neural crest stem cells in the dental pulp, both in adults’ molar and in baby teeth (dental pulp stem cells and stem cells from human exfoliated deciduous teeth, respectively) prompted researchers to investigate their utility as therapy in nervous system disorders. In this review, we recapitulate the advancements on the application of these stem cells in preclinical models of neurodegenerative diseases, highlighting differences and analogies in their maintenance, differentiation, and potential clinical application.

Abstract Background Premature exfoliation of the deciduous teeth is a common manifestation in childhood patients with hypophosphatasia (HPP), which is an autosomal inherited disease caused by ALPL mutations. Dysplasia of the cementum, dentin, and alveolar bone has been proposed to be the main reasons for the exfoliation of teeth, while the extraordinarily complex intracellular mechanisms remain elusive. Dental pulp stem cells (DPSCs) have been demonstrated to successfully regenerate functional pulp-dentin-like tissue. Dental pulp cells derived from HPP patients impaired mineralization; however, insight into the deeper mechanism is still unclear. Methods The effects of ALPL on odontoblastic differentiation of DPSCs from HPP patient were assessed by Alizarin Red staining, immunofluorescent staining, Western blot and RT-PCR, and micro-CT assays. Result Here, we found DPSCs from HPP patient exhibited low ALP activity and impaired odontoblastic differentiation. Meanwhile, we found that loss of function of ALPL reduced phosphorylation of GSK3β in DPSCs. While GSK3β rephosphorylation improved odontoblastic differentiation of HPP DPSCs with LiCl treatment. Finally, we demonstrated systemic LiCl injection ameliorated tooth-associated defects in ALPL+/− mice by enhanced phosphorylation of GSK3β in the teeth. Conclusions Our study indicates that ALPL regulates odontoblastic differentiation of DPSCs and provides useful information for understanding how ALPL deficiency led to tooth dysplasia and, ultimately, may inform efforts at improvement tooth defects in HPP patients.

Bioprinting technology promotes innovation of fabricating tissue engineered constructs. Dental pulp stem cells (DPSCs) have significant advantages over classical bone mesenchymal stem cells (BMSCs) and are a promising seed cell candidate for bone engineering bioprinting. However, current reports about bioprinted DPSCs for bone regeneration are incomprehensive. The objective of this study was to investigate the osteogenic potential of DPSCs in methacrylate gelatin (GelMA) hydrogels bioprinted scaffolds in vitro and in vivo. Firstly, we successfully bioprinted GelMA with different concentrations embedded with or without DPSCs. Printability, physical features and biological properties of the bioprinted constructs were evaluated. Then, osteogenic differentiation levels of DPSCs in bioprinted constructs with various concentrated GelMA were compared. Finally, effects of bioprinted constructs on cranial bone regeneration were evaluated in vivo. The results of our study demonstrated that 10% GelMA had higher compression modulus, smaller pores, lower swelling and degradation rate than 3% GelMA. Twenty-eight days after printing, DPSCs in three groups of bioprinted structures still maintained high cell activities (>90%). Moreover, DPSCs in 10% GelMA showed an upregulated expression of osteogenic markers and a highly activated ephrinB2/EphB4 signaling, a signaling involved in bone homeostasis. In vivo experiments showed that DPSCs survived at a higher rate in 10% GelMA, and more new bones were observed in DPSC-laden 10% GelMA group, compared with GelMA of other concentrations. In conclusion, bioprinted DPSC-laden 10% GelMA might be more appropriate for bone regeneration application, in contrast to GelMA with other concentrations. ● 10% GelMA bioprinted scaffolds loaded with DPSCs have good osteogenic potential. ● Activation of ephrinB2/EphB4 signaling promoted osteogenic differentiation of DPSCs in 10% GelMA. ● The 10% GelMA bioprinted scaffold loaded with DPSCs has good bone regeneration effect in vivo. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Dental pulp stem cells (DPSCs) are adult stem cells of neural crest origin, are readily available, have good self-renewal and multidirectional differentiation properties, can differentiate into a variety of cells, are abundant, less harmful to donate, have no ethical issues, low immunogenicity, and therefore, are widely used as seed cells in the field of tissue engineering and regenerative medicine. MicroRNA (miRNA) is a single- stranded non-coding small RNA consisting of about 22 nucleotides, which plays an important regulatory role in various aspects of cellular activities, such as proliferation, differentiation, and apoptosis. In this paper, we review the regulatory role of small RNA in the differentiation of DPSCs and its mechanism in the past 5 years. This paper aims to reveal the important role of miRNAs in differentiation in DPSCs. MATERIALS AND METHODS: MicroRNAs (miRNAs), differentiation, and DPSCs were extensively searched in three databases from 2014 to 2021. These databases include PubMed, Cochrane Library, Embase. RESULTS: Our study reviews the microRNAs (miR-145, miR-143-3p, miR-140-5p, miR-488, miR- 218, miR-125a-3p, miR-27a-5p, miR-223, miR-21, miR-143, miR-215, miR-219a-1-3p, miR-31, miR- 496, miR-218, miR-24-3p, miR-146a-5p, miR-196a, miR-188-3p, miR-424, miR-378a, miR-135, miR- 124) in the differentiation of DPSCs. CONCLUSIONS: A large body of evidence supports the involvement of miRNAs in differentiation associated with mesenchymal stem cells (MSCs), although the mechanisms involved are not yet clear. Most of the current studies are from in vitro studies, but the ultimate goal is to apply these studies to the clinic, and studies involving in vivo models are needed. [ABSTRACT FROM AUTHOR]

This study evaluated exposed dogs' dental pulp response after direct capping with autologous dental pulp stem cells (DPSCs) and compared the results with MTA (Angelus, Brazil). Sixty teeth from six mongrel dogs were included (n = 60), allocated into four groups; negative control (n = 6), positive control (n = 6), MTA (n = 24), and DPSCs (n = 24). DPSCs were isolated and cultured from extracted first premolars (1premolar/dog). The teeth were prepared for histological and immunohistochemical analyses. Statistical analysis was calculated using ANOVA tests, P ≤ 0.05. MTA induced the formation of an almost complete calcific bridge. DPSCs successfully regenerated the injured pulp and dentin. The comparison between the capping groups and time intervals had a statistically significant effect, as well as, the interaction between the two variables. DPSCs had greater ability in the reparative and regenerative process of dentin and odontoblastic differentiation by having a significantly stronger TGFβ1 expression than MTA. [Display omitted] [ABSTRACT FROM AUTHOR]

SHED and DPSC have stem cell regenerative potential, but comparative research on their cytokine profile is rare. This study aimed to investigate and compare cytokine profiles secreted from stem cells from human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSCs). SHED-conditioned medium (CM) and DPSC-CM were extracted using seven primary and permanent teeth each. Cytokine membrane array was performed for each CM to quantify and compare the secretomes of 120 cytokines. Enzyme-linked immunosorbent assay, immunocytochemistry, and immunohistochemistry analysis were performed to demonstrate cytokine membrane array analysis. Significant differences were observed in the expression levels of 68 cytokines–27 and 41 cytokines were 1.3-fold more strongly expressed in SHED-CM and DPSC-CM, respectively. Cytokines involved in immunomodulation, odontogenesis and osteogenesis were more strongly expressed in SHED-CM. Cytokines involved in angiogenesis were detected more strongly in DPSCs-CM. SHED and DPSCs have distinctive cytokine profiles and characteristics in terms of their stem cell regenerative potential. These observations suggest that SHED may have a better cytokine profile related to inflammatory, proliferative, osteogenic, and odontogenic potential. [ABSTRACT FROM AUTHOR]

Background: Dental pulp stem cells (DPSCs) are low immunogenic and hold immunomodulatory properties that, along with their well-established multi-potency, might enhance their potential application in autoimmune and inflammatory diseases. The present study focused on the ability of DPSCs to modulate the inflammatory microenvironment through PD1/PD-L1 pathway. Methods: Inflammatory microenvironment was created in vitro by the activation of T cells isolated from healthy donors and rheumatoid arthritis (RA) patients with anti-CD3 and anti-CD28 antibodies. Direct and indirect co-cultures between DPSCs and PBMCs were carried out to evaluate the activation of immunomodulatory checkpoints in DPSCs and the inflammatory pattern in PBMCs. Results: Our data suggest that the inflammatory stimuli trigger DPSCs immunoregulatory functions that can be exerted by both direct and indirect contact. As demonstrated by using a selective PD-L1 inhibitor, DPSCs were able to activate compensatory pathways targeting to orchestrate the inflammatory process by modulating pro-inflammatory cytokines in pre-activated T lymphocytes. The involvement of PD-L1 mechanism was also observed in autologous inflammatory status (pulpitis) and after direct exposure to pre-activated T cells from RA patients suggesting that immunomodulatory/anti-inflammatory properties are strictly related to their stemness status. Conclusions: Our findings point out that the communication with the inflammatory microenvironment is essential in licensing their immunomodulatory properties. [ABSTRACT FROM AUTHOR]

Abstract Background Alveolar bone loss is a frequent occurrence. Dental pulp stem cells (DPSCs) which have invasive accessibility and high osteogenic potential is a promising source for cell-based bone regeneration. EphrinB2 is involved in bone homeostasis and osteogenesis. The aim of this study was to investigate the effect and mechanism of ephrinB2 overexpression on osteogenic differentiation of DPSCs and bone defect repair. Methods EphrinB2 expression was analyzed during osteogenic induction of human DPSCs (hDPSCs). Endogenous ephrinB2 expression in hDPSCs was then upregulated using EfnB2 lentiviral vectors. The effect of ephrinB2 overexpression on osteogenic differentiation capacity of hDPSCs was investigated in vitro, and activation of ephrinB2-EphB4 bidirectional signaling in ephrinB2-overexpressing hDPSCs was detected. In vivo, a canine alveolar bone defect model was established and canine DPSCs (cDPSCs) were cultured, characterized, EfnB2-tranfected, and combined with a PuraMatrix scaffold. Micro-CT analysis was performed to evaluate the therapeutic effect of ephrinB2-overexpressing cDPSCs on bone defect repair. Results EphrinB2 was upregulated after osteogenic induction of hDPSCs. EphrinB2 overexpression enhanced osteogenic differentiation capacity of hDPSCs in vitro. Moreover, p-ephrinB2 instead of p-EphB4 was upregulated by ephrinB2 overexpression, and activation of ephrinB2-mediated reverse signaling promoted osteogenic differentiation of hDPSCs. In a canine bone defect model, ephrinB2 overexpression in cDPSCs significantly improved trabecular bone volume per tissue volume (BV/TV) and trabecular thickness, as demonstrated by radiographic analysis. Conclusions EphrinB2 overexpression enhanced osteogenic potential of DPSCs partially via upregulation of ephrinB2-mediated reverse signaling and effectively promoted alveolar bone defect repair.