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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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Periodontitis is a common chronic inflammatory disease, which causes the destruction of both the soft and mineralized tissues. However, current treatments such as bone graft materials, barrier membranes, and protein products all have difficulties in regenerating the complete periodontal tissue structure. Stem cell-based tissue engineering has now emerged as one of the most effective treatments for the patients suffering from periodontal diseases. Plants not only can be substrates for life processes, but also contain hormones or functional molecules. Numbers of preclinical studies have revealed that products from plant can be successfully applied in modulating proliferation and differentiation of human mesenchymal stem cells. Plant-derived substances can induce stem cells osteogenic differentiation, and they also possess angiogenic potency. Furthermore, in the field of tissue engineering, plant-derived compounds or plant extracts can be incorporated with biomaterials or utilized as biomaterials for cell transplantation. So it is speculated that botanical products may become a new perspective in stem cell-based periodontal regeneration. However, the lack of achieving predict clinical efficacy and quality control has been the major impediment to its extensive application. This review gives an overview of the prospect of applying different plant-derived substances in various human mesenchymal stem cells-based periodontal regeneration.

As the key regulator of hard tissue metabolism in both men and women, estrogen regulates the processes necessary for cell growth, proliferation, and differentiation through estrogen receptor (ER). Estrogen deficiency usually causes systemic osteoporosis not only in long bones but also in jaw bones, and exogenous estrogen can enhance the osteogenic potential of mesenchymal stem cells. Dental mesenchymal stem cells (DMSCs) represent a group of stem cells isolated from different parts of the tooth, including dental pulps, apical papillae and periodontal ligaments. A number of studies have proved that estrogen plays an important role in the proliferation, differentiation and tissue regeneration of human DMSCs. Thus, this review will focus on the effects of estrogen on proliferation, apoptosis, and differentiation of dental stem cells, discuss evidence from studies in rodents that estrogen plays an important role in dental morphogenesis as well as periodontal remodeling, and suggest directions for future studies in estrogen-related tooth regeneration.

Introduction: Estrogen plays an important role in the osteogenic differentiation of mesenchymal stem cells, while stem cells from apical papilla (SCAP) can contribute to the formation of dentin/bone-like tissues. To date, the effects of estrogen on the differentiation of SCAP remain unclear., Methods: SCAP was isolated and treated with 10⁻⁷ M 17beta-estradiol (E2). The odonto/osteogenic potency and the involvement of mitogen-activated protein kinase (MAPK) signaling pathway were subsequently investigated by using methyl-thiazolyl-tetrazolium (MTT) assay, and other methods., Results: MTT and flow cytometry results demonstrated that E2 treatment had no effect on the proliferation of SCAP in vitro, while alkaline phosphatase (ALP) assay and alizarin red staining showed that E2 can significantly promote ALP activity and mineralization ability in SCAP. Real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot assay revealed that the odonto/osteogenic markers (ALP, DMP1/DMP1, DSPP/DSP, RUNX2/RUNX2, OSX/OSX and OCN/OCN) were significantly upregulated in E2-treated SCAP. In addition, the expression of phosphor-p38 and phosphor-JNK in these stem cells was enhanced by E2 treatment, as was the expression of the nuclear downstream transcription factors including phosphor-Sp1, phosphor-Elk-1, phosphor-c-Jun and phosphor-c-Fos, indicating the activation of MAPK signaling pathway during the odonto/osteogenic differentiation of E2-treated SCAP. Conversely, the differentiation of E2-treated SCAP was inhibited in the presence of MAPK specific inhibitors., Conclusions: The ondonto/osteogenic differentiation of SCAP is enhanced by 10⁻⁷ M 17beta-estradiol via the activation of MAPK signaling pathway.

Objective: To determine the effects of estrogen deficiency on the proliferation and osteogenic differentiation of mandibular bone marrow stromal cells (mBMSC)., Methods: Ten 8-week-old female SD rats were randomly divided into two groups, ovariectomized group (OVX, n = 5) and sham-operation group (n = 5). All rats were anesthetized and both ovaries of OVX-rats were gently removed. Sham-operation rats were treated with the same incisions to expose the ovaries but without any hurt to them. One month after the operation, the mandibular bones were gently separated and mBMSC were isolated. Methyl thiazolyl tetrazolium (MTT) assay, alkaline phosphotase (ALP) activity, alizarin red staining, real-time reverse transcription (RT)-PCR and Western blotting were respectively used to examine the proliferative activity and osteogenic potential of mBMSC., Results: MTT results showed that OVX-mBMSC exhibited the decreased proliferative activity as compared with Sham-mBMSC. ALP activity of OVX-mBMSC [(0.710±0.011) Sigma unit/protein] was lower than that of Sham-mBMSC [(1.512±0.021) Sigma unit/protein] (P < 0.01). Alizarin red staining showed that OVX-mBMSC formed less calcified nodules than Sham-mBMSC. Ca(2+) concentration analysis showed Ca(2+) of OVX-mBMSC [(0.433±0.045) µg/g] was less than Sham-mBMSC [(1.453±0.131) µg/g] (P < 0.01). Real-time RT-PCR and Western blotting results showed that the expression of osteogenic markers (Alp, Runx2/RUNX2, Osx/OSX, Ocn/OCN) in OVX-mBMSC was significantly inhibited as compared with Sham-mBMSC (P < 0.05)., Conclusions: Estrogen deficiency significantly inhibits the proliferation and osteogenic capacity of mBMSC.

Dentin, the predominant mineralized tissue of the tooth, comprises an extracellular matrix of collagen and a heterogeneous mixture of non-collagenous components, many of which have cellular signaling properties. These properties may be important in signaling stem cell involvement in tissue regeneration following injury and the present study investigates their morphogenic effects on differentiation of Bone Marrow Stromal Stem Cells (BMMSCs) in vitro. Non-collagenous dentin matrix proteins (DMPs) were isolated from healthy human teeth and their effects on BMMSCs behavior examined during in vitro culture. In vitro, DMPs enhanced alkaline phosphatase activity and mineralization in BMMSCs cultures as well as increasing the expression of dentinogenic and osteogenic differentiation markers (including runt-related transcription factor 2, osterix, bone sialoprotein, dentin sialophosphoprotein and osteocalcin) at both transcript and protein levels, with 10 μg/mL DMPs being the optimal stimulatory concentration. Expression of phosphor-ERK/phosphor-P38 in BMMSCs was up-regulated by DMPs and, in the presence of the ERK1/2- and p38-specific inhibitors, the differentiation of BMMSCs was inhibited. These data indicate that DMPs promote the dentinogenic/osteogenic differentiation of BMMSCs via the ERK/p38 MAPK pathways.

Objective: To assess the odontogenic potential of bone marrow mesenchymal stem cells (BMMSCs) to differentiate into odontoblast-like cells under the morphogenetic influence of dentine matrix as a possible basis for new stem cell-mediated therapeutic approaches to pulp diseases., Design: BMMSCs were harvested from the whole bone marrow and cells at passages 3-5 were used for subsequent experiments. For in vitro studies, 1×10(4) cells were seeded on the surface of dentine slabs and co-cultured for 2 weeks in 24-well plates, then fixed, decalcified, embedded in paraffin and serial sections were processed for analyses. Haematoxylin-eosin (HE) staining was used for the morphological analysis of BMMSCs on the dentine slabs. The protein expression of dentine sialoprotein (DSP) in co-cultured BMMSCs was detected by immunohistochemical (IHC) staining. For in vivo studies, 5×10(6) cells were collected as cell pellets, seeded onto dentine slices and transplanted into renal capsules for 6 weeks. Histological analyses of harvested tissues were performed as described for the in vitro studies. Total RNA and protein were extracted from harvested tissues and Dspp/DSP expression was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively., Results: After 2 weeks of co-culture with dentine slabs, BMMSCs demonstrated good viability in terms of morphological appearance and some showed polarization and extension of their cytoplasmic processes into dentine tubules with DSP expression. In vivo study demonstrated similar morphological changes and DSP expression in cells adjacent to dentine. RT-PCR and Western blot also demonstrated that the expression of Dspp/DSP in the co-cultured BMMSCs groups was higher than in the control groups., Conclusion: Dentine matrix can signal morphogenic induction of differentiation of BMMSCs into odontoblast-like cells in vivo and in vitro., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

PDLSC-mediated periodontal tissue regeneration is considered a promising method for periodontitis treatment, but the supply of PDLSCs is limited. As a potential alternative, WJCMSCs are readily available; however, there is a lack of evidence proving that WJCMSCs are suitable for periodontal tissue regeneration. In this study, we investigated the characteristics of WJCMSCs and PDLSCs. We found the osteo-/dentinogenic, adipogenic and chondrogenic differentiation potentials of PDLSCs were more powerful than those of WJCMSCs. Microarray analysis discovered that 903 genes were significantly down-regulated and 726 genes up-regulated in WJCMSCs compared with PDLSCs. Based on the microarray data, we found that several genes may be associated with MSCs characteristics. Further bioinformatic analysis identified that TGF-β and WNT signaling pathways, and several genes, including STAT5B and ITGA4, may play key roles in MSCs. Our results indicate that the differentiation potentials of WJCMSCs are far less than those of PDLSCs, and that unmodified WJCMSCs may not be good seeding cells for periodontal tissue regeneration. Our results also help to elucidate the differentiation mechanisms in MSCs and to find the key factors to prompt WJCMSC-mediated periodontal tissue regeneration., (Copyright © 2012 S. Karger AG, Basel.)

Multiple myeloma is a common hematological malignancy that leads to the occurrence of bone lesions. The combination of traditional cytotoxic and novel agents is usually taken to treat multiple myeloma-related bone diseases. However, the curative effect is not very satisfactory. Stem cell-based therapy has been recently introduced and investigated, which represents a new frontier in the treatment of multiple myeloma. In a recent interesting study, Dr Atsuta and colleagues provide a new insight into the effects of mesenchymal stem cells on multiple myeloma via Fas/Fas ligand pathway, which rekindles the fire of hope for those patients suffering from multiple myeloma.

The BCL-6 co-repressor (BCOR) represses gene transcription by interacting with BCL-6 (Refs 1, 2). BCOR mutation is responsible for oculo-facio-cardio-dental (OFCD) syndrome, which is characterized by canine teeth with extremely long roots, congenital cataracts, craniofacial defects and congenital heart disease. Here we show that BCOR mutation increased the osteo-dentinogenic potential of mesenchymal stem cells (MSCs) isolated from a patient with OFCD, providing a molecular explanation for abnormal root growth. AP-2alpha was identified as a repressive target of BCOR, and BCOR mutation resulted in abnormal activation of AP-2alpha. Gain- and loss-of-function assays suggest that AP-2alpha is a key factor that mediates the increased osteo-dentinogenic capacity of MSCs. Moreover, we found that BCOR maintained tissue homeostasis and gene silencing through epigenetic mechanisms. BCOR mutation increased histone H3K4 and H3K36 methylation in MSCs, thereby reactivating transcription of silenced target genes. By studying a rare human genetic disease, we have unravelled an epigenetic mechanism for control of human adult stem cell function.

Two crucial growth factors, FGF2 and TGFbeta1, were investigated in this study to determine their inductive effects on the odontoblastic differentiation of human dental pulp stem cells (DPSCs) in vitro. DPSCs were isolated by immunomagnetic bead selection using the STRO-1 antibody, and then co-cultured respectively with FGF2, TGFbeta1 and FGF2+TGFbeta1. The results showed that FGF2 can exert a significant effect on the cell proliferation, while TGFbeta1 or FGF2+TGFbeta1 can initiate an odontoblast-like differentiation of DPSCs. Moreover, FGF2 can synergistically upregulate the effects of TGFbeta1 on the odontoblastic differentiation of DPSCs, as indicated by the increased alkaline phosphatase activity, the polarized cell appearance and secretary ultrastructural features, the formation of mineralized nodules and the gene/protein expression of dentin sialoprotein and dentin matrix protein-1. Together, FGF2 acted primarily on the cell proliferation, while TGFbeta1 and FGF2+TGFbeta1 mainly stimulated the odontoblastic differentiation of DPSCs. This study provides interesting progress in the odontoblastic differentiation of DPSCs induced by FGF2 and TGFbeta1.

Aim: Human stem cells from the apical papilla (SCAPs) are an appealing stem cell source for tissue regeneration engineering. Circular RNAs (circRNAs) are known to exert pivotal regulatory functions in various cell differentiation processes, including osteogenesis of mesenchymal stem cells. However, few studies have shown the potential mechanism of circRNAs in the odonto/osteogenic differentiation of SCAPs. Herein, we identified a novel circRNA, circ‐ZNF236 (hsa_circ_0000857) and found that it was remarkably upregulated during the SCAPs committed differentiation. Thus, in this study, we showed the significance of circ‐ZNF236 in the odonto/osteogenic differentiation of SCAPs and its underlying regulatory mechanisms. Methodology: The circular structure of circ‐ZNF236 was identified via Sanger sequencing, amplification of convergent and divergent primers. The proliferation of SCAPs was detected by CCK‐8, flow cytometry analysis and EdU incorporation assay. Western blotting, qRT‐PCR, Alkaline phosphatase (ALP) and Alizarin red staining (ARS) were performed to explore the regulatory effect of circ‐ZNF236/miR‐218‐5p/LGR4 axis in the odonto/osteogenic differentiation of SCAPs in vitro. Fluorescence in situ hybridization, as well as dual‐luciferase reporting assays, revealed that circ‐ZNF236 binds to miR‐218‐5p. Transmission electron microscopy (TEM) and mRFP‐GFP‐LC3 lentivirus were performed to detect the activation of autophagy. Results: Circ‐ZNF236 was identified as a highly stable circRNA with a covalent closed loop structure. Circ‐ZNF236 had no detectable influence on cell proliferation but positively regulated SCAPs odonto/osteogenic differentiation. Furthermore, circ‐ZNF236 was confirmed as a sponge of miR‐218‐5p in SCAPs, while miR‐218‐5p targets LGR4 mRNA at its 3′‐UTR. Subsequent rescue experiments revealed that circ‐ZNF236 regulates odonto/osteogenic differentiation by miR‐218‐5p/LGR4 in SCAPs. Importantly, circ‐ZNF236 activated autophagy, and the activation of autophagy strengthened the committed differentiation capability of SCAPs. Subsequently, in vivo experiments showed that SCAPs overexpressing circ‐ZNF236 promoted bone formation in a rat skull defect model. Conclusions: Circ‐ZNF236 could activate autophagy through increasing LGR4 expression, thus positively regulating SCAPs odonto/osteogenic differentiation. Our findings suggested that circ‐ZNF236 might represent a novel therapeutic target to prompt the odonto/osteogenic differentiation of SCAPs. [ABSTRACT FROM AUTHOR]

Let-7 miRNA family has been proved as a key regulator of mesenchymal stem cells' (MSCs') biological features. However, whether let-7b could affect the differentiation or proliferation of periodontal ligament stem cells (PDLSCs) is still unknown. Here, we found that the expression of hsa-let-7b was visibly downregulated after mineralization induction of PDLSCs. After transfected with hsa-let-7b mimics or inhibitor reagent, the proliferation ability of PDLSCs was detected by cell counting kit-8 (CCK-8), flow cytometry, and 5-ethynyl-2-deoxyuridine (EdU) assay. On the other hand, the osteogenic differentiation capacity was detected by alkaline phosphatase (ALP) staining and activity, alizarin red staining, Western blot, and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). We verified that hsa-let-7b did not significantly impact the proliferation ability of PDLSCs, but it could curb the osteogenic differentiation of PDLSCs. Besides, we predicted CTHRC1 acts as the downstream gene of hsa-let-7b to affect this process. Moreover, the combination of CTHRC1 and hsa-let-7b was verified by dual luciferase reporter assay. Our results demonstrated that the osteogenic differentiation of PDLSCs was enhanced after inhibiting hsa-let-7b, while was weakened after cotransfection with Si-CTHRC1. Collectively, hsa-let-7b can repress the osteogenic differentiation of PDLSCs by regulating CTHRC1. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hyperoside, as an active compound, widely exists in a large number of Chinese herbal medicines and has been reported to possess anti‐inflammatory and diuretic properties. However, the effects and underlying mechanisms of hyperoside on periodontitis have not been previously reported. In this study, we found that hyperoside ameliorates symptoms of periodontitis in a rat model, with improvements in alveolar bone resorption, relief of inflammatory infiltration, increase in orderly arrangement of collagen fibers and increase of osteogenic differentiation. In addition, hyperoside promoted proliferation, up‐regulated EdU‐positive cells, decreased cell‐cycle distribution and increased the protein expression of Ki67 and PCNA in rat bone mesenchymal stem cells (rBMSCs), as revealed by Cell Counting Kit‐8, EdU, flow cytometry and western blot analysis. Moreover, hyperoside significantly promoted osteogenic differentiation, as shown by quantitative RT‐PCR, western blot and alizarin red staining assays. Furthermore, hyperoside activated the nuclear factor‐κB (NF‐κB) signaling pathway in rBMSCs, similar to the results observed in vivo. Finally, BMS345541, an inhibitor of the NF‐κB signaling pathway, could reverse the effects of hyperoside on the biological functions in rBMSCs. In conclusion, our results suggest that hyperoside has potential therapeutic properties against periodontitis via promotion of proliferation and osteogenic differentiation of rBMSCs via activation of the NF‐κB signaling pathway. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Results: [ABSTRACT FROM AUTHOR]