Exosomes 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. Exosomes 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. Endocytosis 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. The 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.

Skin wound healing is a common challenging clinical issue which requires advanced treatment strategies. The present study investigated the therapeutic effects of exosomes derived from dental pulp stem cells (DPSC‑Exos) on cutaneous wound healing and the underlying mechanisms. The effects of DPSC‑Exos on cutaneous wound healing in mice were examined by measuring wound closure rates, and using histological and immunohistochemical analysis. A series of functional assays were performed to evaluate the effects of DPSC‑Exos on the angiogenic activities of human umbilical vein endothelial cells (HUVECs) in vitro. Tandem mass tag‑based quantitative proteomics analysis of DPSCs and DPSC‑Exos was performed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were used to evaluate the biological functions and pathways for the differentially expressed proteins in DPSC‑Exos. Western blot analysis was used to assess the protein levels of cell division control protein 42 (Cdc42) and p38 in DPSC‑Exos and in HUVECs subjected to DPSC‑Exos‑induced angiogenesis. SB203580, a p38 mitogen‑activated protein kinase (MAPK) signaling pathway inhibitor, was employed to verify the role of the p38 MAPK pathway in vitro and in vivo. Histological and immunohistochemical staining revealed that the DPSC‑Exos accelerated wound healing by promoting neovascularization. The DPSC‑Exos promoted the migration, proliferation and capillary formation capacity of HUVECs. Proteomics data demonstrated that proteins contained in DPSC‑Exos regulated vasculature development and angiogenesis. Pathway analysis revealed that proteins expressed in DPSC‑Exos were involved in several pathways, including MAPK pathway. Western blot analysis demonstrated that the DPSC‑Exos increased the protein levels of Cdc42 and phosphorylation of p38 in HUVECs. SB203580 suppressed the angiogenesis induced by DPSC‑Exos. On the whole, the present study demonstrates that DPSC‑Exos accelerate cutaneous wound healing by enhancing the angiogenic properties of HUVECs via the Cdc42/p38 MAPK signaling pathway.

Background: To investigate the odonto-immunomodulatory properties of dental pulp stem cell-derived small extracellular vesicles (DPSCs-sEV), which promote odontogenesis by switching macrophages toward the pro-healing M2 phenotype. Methods: MicroRNA sequencing was carried out for microRNA profiling of DPSCs-sEV. Automated Western blot, qPCR, ELISA, and flow cytometry were performed to identify the functions of microRNA-enriched DPSCs-sEV in macrophages. A luciferase reporter gene assay was carried out to confirm exosomal miR-125a-3p’s direct target gene. DPSCs-sEV-stimulated macrophage-conditioned media were used to promote odontogenesis in DPSCs and explore the mechanism of immune response in DPSCs-SEV-stimulated odontogenesis. DPSCs-sEV were injected into the exposed pulp tissue of rat incisor to investigate the odonto-immunomodulatory properties of DPSCs-sEV in vivo. Results: DPSCs-sEV switched macrophages to the pro-healing M2 phenotype by inhibiting TLR and NFκΒ signaling. MicroRNA sequencing found 81 microRNAs significantly altered in DPSCS-sEV, with miR-125a-3p showing a 12-fold upregulation. Exosomal miR-125a-3p switched macrophages toward the M2 phenotype via inhibiting NFκΒ and TLR signaling via direct IKBKB targeting. Interestingly, DPSCs-sEV and the encapsulated miR-125a-3p enhanced BMP2 release in macrophages, promoting odontogenesis in DPSCs through BMP2 pathway activation. The rat study confirmed that DPSCs-sEV could be used as ideal biomimetic tools to enhance odontogenesis by switching macrophages toward pro-healing M2 cells. Conclusions: We firstly defined the odonto-immunomodulatory properties of microRNA-enriched DPSCs-sEV, which could be used as ideal biomimetic tools to enhance odontogenesis by switching macrophages toward the pro-healing M2 phenotype.

Background: Magnesium (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. Methods: DPSCs 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. Results: Mg²⁺-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. Conclusions: Mg²⁺-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.

Bone marrow mesenchymal stem cells (BMMSCs) are suitable cell sources for dental pulp regeneration, but the mechanism of BMMSCs differentiation into odontogenic lineage remains unknown. The aim of the present study was to reveal the role of magnesium transporter protein 1 (MagT1) and MAPK pathways in the odontogenic differentiation of BMMSCs. The RNA sequencing (RNA-seq) was performed to explore the altered transcriptome of BMMSCs undergoing odontogenic differentiation induced by tooth germ cell-condition medium (TGC-CM). Pathway analysis was conducted to explore enriched pathways of the differential expression signature. Automated western blot, real-time PCR, shRNA lentivirus, and flow cytometry were used to detect the function of MagTl and MAPK pathway in odontogenic differentiation of BMMSCs. RNA-seq identified 622 differentially expressed genes associated with odontogenic differentiation of BMMSCs induced by TGC-CM, some of which were responsible for MAPK pathway. Consistently, we verified that TGC-CM induced odontogenic differentiation of BMMSCs through activating ERK/MAPK pathway, and the inactivation of ERK/MAPK pathway inhibited the odontogenic differentiation induced by TGC-CM. We also found MagT1 protein was significantly increased during odontogenic differentiation of BMMSCs induced by TGC-CMM, in accordance, MagT1 knockdown significantly decreased the extent of mineralized nodules and the protein levels of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSP). Flow cytometry showed that intracellular Mg2+ was significantly reduced in MagT1-knockdown BMMSCs, indicating the suppression of MagT1 inhibited odontogenic differentiation of BMMSCs by decreasing intracellular Mg2+. Finally, we performed RNA-seq to explore the altered transcriptome of MagT1-knockdown BMMSCs undergoing odontogenic differentiation and identified 281 differentially expressed genes, some of which were involved in MAPK pathway. Consistently, automated western blot analysis found the ERK/MAPK pathway was inhibited in MagT1-knockdown BMMSCs during odontogenic differentiation, indicating that suppression of MagT1 inhibited odontogenic differentiation of BMMSCs via ERK/MAPK pathway. This study identified the significant alteration of transcriptome in BMMSCs undergoing odontogenic differentiation induced by TGC-CM. We clarified the pivotal role of MagT1 and ERK/MAPK pathway in odontogenic differentiation of BMMSCs, and suppression of MagT1 inhibited the odontogenic differentiation of BMMSCs by decreasing the intracellular Mg2+ and inactivating ERK/MAPK pathway.

Background: Some studies have reported biological linkages between periodontitis and esophageal cancer, prostate cancer, kidney cancer, hematological malignancy, and melanoma of the skin. This meta-analysis aimed to assess the relationship between periodontitis and the aforementioned five cancers.