miRNA-124-3p.1 Inhibits the Osteogenic and Odontogenic Differentiation of SCAPs via MACF1/smad7 Axis

Background: Previous research has indicated that altered expression of micro-RNAs (miRNAs) is in connection with differentiation of stem cells from apical papillae (SCAPs). We investigated the mechanisms that miR-124-3p.1 inhibited osteogenic and odontogenic differentiation of SCAPs. Methods: SCAPs were isolated from dental apical papilla. MiR-124-3p.1 mimic and inhibitor were used for overexpression and knockdown assays. For overexpression and knockdown of microtubule actin cross‐linking factor 1 (MACF1), lentivirus infection and siRNA transfection were performed. Luciferase reporter assay was performed to determine the relationship between miR-124-3p.1 and MACF1. The osteogenic and odontogenic differentiation potential was analyzed by alkaline phosphatase activity analysis (ALP), alizarin red S (ARS) staining, quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR), western blot and immunofluorescence (IF) staining. Results: We observed a time dependent decrease of miR‐124‐3p.1 level in mineralization induction of SCAPs. Further study found that miR‐124‐3p.1 exhibited an inhibitory effect on SCAPs osteo/odontogenic differentiation. Similarly, we found that the overexpression of miR‐124‐3p.1 dramatically inhibited MACF1 protein level in SCAPs and knockdown of miR‐124‐3p.1 significantly increased MACF1 protein level in SCAPs. Moreover, MACF1 was verified as the targeting of miR‐124‐3p.1. Meanwhile, the expression of MACF1 was related to smad7 nuclear translocation.Conclusion: Collectively, diverse data demonstrated that miR‐124‐3p.1 is a regulator of MACF1/smad7, playing plays an important role in osteogenic and odontogenic differentiation of SCAPs via MACF1/smad7 axis.

Tailoring Silicon Nitride Surface Chemistry for Facilitating Odontogenic Differentiation of Rat Dental Pulp Cells

Silicon nitride (Si3N4) can facilitate bone formation; hence, it is used as a biomaterial in orthopedics. Nevertheless, its usability for dentistry is unexplored. The aim of the present study was to investigate the effect of Si3N4 granules for the proliferation and odontogenic differentiation of rat dental pulp cells (rDPCs). Four different types of Si3N4 granules were prepared, which underwent different treatments to form pristine as-synthesized Si3N4, chemically treated Si3N4, thermally treated Si3N4, and Si3N4 sintered with 3 wt.% yttrium oxide (Y2O3). rDPCs were cultured on or around the Si3N4 granular beds. Compared with the other three types of Si3N4 granules, the sintered Si3N4 granules significantly promoted cellular attachment, upregulated the expression of odontogenic marker genes (Dentin Matrix Acidic Phosphoprotein 1 and Dentin Sialophosphoprotein) in the early phase, and enhanced the formation of mineralization nodules. Furthermore, the water contact angle of sintered Si3N4 was also greatly increased to 40°. These results suggest that the sintering process for Si3N4 with Y2O3 positively altered the surface properties of pristine as-synthesized Si3N4 granules, thereby facilitating the odontogenic differentiation of rDPCs. Thus, the introduction of a sintering treatment for Si3N4 granules is likely to facilitate their use in the clinical application of dentistry.

miR-140-3p enhanced the osteo/odontogenic differentiation of DPSCs via inhibiting KMT5B under hypoxia condition

Human dental pulp stem cells (DPSCs) have emerged as an important source of stem cells in the tissue engineering, and hypoxia will change various innate characteristics of DPSCs and then affect dental tissue regeneration. Nevertheless, little is known about the complicated molecular mechanisms. In this study, we aimed to investigate the influence and mechanism of miR-140-3p on DPSCs under hypoxia condition. Hypoxia was induced in DPSCs by Cobalt chloride (CoCl2) treatment. The osteo/dentinogenic differentiation capacity of DPSCs was assessed by alkaline phosphatase (ALP) activity, Alizarin Red S staining and main osteo/dentinogenic markers. A luciferase reporter gene assay was performed to verify the downstream target gene of miR-140-3p. This research exhibited that miR-140-3p promoted osteo/dentinogenic differentiation of DPSCs under normoxia environment. Furthermore, miR-140-3p rescued the CoCl2-induced decreased osteo/odontogenic differentiation potentials in DPSCs. Besides, we investigated that miR-140-3p directly targeted lysine methyltransferase 5B (KMT5B). Surprisingly, we found inhibition of KMT5B obviously enhanced osteo/dentinogenic differentiation of DPSCs both under normoxia and hypoxia conditions. In conclusion, our study revealed the role and mechanism of miR-140-3p for regulating osteo/dentinogenic differentiation of DPSCs under hypoxia, and discovered that miR-140-3p and KMT5B might be important targets for DPSC-mediated tooth or bone tissue regeneration.

Novel Bioactive Adhesive Monomer CMET Promotes Odontogenic Differentiation and Dentin Regeneration

This study aimed to evaluate the in vitro effect of the novel bioactive adhesive monomer CMET, a calcium salt of 4-methacryloxyethyl trimellitate acid (4-MET), on human dental pulp stem cells (hDPSCs) and its capacity to induce tertiary dentin formation in a rat pulp injury model. Aqueous solutions of four tested materials [4-MET, CMET, Ca(OH)2, and mineral trioxide aggregate (MTA)] were added to the culture medium upon confluence, and solvent (dH2O) was used as a control. Cell proliferation was assessed using the Cell Counting Kit-8 assay, and cell differentiation was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. The mineralization-inducing capacity was evaluated using alizarin red S staining and an alkaline phosphatase activity assay. For an in vivo experiment, a mechanical pulp exposure model was prepared on Wistar rats; damaged pulp was capped with Ca(OH)2 or CMET. Cavities were sealed with composite resin, and specimens were assessed after 14 and 28 days. The in vitro results showed that CMET exhibited the lowest cytotoxicity and highest odontogenic differentiation capacity among all tested materials. The favorable outcome on cell mineralization after treatment with CMET involved p38 and c-Jun N-terminal kinases signaling. The nuclear factor kappa B pathway was involved in the CMET-induced mRNA expression of odontogenic markers. Similar to Ca(OH)2, CMET produced a continuous hard tissue bridge at the pulp exposure site, but treatment with only CMET produced a regular dentinal tubule pattern. The findings suggest that (1) the evaluated novel bioactive adhesive monomer provides favorable biocompatibility and odontogenic induction capacity and that (2) CMET might be a very promising adjunctive for pulp-capping materials.

Effect of mesoporous bioactive glass on odontogenic differentiation of human dental pulp stem cells

Healthy pulp tissue plays an important role in normal function and long-term retention of teeth. Mesoporous bioactive glass (MBG) as a kind of regenerative biomaterials shows the potential in preserving the vital pulp. In this study, MBG prepared by organic template method combined with sol-gel method were used in human dental pulp cell culture and ectopic mineralization experiment. Real-Time PCR was used to explore its ability to induce odontogenic differentiation of dental pulp cells. MBG and rat crowns were implanted under the skin of nude mice for 4 weeks to observe the formation of pulp dentin complex. We found that MBG can release Si and Ca ions and has a strong mineralization activity in vitro. The co-culture of MBG with human dental pulp cells promoted the expression of DMP-1 (dentin matrix protein-1) and ALP (alkalinephosphatase) without affecting cell proliferation. After 4 weeks of subcutaneous implantation in nude mice, the formation of hard tissue with regular structure under the material could be seen, and the structure was similar to dentin tubules. These results indicate that MBG can induce the differentiation of dental pulp cells and the formation of dental pulp-dentin complex and has the potential to promote the repair and regeneration of dental pulp injuries.

Functionalized self-assembled peptide RAD/Dentonin hydrogel scaffold promotes dental pulp regeneration

RADA16-Ⅰ is an ion-complementary self-assembled peptide with a regular folded secondary conformation and can be assembled into an ordered nanostructure. Dentonin is an extracellular matrix phosphate glycoprotein functional peptide motif-containing RGD and SGDG motifs. In this experiment, we propose to combine RAD and Dentonin to form a functionalized self-assembled peptide RAD/Dentonin hydrogel scaffold. Furthermore, we expect that the RAD with the addition of functional motif Dentonin can promote pulp regeneration. The study analyzed the physicochemical properties of RAD/Dentonin through Circular dichroism, Morphology scanning, and Rheology. Besides, we examined the scaffold’s biocompatibility by Immunofluorescent staining, CCK-8 method, Live/Dead fluorescent staining, and 3D reconstruction. Finally, we applied ALP activity assay, RT-qPCR, and Alizarin red S staining to detect the effect of RAD/Dentonin on the odontogenic differentiation of human dental pulp stem cells (hDPSCs). The results showed that RAD/Dentonin spontaneously assembles into a hydrogel with a β-sheet-based nanofiber network structure. In vitro, RAD/Dentonin has superior biocompatibility and enhances adhesive proliferation, migration, odontogenic differentiation, and mineralization deposition of hDPSCs. In conclusion, the novel self-assembled peptide RAD/Dentonin is a new scaffold material suitable for cell culture and has promising applications as a scaffold for endodontic tissue engineering.

miR-6807-5p Inhibited the Odontogenic Differentiation of Human Dental Pulp Stem Cells Through Directly Targeting METTL7A

Background: Tooth tissue regeneration mediated by mesenchymal stem cells (MSCs) has become the most ideal treatment. Although the known regulatory mechanism and some achievements have been discovered, directional differentiation cannot effectively induce regeneration of tooth tissue. In this study, we intended to explore the function and mechanism of miR-6807-5p and its target gene METTL7A in odontogenic differentiation.Methods: In this study, human dental pulp stem cells (DPSCs) were used. Alkaline phosphatase (ALP), Alizarin red staining (ARS), and calcium ion quantification were used to detect the odontogenic differentiation of miR-6807-5p and METTL7A. Real-time RT-PCR, western blot, dual-luciferase reporter assay, and pull-down assay with biotinylated miRNA were used to confirm that METTL7A was the downstream gene of miR-6807-5p. Protein mass spectrometry and co-immunoprecipitation (Co-IP) were used to detect that SNRNP200 was the co-binding protein of METTL7A.Results: After mineralized induction, the odontogenic differentiation was enhanced in the miR-6807-5p-knockdown group and weakened in the miR-6807-5p-overexpressed group compared with the control group. METTL7A was the downstream target of miR-6807-5p. After mineralized induction, the odontogenic differentiation was weakened in the METTL7A-knockdown group and enhanced in the METTL7A-overexpressed group compared with the control group. SNRNP200 was the co-binding protein of METTL7A. The knockdown of SNRNP200 inhibited the odontogenic differentiation of DPSCs.Conclusion: This study verified that miR-6807-5p inhibited the odontogenic differentiation of DPSCs. The binding site of miR-6807-5p was the 3′UTR region of METTL7A, which was silenced by miR-6807-5p. METTL7A promoted the odontogenic differentiation of DPSCs. SNRNP200, a co-binding protein of METTL7A, promoted the odontogenic differentiation of DPSCs.

Differential expression of long noncoding RNAs in SDF-1α-induced odontogenic differentiation of human dental pulp stem cells

SDF-1α cotreatment was shown to have synergistic effects on BMP-2-induced odontogenic differentiation of human apical dental papillary stem cells (SCAP) both in vitro and in vivo. Long noncoding RNAs (lncRNAs) have an important role in the odontogenic differentiation of dental pulp stem cells (DPSCs). We examined the altered expression of lncRNAs in SDF-1α-induced odontogenic differentiation of DPSCs by lncRNA microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses. Alterations in lncRNA expression during odontogenic differentiation of DPSCs were identified. Moreover, bioinformatic analysis [Gene Ontology (GO) analysis and coding-noncoding gene coexpression (CNC) analysis] was conducted to predict the interactions of lncRNAs and identify core regulatory factors in SDF-1α-induced odontogenic differentiation of DPSCs. The microarray analysis identified 206 differentially expressed lncRNAs (134 lncRNAs with upregulated expression and 72 with downregulated expression) at 7 days post‑treatment. The data demonstrated that one lncRNA, AC080037.1, regulates SDF-1α-induced odontogenic differentiation of DPSCs. Our data showed that lncRNA AC080037.1 siRNA suppresses DPSCs migration and the expression of Rho GTPase induced by SDF-1α. Moreover, AC080037.1 knockdown significantly affected SDF-1α- and BMP-2-induced mineralized nodule formation and strongly suppressed Runt-related factor-2 (RUNX-2), DMP-1 and DSPP expression in DPSCs. Our results highlighted the significant involvement of one lncRNA, AC080037.1, in the positive regulation of the osteo/odontogenic differentiation of DPSCs and indicated that lncRNA AC080037.1 could be a potential target in regenerative endodontics.These findings reveal how lncRNAs are involved in regulating the SDF-1α-induced odontogenic differentiation of DPSCs, which may further advance translational studies of pulp tissue engineering.

PD-1 Suppresses the Osteogenic and Odontogenic Differentiation of Stem Cells From Dental Apical Papilla via Targeting SHP2/NF-κB axis

BackgroundStem cells from the apical papilla (SCAPs) are important for tooth root development and regeneration of root dentin. Here, we examined the expression of programmed cell death protein-1 (PD-1) in SCAPs and investigated the effect of PD-1 on odontogenic and osteogenic differentiation and the relationship between PD-1 and cell differentiation and SHP2/NF-κB signals.MethodsSCAPs were obtained culture in the related medium. The proliferation ability was evaluated by cell counting kit 8 and 5‐ethynyl‐20‐deoxyuridine (EdU) assay. Alkaline phosphatase (ALP) activity assay, ALP staining, western blot, real-time RT-PCR, Alizarin Red S staining, and immunofluorescence staining were performed to explore the osteo/odontogenic potential and the involvement of SHP2/NF-κB pathways. Besides, we transplanted SCAPs component into mouse calvaria defects to evaluate osteogenesis in vivo. ResultsWe found that human SCAPs expressed PD-1 for the first time. PD-1 knockdown enhanced the osteo/odontogenic differentiation of SCAPs by suppressing SHP2 pathway and activating NF-κB pathway. Overexpression of PD-1 inhibited the osteogenesis and odontogenesis of SCAPs via activation of SHP2 signal and inhibition of NF-κB pathway. ConclusionPD-1 activated SHP2 signal to block NF-κB signal and then played a vital role in osteo/odontogenic differentiation of SCAPs.