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.

Effect of Biodentine Coated with Emdogain on Proliferation and Differentiation of Stem Cells from the Apical Papilla

Background: This study assessed the effect of Biodentine coated with Emdogain (Biodentine/Emdogain) on proliferation and differentiation of stem cells from the apical papilla (SCAP). Methods and Results: In this in vitro, experimental study, SCAP were isolated from two immature impacted third molars and cultured. After ensuring the stemness of the cells by assessing their cell surface markers, they were exposed to Biodentine, Emdogain, and Biodentine/Emdogain for 24 and 72 hours. The control cells did not receive any intervention. Cell viability was evaluated by the methyl thiazolyl tetrazolium (MTT) assay. Expression of odontogenic differentiation genes was analyzed by the quantitative reverse transcription polymerase chain reaction (qRT-PCR). Alkaline phosphatase (ALP) activity was quantified by the respective kit. Data were analyzed by one-way ANOVA, t-test, and Mann-Whitney test (α=0.05). Cell viability did not change after 24 hours of exposure to biomaterials. At 72 hours, the viability of the cells exposed to Biodentine and Biodentine/Emdogain decreased compared with the control group. The expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP) genes, and ALP activity significantly increased in all three experimental groups, compared with the control group at both 24 and 72 hours; this increase was significantly greater in Biodentine/Emdogain group. The number of mineralized nodules significantly increased in all groups after 72 hours with a greater rate in Biodentine/Emdogain group.Conclusions: All biomaterials increased the differentiation of SCAP, expression of odontogenic genes, and ALP activity, but Biodentine/Emdogain was significantly more effective for this purpose.

Calcium silicate-based cements affect the cell viability and the release of TGF-β1 from apical papilla cells

This study investigated the cytotoxicity and release of Transforming Growth Factor Beta 1 (TGF-β1) from cultured human apical papilla cells (APCs) after application of four bioactive materials. Culture of APCs was established and used for cytotoxic and quantitative assays. Extracts of Biodentine, Bio-C Repair, MTA Repair and White MTA were prepared and diluted (1, 1:4 and 1:16) and used for MTT assays up to 72 h. Total TGF-β1 was quantified by ELISA. Data were analyzed by ANOVA and Tukey’s test (α = 0.05). For Biodentine, at 24 h and 48 h, cell viability was lower than control (p < 0.05). At 72 h, only undiluted extract of Biodentine were cytotoxic (p < 0.05). At 24 h, a cytotoxic effect was found for undiluted and 1:4 dilution of Bio-C Repair (p < 0.05). At 48 h, however, Bio-C Repair at 1:4 and 1:8 dilution showed higher cell viability (p < 0.05). At 24 and 48 h, the cell viability for undiluted MTA Repair were higher than control (p < 0.05). For White MTA, at 24 and 48 h, all dilutions were cytotoxic (p < 0.05). All cements led to reduced release of total TGF-β1 from the APCs (p < 0.05). In conclusion, cell viability varied depending on the material and dilution. Only Bio-C repair and MTA repair led to higher cell viability of APCs. All materials induced a decrease in the release of total TGF-β1 from the APCs.

Translational and Clinical Applications of Dental Stem Cell-Derived Exosomes

Mesenchymal stem cells (MSCs) are promising seed cells in tissue repair and regeneration due to their featured properties of self-renewal and multipotency. However, a growing body of evidence has demonstrated that MSCs exert biological functions mainly through secreting exosomes. Exosomes, which contain RNA, proteins, lipids, and metabolites, are new players in regulating many fundamental processes and play important roles in regenerative medicine. Exosomes not only mimic the effects of their parent cells but also possess many advantages such as high drug loading capacity, low immunogenicity, excellent biocompatibility, and low side effects. Currently, a total of 6 different dental stem cells (DSCs) including dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHEDs), periodontal ligament stem cells (PDLSCs), dental follicle progenitor cells (DFPCs), stem cells from apical papilla (SCAPs) and gingival mesenchymal stem cells (GMSCs) have been isolated and identified. DSC-derived exosomes (DSC-Exos) are actively involved in intercellular communication, anti-inflammation, osteogenesis, angiogenesis, immunomodulation, nurturing neurons, and promoting tumor cell apoptosis. In this review, we will critically review the emerging role and clinical application potential of DSC-Exos.

Effect of 1,25-Dihydroxyvitamin D3 on Stem Cells from Human Apical Papilla: Adhesion, Spreading, Proliferation, and Osteogenic Differentiation

Currently, it still remains a difficult problem to treat apical insufficiency of young permanent teeth resulted from pulp necrosis or periapical periodontitis. Previous studies have demonstrated that the treatment of revascularization using stem cells from apical papilla (SCAPs) results in increased root length and thickness of traumatized immature teeth and necrotic pulp. In this study, we investigated the role of 1,25-dihydroxyvitamin D3 in regulating the adhesion, spreading, proliferation, and osteogenic differentiation of SCAP, laying the foundation for subsequent clinical drug development. The immature tooth samples were collected in clinical treatment. SCAPs with stable passage ability were isolated and cultured. The multidifferentiation potential was determined by directed induction culture, while the stem cell characteristics were identified by flow cytometry. There were three groups: group A—SCAPs general culture group; group B—SCAPs osteogenesis induction culture group; and group C—SCAPs osteogenesis induction culture+1,25-dihydroxyvitamin D3 group, and the groups were compared statistically. The proliferation of SCAPs in each groups was detected through CCK-8 assay. RT-qPCR was used to detect the transcription levels of Runx2, ALP, Col I, and OCN of SCAPs in each groups. Results exhibited that the isolated SCAPs had multidifferentiation potential and stem cell characteristics. After 24 h culturing, cells in group C spread better than those in groups A and B. The proliferation activity of SCAPs factored by CCK-8 ranked as group C > group B > group A , while the transcription levels of Runx2, ALP, Col I, and OCN leveled as group C > group B > group A . These results suggested that 1,25-dihydroxyvitamin D3 can significantly promote the adhesion, spreading, and proliferation of SACPs and improve the osteogenic differentiation of SCAPs by means of regulating upward the transcription level of osteogenic differentiation marker.