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.

Background: Pulpitis is an inflammatory disease, the grade of which is classified according to the level of inflammation. Traditional methods of evaluating the status of dental pulp tissue in clinical practice have limitations. The rapid and accurate diagnosis of pulpitis is essential for determining the appropriate treatment. By integrating different datasets from the Gene Expression Omnibus (GEO) database, we analysed a merged expression matrix of pulpitis, aiming to identify biological pathways and diagnostic biomarkers of pulpitis. Methods: By integrating two datasets (GSE77459 and GSE92681) in the GEO database using the sva and limma packages of R, differentially expressed genes (DEGs) of pulpitis were identified. Then, the DEGs were analysed to identify biological pathways of dental pulp inflammation with Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Set Enrichment Analysis (GSEA). Protein–protein interaction (PPI) networks and modules were constructed to identify hub genes with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape. Results: A total of 470 DEGs comprising 394 upregulated and 76 downregulated genes were found in pulpitis tissue. GO analysis revealed that the DEGs were enriched in biological processes related to inflammation, and the enriched pathways in the KEGG pathway analysis were cytokine-cytokine receptor interaction, chemokine signalling pathway and NF-κB signalling pathway. The GSEA results provided further functional annotations, including complement system, IL6/JAK/STAT3 signalling pathway and inflammatory response pathways. According to the degrees of nodes in the PPI network, 10 hub genes were identified, and 8 diagnostic biomarker candidates were screened: PTPRC, CD86, CCL2, IL6, TLR8, MMP9, CXCL8 and ICAM1. Conclusions: With bioinformatics analysis of merged datasets, biomarker candidates of pulpitis were screened and the findings may be as reference to develop a new method of pulpitis diagnosis.

Background: Long noncoding RNAs (lncRNAs) play an important role in the multiple differentiations of mesenchymal stem cells (MSCs). However, few studies have focused on the regulatory mechanism of lncRNAs in the odontogenic differentiation of human dental pulp stem cells (hDPSCs).Methods: hDPSCs were induced to differentiate into odontoblasts in vitro, and the expression profiles of lncRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in differentiated and undifferentiated cells were obtained by microarray. Bioinformatics analyses including Gene Ontology (GO) analysis, pathway analysis, and binding site prediction were performed for functional annotation of lncRNA. miRNA/odontogenesis-related gene networks and lncRNA-associated ceRNA networks were constructed. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to verify the expression of selected genes. RNA fluorescence in situ hybridization (FISH), qRT-PCR, and western blot analysis were used to explore the location and function of lncRNA-G043225. Dual-luciferase reporter assay was performed to confirm the binding sites of miR-588 with G043225 and Fibrillin 1 (FBN1).Results: We identified 132 lncRNAs, 114 miRNAs, and 172 mRNAs were differentially expressed. GO analysis demonstrated that regulation of the neurogenic locus notch homolog (Notch), Wnt, and epidermal growth factor receptor (ERBB) signaling pathways and activation of mitogen-activated protein kinase (MAPK) activity were related to odontogenic differentiation. Pathway analysis indicated that the most significant pathway was the forkhead box O (FoxO) signaling pathway, which is related to odontogenic differentiation. Two odontogenesis-related gene-centered lncRNA-associated ceRNA networks were successfully constructed. The qRT-PCR validation results were consistent with the microarray analysis. G043225 mainly locating in cytoplasm was proved to promote the odontogenic differentiation of hDPSCs via miR-588 and FBN1.Conclusion: This is the first study revealing lncRNA-associated ceRNA network during odontogenic differentiation of hDPSCs using microarray, and it could provide clues to explore the mechanism of action at the RNA-RNA level as well as novel treatments for dentin regeneration based on stem cells.

Background. Exosomes from human dental pulp stem cells (hDPSCs) were indicated to play a positive role in vascular regeneration processes. But the angiogenic capabilities of exosomes from inflammatory hDPSCs and the underlying mechanism remain unknown. In this study, the inflammatory factor lipopolysaccharide (LPS) was used to stimulate hDPSCs, and exosomes were extracted from these hDPSCs. The proangiogenic potential of exosomes was examined, and the underlying mechanism was studied. Method. Exosomes were isolated from hDPSCs with or without LPS stimulation (N-EXO and LPS-EXO) and cocultured with human umbilical vein endothelial cells (HUVECs). The proangiogenic potential of exosomes was evaluated by endothelial cell proliferation, migration, and tube formation abilities in vitro. To investigate the proangiogenic mechanism of LPS-EXO, microRNA sequencing was performed to explore the microRNA profile of N-EXO and LPS-EXO. Gene Ontology (GO) analysis was used to study the functions of the predicted target genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to estimate the signaling pathways associated with the inflammation-induced angiogenesis process. Result. Compared to the uptake of N-EXO, uptake of LPS-EXO activated the angiogenic potential of HUVECs by promoting the proliferation, migration, and tube formation abilities in vitro. The mRNA expression levels of vascular endothelial growth factor (VEGF) and kinase-insert domain-containing receptor (KDR) in the LPS-EXO group were significantly higher than those in the N-EXO group. MicroRNA sequencing showed that 10 microRNAs were significantly changed in LPS-EXO. Pathway analysis showed that the genes targeted by differentially expressed microRNAs were involved in multiple angiogenesis-related pathways. Conclusion. This study revealed that exosomes derived from inflammatory hDPSCs possessed better proangiogenic potential in vitro. This is the first time to explore the role of exosomal microRNA from hDPSCs in inflammation-induced angiogenesis. This finding sheds new light on the effect of inflammation-stimulated hDPSCs on tissue regeneration.