Mechanical Memory and NF-κB Signaling in Dental Pulp Stem Cell Odontogenic Differentiation.

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.

BackgroundMagnesium (Mg2+)-enriched microenvironment promotes odontogenic differentiation in human dental pulp stem cells (DPSCs), but the regulatory mechanisms remain undefined. The aim of this work was to assess magnesium’s function in the above process and to explore the associated signaling pathway.MethodsDPSCs underwent culture in odontogenic medium with the addition of 0, 1, 5, or 10 mM MgCl2. Intracellular Mg2+ levels in DPSCs were evaluated flow cytometrically using Mag-Fluo-4-AM. Mg2+-entry was inhibited by TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB). RNA-Sequencing was carried out for assessing transcriptome alterations in DPSCs during odontogenic differentiation associated with high extracellular Mg2+. KEGG pathway analysis was performed to determine pathways related to the retrieved differentially expressed genes (DEGs). Immunoblot was performed for assessing magnesium’s role and exploring ERK/BMP2/Smads signaling.ResultsMg2+-enriched microenvironment promoted odontogenic differentiation in DPSCs via intracellular Mg2+ increase. Consistently, the positive effect of high extracellular Mg2+ on odontogenic differentiation in DPSCs was blocked by 2-APB, which reduced Mg2+ entry. RNA-sequencing identified 734 DEGs related to odontogenic differentiation in DPSCs in the presence of high extracellular Mg2+. These DEGs participated in many cascades such as MAPK and TGF-β pathways. Consistently, ERK and BMP2/Smads pathways were activated in DPSCs treated with high extracellular Mg2+. In agreement, ERK signaling inhibition by U0126 blunted the effect of high extracellular Mg2+ on mineralization and odontogenic differentiation in DPSCs. Interestingly, BMP2, BMPR1, and phosphorylated Smad1/5/9 were significantly decreased by U0126, indicating that BMP2/Smads acted as downstream of ERK.ConclusionsMg2+-enriched microenvironment promotes odontogenic differentiation in DPSCs by activating ERK/BMP2/Smads signaling via intracellular Mg2+ increase. This study revealed that Mg2+-enriched microenvironment could be used as a new strategy for dental pulp regeneration.

Cav1.2 of L-type Calcium Channel Is a Key Factor for the Differentiation of Dental Pulp Stem Cells

Odontogenic differentiation of dental pulp-derived stem cells on tricalcium phosphate scaffolds

The multipotent nature of dental pulp stem cells (DPSCs) promises regenerative endodontic potentials. Alterations in microenvironment have been shown to control the differentiation phenotypes of DPSCs. Understanding the biological mechanisms and finding the optimal DPSC differentiation protocols are crucial for successful DPSC engineering strategies in pulp and dentin healing. The aim of this study is to identify the role of p38 mitogen-activated protein kinase (p38) under normal and oxygen-deprived conditions (2%) to reveal its effect on odontogenic DPSC differentiation. Human DPSCs were isolated from healthy molars and underwent odontogenic differentiation in regular and osteogenic media treated with SB203580, a p38 inhibitor, for 72 hours, and then swapped with osteogenic media for 21 days under hypoxic condition. Immunochemistry and PCR analysis for the various odontogenic differentiation genes and proteins were performed. Our PCR data demonstrate that p38 inhibition resulted in a significant upregulation in odontogenic gene expressions such as DMP-1, DSPP, RUNX, and OSX in normal conditions. Under hypoxia, this effect was reversed. These results were further supported by DSPP immunohistochemistry. The DSPP expression under hypoxia was significantly weaker compared to the control. Our results indicate that p38 represents a negative regulator of the odontogenic DPSC differentiation in normoxia. Under hypoxia, p38 exerts a positive function of DPSC differentiation. Taken together, we identified the p38 and oxygen level as crucial factors to control odontogenic DPSC differentiation providing their essential roles in designing for successful pulp-dentin complex engineering strategies.

Aim Inflammation is a complex host response to harmful infection or injury, and it seems to play a crucial role in tissue regeneration both positively and negatively. We have previously demonstrated that the activation of the complement C5a pathway affects dentin-pulp regeneration. However, limited information is available to understand the role of the complement C5a system related to inflammation-mediated dentinogenesis. The aim of this study was to determine the role of complement C5a receptor (C5aR) in regulating lipopolysaccharide (LPS)-induced odontogenic differentiation of dental pulp stem cells (DPSCs). Material and Methods Human DPSCs were subjected to LPS-stimulated odontogenic differentiation in dentinogenic media treated with the C5aR agonist and antagonist. A putative downstream pathway of the C5aR was examined using a p38 mitogen-activated protein kinase (p38) inhibitor (SB203580). Results Our data demonstrated that inflammation induced by the LPS treatment potentiated DPSC odontogenic differentiation and that this is C5aR dependent. C5aR signaling controlled the LPS-stimulated dentinogenesis by regulating the expression of odontogenic lineage markers like dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP-1). Moreover, the LPS treatment increased the total p38, and the active form of p38 expression, and treatment with SB203580 abolished the LPS-induced DSPP and DMP-1 increase. Conclusions These data suggest a significant role of C5aR and its putative downstream molecule p38 in the LPS-induced odontogenic DPSCs differentiation. This study highlights the regulatory pathway of complement C5aR/p38 and a possible therapeutic approach for improving the efficiency of dentin regeneration during inflammation.

Dental pulp stem cells (DPSCs) regulate immune responses; however, their heterogeneity in deep caries remains unclear. This study aimed at investigating the role of intercellular adhesion molecule 1-positive DPSCs (ICAM1+ DPSCs) within the immune microenvironment of deep carious pulp tissue to develop therapeutic strategies. Single-cell sequencing was used to compare cellular profiles between deep caries and healthy pulp tissues. ICAM1+ DPSCs were quantified using immunofluorescence/flow cytometry in human/mouse models and sorted for functional analyses. Odontogenic differentiation was assessed using alkaline phosphatase/Alizarin Red staining, while inflammatory mediator production was assessed using RT-qPCR, Western Blot, ELISA, SCENIC and RNA-seq. THP-1 was cultured in conditioned media from ICAM1+ DPSCs and ICAM1- DPSCs. RT-qPCR, Western Blot and flow cytometry were used to assess the proportion of proinflammatory to reparative THP-1. Macrophage-derived cytokines (IL-1β/4/6/10 and TNF-α) were tested for DPSCs to ICAM1+ differentiation induction. Cellular profiling showed a significant increase in ICAM1+ DPSCs and proinflammatory monocytes in deep carious dental pulp tissue, with ICAM1+ DPSCs closely interacting with mononuclear macrophages. Immunofluorescence and flow cytometry confirmed the increase in ICAM1+ DPSCs in deep caries in the affected human and mouse pulp tissue. Alkaline phosphatase and Alizarin Red staining, SCENIC, RT-qPCR, Western Blot and ELISA revealed decreased odontogenic differentiation in ICAM1+ DPSCs and increased expression of CEBPD, IL-6, CCL2 and CXCL10 in ICAM1+ DPSC cells. RT-qPCR, Western Blot and flow cytometry indicated an elevated proinflammatory to reparative THP-1 ratio for THP-1 that was cultured in ICAM1+ DPSC-conditioned media for 1-3 days. During deep caries progression, TNF-α drives the transformation of DPSCs into inflammatory ICAM1+ DPSCs. This subcluster exhibits impaired odontogenic differentiation capacity, secretes proinflammatory cytokines and chemokines, and enhances macrophage inflammatory activity, contributing to the advancement of deep caries lesions.

BackgroundExosomes derived from dental pulp stem cells (DPSCs) can be used as biomimetic tools to induce odontogenic differentiation of stem cells, but the regulatory mechanisms and functions of exosome-encapsulated microRNAs are still unknown. The present study aimed to clarify the role of microRNAs contained in the exosomes derived from human DPSCs and their potential signaling cascade in odontogenic differentiation.MethodsExosomes were isolated from human DPSCs cultured undergrowth and odontogenic differentiation conditions, named UN-Exo and OD-Exo, respectively. The microRNA sequencing was performed to explore the microRNA profile contained in UN-Exo and OD-Exo. Pathway analysis was taken to detect enriched pathways associated with the predicted target genes of microRNAs. The regulatory roles of a highly expressed microRNA in OD-Exo were investigated through its inhibition or overexpression (miRNA inhibitors and miRNA mimics). Automated western blot was used to identify the function of exosomal microRNA and the roles of TGFβ1/smads pathway in odontogenic differentiation of DPSCs. A luciferase reporter gene assay was used to verify the direct target gene of exosomal miR-27a-5p.ResultsEndocytosis of OD-Exo triggered odontogenic differentiation of DPSCs by upregulating DSP, DMP-1, ALP, and RUNX2 proteins. MicroRNA sequencing showed that 28 microRNAs significantly changed in OD-Exo, of which 7 increased and 21 decreased. Pathway analysis showed genes targeted by differentially expressed microRNAs were involved in multiple signal transductions, including TGFβ pathway. 16 genes targeted by 15 differentially expressed microRNAs were involved in TGFβ signaling. Consistently, automated western blot found that OD-Exo activated TGFβ1 pathway by upregulating TGFβ1, TGFR1, p-Smad2/3, and Smad4 in DPSCs. Accordingly, once the TGFβ1 signaling pathway was inhibited by SB525334, protein levels of p-Smad2/3, DSP, and DMP-1 were significantly decreased in DPSCs treated with OD-Exo. MiR-27a-5p was expressed 11 times higher in OD-Exo, while miR-27a-5p promoted odontogenic differentiation of DPSCs and significantly upregulated TGFβ1, TGFR1, p-Smad2/3, and Smad4 by downregulating the inhibitory molecule LTBP1.ConclusionsThe microRNA expression profiles of exosomes derived from DPSCs were identified. OD-Exo isolated under odontogenic conditions were better inducers of DPSC differentiation. Exosomal microRNAs promoted odontogenic differentiation via TGFβ1/smads signaling pathway by downregulating LTBP1.

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.

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.

Bone marrow stem cells (BMSCs), dental pulp stem cells (DPSCs), and periodontal ligament stem cells (PDLSCs) represent key stem cell sources for regenerative endodontic therapy. Recent studies indicate that these 3 cell types exhibit similar mineralization potential during mineralized induction. This study aimed to compare the mineralization potential (including odontogenic and osteogenic potential) of these 3 mouse-derived cells and identify shared or related circular RNA-mediated regulatory mechanisms. In this study, we observed high expression levels of osteogenic differentiation markers alkaline phosphatase (ALP), RUNX2, and osteocalcin (OCN) in both BMSCs and PDLSCs during mineralization, while key markers for odontoblastic differentiation (DSPP and DMP-1) were significantly upregulated, specifically in DPSCs. Bioinformatics and experimental validation identified circ_015350 as consistently upregulated during mineralization. Functional studies demonstrated that circ_015350 knockdown reduced mineralization markers: ALP and OCN in BMSCs/PDLSCs, while primarily affecting DMP-1 and DSPP in DPSCs. Conversely, circ_015350 overexpression enhanced odonto/osteogenic markers across all cell types, with particularly strong DMP-1 and DSPP elevation in DPSCs. Bioinformatics analysis predicted circ_015350 interactions with 10 microRNAs and 89 RNA-binding proteins, along with involvement in Hippo, PI3K-Akt, and AMPK pathways. Our findings reveal distinct differentiation potential: BMSCs showed greater osteogenic capacity, while DPSCs displayed stronger odontogenic potential. Circ_015350 emerged as a key regulator promoting both odontogenic and osteogenic differentiation in all 3 stem cell types, with particularly pronounced effects on odontogenic differentiation. These results suggest circ_015350 as a potential therapeutic target for dental tissue regeneration, although further investigation is needed to fully elucidate its downstream regulatory mechanisms.

Background/purpose The regeneration of dental-related tissue is a major problem in dentistry. Thus, it is beneficial to develop dental constructs that are fabricated with dental pulp stem cells (DPSCs) and an appropriate scaffold. The present study investigates the level of odontogenic differentiation of human DPSCs on tricalcium phosphate (TCP) scaffolds. Materials and methods We isolated pulp stem cells from human third molars and culture-expanded them through several successive subcultures. The cells from passage 3 were then loaded onto TCP scaffolds and treated with odontogenic supplements (OSs) that included vitamin D3 for a period of 21 days. DPSCs cultivated on TCP without OS, a monolayer culture treated with OS, and normal pulp tissue were the controls. We compared the groups in terms of odontogenic differentiation markers. Results Alkaline phosphatase (ALP) activity and the amount of culture mineralization, as well as the expression levels of dentin sialophosphoprotein ( DSPP ) and dentin matrix acidic phosphoprotein 1 ( DMP1 ) genes tended to be significantly higher in the three-dimensional (3D) cultures treated with OS compared to those 3D cultures without OS and the monolayer culture with OS (P Conclusion The 3D culture system improves odontogenic differentiation of DPSCs. The differentiation level of the cells in 3D culture is significantly lower than that of odontoblasts present in pulp tissue. TCP biomaterial possesses an odontogenic-inducing property.

ContextThe main target in vital pulp therapy and regenerative procedures is to preserve or regenerate the pulp vitality via progenitor stem cells differentiation into secretory terminal cells. This differentiation was suggested to be triggered by the direct contact with the capping material.AimThe aim of this study was to evaluate and compare the effect of three bioactive materials on the odontogenic differentiation potential of human dental pulp stem cells using two different culture mediums.Patients and methodsNanohydroxyapatite, mineral trioxide aggregate and calcium enriched mixture cements were mixed and molded into equal sized cylinders. Isolated dental pulp stem cells from human third molars were characterized and then, the cultured cells were classified according to biomaterials supplementation in odontogenic differentiation medium or in growth medium. Cells without biomaterial supplementation in differentiation medium or in growth medium were served as positive and negative control respectively. After 14 days of incubation, alizarin red staining test was carried out to detect the presence of mineralized nodules in addition to measuring the relative expressions of the odontogenic differentiation genes in the cells by quantitative real-time reverse-transcription PCRs.ResultsBiomaterials cultured with odontogenically induced dental pulp stem cells had more significantly odontogenic differentiation potential than those cultured with uninduced dental pulp stem cells or than dental pulp stem cells cultured with differentiation medium only.ConclusionAll tested materials can promote the odontogenic differentiation of dental pulp stem cells. Therefore, they can be considered as bioactive materials for pulp capping and regenerative applications.

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.

Extracellular IL-37 promotes osteogenic and odontogenic differentiation of human dental pulp stem cells via autophagy