METTL3-mediated pre-miR-665/DLX3 m6A methylation facilitates the committed differentiation of stem cells from apical papilla.

Methyltransferase-like 3 (METTL3) is a crucial element of N6-methyladenosine (m6A) modifications and has been extensively studied for its involvement in diverse biological and pathological processes. In this study, we explored how METTL3 affects the differentiation of stem cells from the apical papilla (SCAPs) into odonto/osteoblastic lineages through gain- and loss-of-function experiments. The m6A modification levels were assessed using m6A dot blot and activity quantification experiments. In addition, we employed Me-RIP microarray experiments to identify specific targets modified by METTL3. Furthermore, we elucidated the molecular mechanism underlying METTL3 function through dual-luciferase reporter gene experiments and rescue experiments. Our findings indicated that METTL3+/- mice exhibited significant root dysplasia and increased bone loss. The m6A level and odonto/osteoblastic differentiation capacity were affected by the overexpression or inhibition of METTL3. This effect was attributed to the acceleration of pre-miR-665 degradation by METTL3-mediated m6A methylation in cooperation with the "reader" protein YTHDF2. Additionally, the targeting of distal-less homeobox 3 (DLX3) by miR-665 and the potential direct regulation of DLX3 expression by METTL3, mediated by the "reader" protein YTHDF1, were demonstrated. Overall, the METTL3/pre-miR-665/DLX3 pathway might provide a new target for SCAP-based tooth root/maxillofacial bone tissue regeneration.

CTP-CM enhances osteogenic differentiation of hPDLSCs via NF-κB pathway.

OBJECTIVE: The conditioned medium of calcined tooth powder (CTP-CM) is a type of biomimetic mineralized material and well contributing to bone healing and bone formation in vivo. However, little is known about the effect of CTP-CM on human periodontal ligament stem cells (hPDLSCs) as well as the underlying mechanisms. METHODS: ALP activity assay was conducted to select the concentration with the highest ALP level, which was used for the following experiments. Cell proliferation was measured by cell counting kit-8 assay and flow cytometry analysis. Expression levels of osteogenic markers in CTP-CM-induced hPDLSCs were evaluated with real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunofluorescence staining, and Western blot. Mineralization of CTP-CM-induced hPDLSCs was evaluated by alizarin red staining. Furthermore, the involvement of NF-κB pathway was examined by immunofluorescence staining and Western blot. RESULTS: 20 µg/ml was selected for the further experiments. Functional studies demonstrated that CTP-CM exerted almost no influence on the proliferation of hPDLSCs and CTP-CM increased the osteogenic differentiation of hPDLSCs. Mechanistically, CTP-CM leads to activation of NF-κB signaling pathway. When treated with BMS345541, the osteogenic differentiation of CTP-CM-treated hPDLSCs was significantly attenuated. CONCLUSION: CTP-CM can promote the osteogenic differentiation of hPDLSCs via activating NF-κB pathway.

Dentin-Derived Inorganic Minerals Promote the Osteogenesis of Bone Marrow-Derived Mesenchymal Stem Cells: Potential Applications for Bone Regeneration.

BACKGROUND: Oral and maxillofacial bone loss is highly prevalent among populations, and nowadays, increased attention has been focused on dentin derivatives serving as desirable graft materials for bone regeneration. In this study, dentin-derived inorganic mineral (DIM) was fabricated with a high-temperature calcination technique and the effects of DIM on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) and the bone formation were elucidated. METHODS: The effects of DIM on BMMSC proliferation and apoptosis capacity were evaluated by CCK-8, flow cytometry, and EdU assays. Alkaline phosphatase (ALP) activity detection, ALP staining, alizarin red staining, and osteogenic marker expression analysis were performed to investigate the influence of DIM on the osteogenic differentiation of BMMSCs, as well as the relevant signal mechanisms. The model of critical-sized defects in the calvarium of rats was constructed for exploring the in vivo efficiency of DIM on bone regeneration. RESULTS: Cell viability assays indicated that DIM had no cytotoxicity. BMMSCs cultured with DIM presented a higher level of osteogenic differentiation ability than those in the control group. The activation in ERK and p38 signals was detected in DIM-treated BMMSCs, and both pathways and osteogenic process were suppressed while using ERK inhibitor U0126 and p38 inhibitor SB203580, respectively. Furthermore, the animal experiments revealed that DIM could dramatically enhance new bone formation compared to the control group. CONCLUSION: DIM could promote BMMSC osteogenic differentiation via triggering the ERK and p38 MAPK signaling pathways and might be a novel predictable material for facilitating bone formation.

Circular RNA SIPA1L1 regulates osteoblastic differentiation of stem cells from apical papilla via miR-204-5p/ALPL pathway.

BACKGROUND: Osteogenesis is a complex biological process which requires the coordination of multiple molecular mechanisms. This research aimed to explore the biological role and underlying regulatory mechanism of circSIPA1L1 during the osteogenic differentiation of stem cells from apical papilla (SCAPs). METHODS: EdU retention assay, flow cytometry assay, and CCK-8 assay were used to evaluate the proliferation capacity of SCAPs. Western blot assay, alkaline phosphatase (ALP), and alizarin red staining (ARS) were conducted to investigate the biological roles of circSIPA1L1 and miR-204-5p. Fluorescence in situ hybridization was applied for circSIPA1L1 localization. Dual-luciferase reporter assay was performed to prove the interaction of circSIPA1L1 and miR-204-5p. RESULTS: CircSIPA1L1 had no significant effect on the proliferative capacity of SCAPs. CircSIPA1L1 promotes osteogenic differentiation of SCAPs by serving as a miRNA sponge for miR-204-5p. Either knockdown of circSIPA1L1 or overexpression of miR-204-5p significantly suppresses osteogenic differentiation of SCAPs. CONCLUSIONS: CircSIPA1L1 upregulates ALPL through targeting miR-204-5p and promotes the osteogenic differentiation of SCAPs.

Circular RNA SIPA1L1 promotes osteogenesis via regulating the miR-617/Smad3 axis in dental pulp stem cells.

BACKGROUND: Bone regeneration is preferred for bone loss caused by tumors, bone defects, fractures, etc. Recently, mesenchymal stem cells are considered as optimistic tools for bone defect therapy. Dental pulp stem cells (DPSCs) are a promising candidate for regenerative medicine and bone regeneration. Our previous study showed that upregulated circSIPA1L1 during osteogenesis of DPSCs is of significance. In this paper, the potential role of circSIPA1L1 in osteogenesis of DPSCs and its underlying mechanisms are explored. METHODS: The circular structure of circSIPA1L1 was identified by Sanger sequencing and PCR. Regulatory effects of circSIPA1L1 and miR-617 on mineral deposition in DPSCs were assessed by alkaline phosphatase (ALP) and alizarin red S (ARS) staining and in vivo bone formation assay were conducted to verify the biological influences of circSIPA1L1 on DPSCs. Western blot was performed to detect the protein expression of Smad3. Localization of circSIPA1L1 and miR-617 was confirmed by FISH. Dual-luciferase reporter assay and rescue experiments were conducted to investigate the role of the circSIPA1L1/miR-617/Smad3 regulatory axis in osteogenesis of DPSCs. RESULTS: Sanger sequencing and back-to-back primer experiments confirmed the closed-loop structure of circSIPA1L1. CircSIPA1L1 could promote the committed differentiation of DPSCs. MiR-617 was predicted to be the target binding circSIPA1L1 through MiRDB, miRTarBase, and TargetScan database analyses, which was further confirmed by dual-luciferase reporter assay. FISH results showed that circSIPA1L1 and miR-617 colocalize in the cytoplasm of DPSCs. MiR-617 exerted an inhibitory effect on the osteogenesis of DPSCs. Knockdown of circSIPA1L1 or upregulation of miR-617 downregulated phosphorylated Smad3. In addition, rescue experiments showed that knockdown of miR-617 reversed the inhibitory effect of circSIPA1L1 on osteogenesis of DPSCs. CONCLUSION: CircRNASIPA1L1 promotes osteogenesis of DPSCs by adsorbing miR-617 and further targeting Smad3.

Remineralization of dentin slices using casein phosphopeptide-amorphous calcium phosphate combined with sodium tripolyphosphate.

BACKGROUND: The remineralization approach mechanically occludes the exposed dentinal tubules mechanically, reduces the permeability of dentinal tubules and eliminates the symptoms of dentin hypersensitivity. The aim of the present study was to investigate the remineralization of demineralized dentin slices using CPP-ACP combined with TPP, and the research hypothesis was that CPP-ACP combined with TPP could result in extrafibrillar and intrafibrillar remineralization of dentin. METHODS: Demineralized dentin slices were prepared and randomly divided into the following groups: A (the CPP-ACP group), B (the CPP-ACP + TPP combination group), C (the artificial saliva group), D (the negative control group), and E (the positive control group). Dentin slice samples from groups A, B and C were remineralized and the remineralization effect was evaluated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray diffraction (XRD). RESULTS: Treatment with CPP-ACP combined with TPP occluded the dentinal tubules and resulted in remineralization of collagen fibrils. The hydroxyapatite crystals formed via remineralization were found to closely resemble the natural dentin components. CONCLUSION: CPP-ACP combined with TPP has a good remineralization effect on demineralized dentin slices.

iRoot SP Promotes Osteo/Odontogenesis of Bone Marrow Mesenchymal Stem Cells via Activation of NF-κB and MAPK Signaling Pathways.

The regeneration of bone and tooth tissues, and related cellular therapies, has attracted widespread attention. Bone marrow mesenchymal stem cells (BMSCs) are potential candidates for such regeneration. iRoot SP is a premixed bioceramic root canal sealer widely used in clinical settings. However, the effect of iRoot SP on the biological features of BMSCs has not been elucidated. In the present study, we found that 0.2 mg/ml iRoot SP conditioned medium promoted osteo/odontogenic differentiation and enhanced mineralization of BMSCs without affecting the proliferative ability. Mechanistically, the NF-κB and MAPK signaling pathways were activated in SP-treated BMSCs, and differentiation was inhibited when cultured with the specific inhibitor. Taken together, these findings demonstrate that iRoot SP promotes osteo/odontogenic differentiation of BMSCs via the NF-κB and MAPK signaling pathways, which could provide a new theoretical basis for clinical applications of iRoot SP and a new therapeutic target for the regeneration of bone and tooth tissue in the future.

Parathyroid hormone enhances the osteo/odontogenic differentiation of dental pulp stem cells via ERK and P38 MAPK pathways.

OBJECTIVE: Parathyroid hormone (PTH) is a main systemic mediator of calcium and phosphate homeostasis in the bone. Dental pulp stem cells (DPSCs) have been extensively studied in the regeneration of bone and tooth tissues. This paper aims to uncover the influences of PTH on the proliferative ability and osteo/odontogenic differentiation of DPSCs, as well as the underlying mechanisms. MATERIALS AND METHODS: The optimal concentration of PTH on DPSCs was determined by alkaline phosphatase (ALP) activity assay, ALP staining and western blot analysis. Proliferative ability and cell cycle distribution of DPSCs were analyzed by Cell counting kit-8, 5-ethynyl-20-deoxyuridine assay, and flow cytometry. Osteo/odontogenic capacity of DPSCs was evaluated and finally, the involvement of mitogen-activated protein kinase (MAPK) pathway was assessed. RESULTS: Purified DPSCs were obtained by enzymatic digestion, which presented a typical fibroblast-like morphology. 10-9 mol/L PTH was concerned as the optimal concentration for DPSCs induction. 10-9 mol/L PTH treatment did not change the proliferative rate of DPSCs (p > .05). Relative expressions of DSPP/DSPP, RUNX2/RUNX2, OSX/OSX, and ALP/ALP were upregulated in PTH-treated DPSCs relative to control group. Particularly, their mRNA/protein levels at Day 7 were markedly higher relative to those at Day 3 (p < .05 or p < .01). Mineralized nodules were formed after PTH induction, and calcium content increased by cetylpyridinium chloride quantitative analysis. Mechanistically, the protein levels of p-ERK and p-P38 significantly increased after PTH treatment, and the inhibitors targeting MAPK were identified that weakened the effects of PTH on the committed differentiation of DPSCs. CONCLUSIONS: PTH enhances the osteo/odontogenic differentiation capacity of DPSCs via ERK and P38 signaling pathways.

MiR-141-3p regulates proliferation and senescence of stem cells from apical papilla by targeting YAP.

Biological phenotypes of mesenchymal stem cells (MSCs) are regulated by a series of biochemical elements, including microRNAs, hormones and growth factors. Our previous study illustrated a significant role of miR-141-3p during the osteogenic differentiation of stem cells from apical papilla (SCAPs). Nevertheless, the functions of miR-141-3p in regulating the proliferative ability and senescence of SCAPs have not been determined. This study identified that overexpression of miR-141-3p inhibited the proliferative ability of SCAPs. Meanwhile, the senescence of SCAPs was ahead of time. Conversely, transfection of miR-141-3p inhibitor promoted the proliferative ability of SCAPs and delayed their senescence. Yes-associated protein (YAP) was predicted as the downstream target gene of miR-141-3p by online softwares (miRDB, miRTarBase, miRWalk, and TargetScan), and was further verified by dual-luciferase reporter gene assay. Additionally, knockdown of YAP inhibited the proliferation and accelerated the senescence of SCAPs. Collectively, these findings proposed a novel direction that miR-141-3p impeded proliferative ability and promoted senescence of SCAPs through post-transcriptionally downregulating YAP.

iRoot BP Plus promotes osteo/odontogenic differentiation of bone marrow mesenchymal stem cells via MAPK pathways and autophagy.

BACKGROUND: iRoot BP Plus is a novel bioceramic endodontic material. Recently, it has been considered as an alternative to MTA which is the most popular scaffold cover during regenerative endodontic therapy. This study aimed to evaluate the effects of iRoot BP Plus on the osteo/odontogenic capacity of bone marrow mesenchymal stem cells (BMMSCs), including the underlying mechanisms. METHODS: BMMSCs were collected by a whole marrow method and treated with iRoot BP Plus-conditioned medium (BP-CM). The proliferation ability was evaluated by cell counting kit 8 and flow cytometry. Complete medium was used as a blank control, and 2 mg/ml MTA-conditioned medium was served as a positive control. 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 MAPK pathways. Besides, autophagy was investigated by western blot, immunofluorescence staining, and transmission electron microscopy.

Differential circular RNA expression profiling during osteogenic differentiation of stem cells from apical papilla.

Aim: This study aimed to investigate the distinct expression pattern of circular RNAs (circRNAs) in stem cells from apical papilla (SCAPs) during osteogenesis. Materials & methods: Isolated SCAPs were cultured in growth medium or osteogenic medium, respectively. Total RNA was extracted and submitted to RNA-sequencing. Expression profiles of circRNAs and constructed circRNA-miRNA-mRNA networks were determined. Results: A total of 333 unregulated circRNAs and 317 downregulated circRNAs in osteogenic differentiation were detected. Bioinformatics analysis identified that several biological pathways may be associated with osteogenic differentiation of SCAPs. Moreover, ten circRNAs, 21 miRNAs and 19 mRNAs were selected to construct competing endogenous RNA networks. Conclusion: This study revealed that expression profiles of circRNAs were significantly altered and specific circRNAs might function as competing endogenous RNAs in SCAPs during osteogenic differentiation.