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Background: In mouse, it was discovered that resveratrol (Res) enhanced osteoporosis (OP) by boosting osteogenesis. Besides, Res can also have an impact on MC3T3-E1 cells, which are crucial for the control of osteogenesis and thus increase osteogenesis. Although some articles have discovered that Res enhanced autophagy to promote the value-added differentiation of MC3T3, it is unclear exactly how this affects the process of osteogenesis in mouse. Therefore, we will show that Res encourages MC3T3-E1 proliferation and differentiation in mouse pre-osteoblasts and further investigate the autophagy-related mechanism for this impact. Methods: (1) MC3T3-E1 cells were separated into blank control group and various concentrations (0.01, 0.1, 1, 10, 100µmol/L) of group in order to determine the ideal Res concentration. In the Res group, Cell Counting Kit-8 (CCK-8) was used to measure the proliferation activity of pre-osteoblasts in mice in each group after resveratrol intervention. Alkaline Phosphatase (ALP) and alizarin red staining were used to gauge the degree of osteogenic differentiation, and RT-qPCR was used to measure the expression levels of Runx2 and OCN in the osteogenic differentiation ability of the cells. (2) In the experiment, four groups were set up: the control group, 3MA group, Res group, and Res + 3MA group. To examine cell mineralization, ALP and alizarin red staining were utilized. RT-qPCR and Western blot detection of cell autophagy activity levels and osteogenic differentiation capacity in each group following intervention. Results: (1) Resveratrol might increase the number of mice pre-osteoblast, with the impact being most pronounced at 10µmol/L (P < 0.05). The nodules developed substantially more often than in the blank control group, and Runx2 and OCN expressions significantly increased (P < 0.05). (2) In contrast to the Res group, after 3MA purine blocked autophagy, the Res + 3MA group's alkaline phosphatase staining and the development of mineralized nodules were reduced. Runx2, OCN, LC3II / LC3I expression decreased, p62 expression increased (P < 0.05). Conclusion: The present study partially or indirectly demonstrated that Res may, through increased autophagy, induce osteogenic differentiation of MC3T3-E1 cells. [ABSTRACT FROM AUTHOR]

Background: While autophagy is essential for stem cells' self-renewal and differentiation, its effect on bone marrow mesenchymal stem cells (BMSCs) remains unclear. This study aimed to investigate the interaction between autophagy and osteogenic differentiation using rapamycin (RAPA), a classical autophagy agonist with osteo-regulatory effects. Methods: Rat BMSC's autophagy was analyzed after osteoinduction (0, 7, 14, and 21 d) by western blotting, immunofluorescence, and real-time quantitative polymerase chain reaction (RT-qPCR). In addition, we evaluated osteogenic differentiation using alizarin red staining, alkaline phosphatase assays, and RT-qPCR/Western blotting quantification of bone sialoprotein, type 1 collagen, alkaline phosphatase, osteopontin, and Runt-related transcription factor 2 mRNA and protein levels. Results: The BMSC's basal autophagy level gradually decreased during osteogenic differentiation with a decrease in BECN1 level and the lipidated (LC3-II) to unlipidated (LC3-I) microtubule-associated protein 1 light chain 3 ratio and an increase in the expression of selective autophagic target p62. In contrast, it increased with increasing RAPA concentration. Furthermore, while 2 nM RAPA promoted BMSC osteogenic differentiation on days 7 and 14, 5 nM RAPA inhibited osteogenesis on days 14 and 21. Inhibition of autophagy by the inhibitor 3-methyladenine could impair RAPA's osteogenesis-enhancing effect on BMSCs. Conclusions: The BMSC's basal autophagy level decreased over time during osteogenic differentiation. However, an appropriate RAPA concentration promoted BMSC osteogenic differentiation via autophagy activation. [ABSTRACT FROM AUTHOR]

BACKGROUND: Studies have shown that the loss of sex combing protein 1 (Asxl1) can lead to the occurrence of bone dysplasia and bone defects, but the relationship between this factor and bone destruction in the microenvironment of apical periodontitis has not been reported. OBJECTIVE: To study the effect of Asxl1 on proliferation and differentiation of osteoblasts in an inflammatory microenvironment. METHODS: MC3T3-E1 cells were excited by lipopolysaccharide to establish an in vitro inflammatory microenvironment. The best concentration and optimal action time of lipopolysaccharide were screened by cell counting kit-8 test. MC3T3-E1 cells were then stimulated with 20 mg/L lipopolysaccharide for 24 hours. The expression levels of Asxl1 protein and mRNA were detected by immunofluorescence and real-time PCR, respectively. After lipopolysaccharide stimulated the formation of inflammatory microenvironment, Asxl1-Si RNA was transfected for 24 hours, cell counting kit-8 was applied to detect the activity of cell proliferation, and real-time PCR was used to detect the expression levels of Asxl1 and osteogenic related genes ALP and RUNX2 mRNA. RESULTS AND CONCLUSION: After lipopolysaccharides stimulation of MC3T3-E1 cells, the expression levels of Asxl1 protein and mRNA were decreased. Under the inflammatory microenvironment, the proliferation activity of MC3T3-E1 cells transfected with Asxl1-Si RNA for 24 hours was significantly decreased, and the expression levels of Asxl1, ALP and RUNX2 mRNA were markedly decreased. These findings indicate that Asxl1 may influence the proliferation and differentiation of osteoblasts by involvement in the process of inflammatory reaction, thereby participating in bone destruction. [ABSTRACT FROM AUTHOR]

BACKGROUND: P75 neurotrophin receptor (P75NTR) is widely expressed in nerve tissues and cells, and plays a dual role in promoting or inhibiting differentiation. P75NTR is also overexpressed in local tissues with fracture nonunion. Therefore, P75NTR is studied for the osteogenic differentiation of bone marrow mesenchymal stem cells, which is of great significance to provide important targets for the clinical treatment of fracture nonunion. OBJECTIVE: To elucidate the effect of P75NTR overexpression on osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro. METHODS: The bilateral femurs of Sprague-Dawley rats were selected, and the rat bone marrow mesenchymal stem cells were extracted by whole bone marrow separation and adherence method and subcultured in vitro. The P75NTR overexpression plasmid GV358-P75NTR expressing enhanced green fluorescent protein was constructed, and the P75NTR overexpression lentiviral vector was collected by empty lentiviral vector packaging. Rat bone marrow mesenchymal stem cells primary cultured in vitro for 10 days were selected, and seeded into culture plates after digestion. P75NTR overexpression lentivirus and related infection reagents were added for subsequent infection experiments. After 7 days of infection, the expression of green fluorescent protein was observed by fluorescence microscope and overexpression of P75NTR protein was detected by western blot. Transfected cells were cultured for 7 days in a conventional medium, followed by culture in the osteogenic differentiation medium. Alkaline phosphatase activity was quantified by enzyme linked immunosorbent assay on the 7th, 10th, and 14th days after osteogenic induction. Formation of mineralized nodules was observed by alizarin red staining on the 7th and 14th days after osteogenic induction. RESULTS AND CONCLUSION: P75NTR overexpression lentiviral vector-infected bone marrow mesenchymal stem cells expressed green fluorescent protein (infection efficiency was about 90%), and the expression of P75NTR protein was significantly increased, which was significantly different from that in the negative control group (P < 0.05). Cell model of P75NTR overexpression was successfully constructed. Compared with the negative control and blank control groups, the alkaline phosphatase activity of the P75NTR overexpression group was significantly decreased at the corresponding time point after osteogenic induction, the number of mineralized nodules was significantly reduced, and cell aggregation and distribution were significantly weakened (P < 0.05). To conclude, P75NTR overexpression negatively regulates the osteogenic differentiation of rat bone marrow mesenchymal stem cells cultured in vitro. Overexpression of P75NTR in local tissues inhibits the osteogenic differentiation of surrounding bone marrow mesenchymal stem cells, which may be an important factor for bone defects or fracture nonunion. [ABSTRACT FROM AUTHOR]

BACKGROUND: Previous studies have confirmed that insulin-like growth factor-binding protein 5 (IGFBP5) is highly expressed in dental stem cells and exerts roles by activating multiple signaling pathways. However, it is unclear whether IGFPB5 is involved in the regulation of mesenchymal stem cell osteogenic differentiation in inflammatory microenvironment. OBJECTIVE: To study the influence and molecular mechanism of IGFBP5 on osteogenic differentiation of periodontal ligament stem cells under inflammatory microenvironment, and to provide a new therapeutic target for periodontal tissue regeneration in inflammatory microenvironment. METHODS: After periodontal ligament stem cells were isolated and collected, their surface antigen markers were detected by flow cytometry. The stable IGFBP5 knock-down cell lines were established, and were cultured in osteogenic differentiation medium containing tumor necrosis factor α and interleukin 17. We then detected alkaline phosphatase activities and mRNA expression of marker genes relating to osteogenic differentiation and histone methyltransferase. The study protocol was implemented in line with the ethic requirements of Tianjin Medical University Hospital of Stomatology. RESULTS AND CONCLUSION: (1) The osteogenic capacity of periodontal ligament stem cells decreased under stimulation of tumor necrosis factor α and interleukin 17. Compared with the blank control group, the activity of alkaline phosphatase and the expressions of osteogenic differentiation related genes OCN and BSP were significantly reduced after osteoblastic induction. (2) Gene silencing of IGFBP5 allowed tumor necrosis factor α and interleukin 17 to further inhibit the osteogenesis of periodontal ligament stem cells, characterized by decreased alkaline phosphatase activity and further decreased expression of osteoblastic differentiation related genes OCN and BSP. (3) Inflammatory factor stimulation and gene silencing of IGFBP5 also affected the mRNA expression of histone demethylases KDM2A, KDM3B, KDM5B and JMJD6. To conclude, IGFBP5 may play a positive role in the osteogenic differentiation of periodontal ligament stem cells. [ABSTRACT FROM AUTHOR]

Objective: To explore the expression of miR-31 and Satb2 gene in the serum of postmenopausal women with osteoporosis (OP)., Methods: 97 postmenopausal women with OP and 100 healthy women were selected as research subjects. MSCs were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. Bone marrow-derived mesenchymal stem cells (BMSCs) were isolated, identified and transfected, and then quantified by alkaline phosphatase (ALP) levels. The expression levels of miR-31 and Satb2 gene mRNA were determined by qRT-PCR. The proteins of RUNX2, OCN and BMP and Wnt/β-catenin pathway-related proteins (GSK-3, Frizzled 1, Lrp5, Lrp6 and β-catenin) were tested by Western blotting., Results: In the OP group, the relative expression of miR-31 was 3.61±0.54, significantly higher than that (1.75±0.27) in the healthy control group (t=9.422, P<0.001). The relative expression of mRNA of Satb2 gene was 0.86±0.12, significantly lower than that (1.35±0.21) in the healthy control group (t=5.897, P<0.001)., Conclusions: The increase in miR-31 expression can down-regulate the Wnt/β-catenin pathway by targeting the expression of Satb2 gene, thereby inhibiting the osteogenic differentiation of BMSCs. This provides an important reference for further understanding the mechanism of OP and identifying targets for early diagnosis and treatment., Competing Interests: The authors have no conflict of interest.

Er-xian Decoction (EXD) is a well-known prescription widely used to prevent and treat climacteric syndrome and osteoporosis in China. BK channel positively affects osteoblast bone formation in vitro. However, it is still unclear whether the effect of EXD on promoting osteoblasts osteogenic differentiation is related to BK channel. The study is aimed at determining whether the EXD-containing serum promotes the proliferation of osteoblasts and their differentiation through BK channel. The chemical compounds of EXD were analyzed by UPLC-Q-TOF/MS. An osteogenic induction medium (OM) was used to induce MC3T3-E1 cells' osteogenic differentiation. The effects of EXD-containing serum and tetraethylammonium (TEA) on the proliferation activity of Mc3t3-e1 cells were detected by CCK-8 assay. ALP activity was determined by an alkaline phosphatase kit. The protein expression (BMP2, OPG, and COL1) was analyzed by Western blot, and the mRNA expression (Runx2, OPG, and BMP2) was assessed by real-time PCR. Alizarin red was used to stain the mineralized region of osteoblasts. In addition, we analyzed the relationship between BK channel and its downstream PTEN/Akt/Foxo1 signaling pathway. 72 compounds were identified by UPLC-Q-TOF/MS analysis in EXD. Mangiferin, ferulic acid, berberine, and icariin were main active components of EXD. EXD-containing serum could enhance the cell viability of MC3T3-E1 cells by decreasing the expression of BKα protein. EXD-containing serum increased ALP activity and calcium nodule formation of Mc3t3-e1 cells, promoted the protein expression of BKα, COL1, BMP2, OPG, and the mRNA expression of RUNX2, OPG, and BMP2, however, these effects can be reversed after adding TEA. In addition, EXD-containing serum could upregulate phosphorylation of Akt and Foxo1 in osteogenic-induced Mc3t3-e1 cells, and lower the expression of PTEN. And these effects of EXD-containing serum could be reduced by TEA. The effect of EXD-containing serum on promoting cell proliferation and osteogenic differentiation of Mc3t3-e1 cells might be linked to the regulation of BK channel. [Display omitted] • 72 chemical components were identified in EXD by UPLC-Q-TOF/MS. • EXD-containing serum promotes Mc3t3-e1 cell proliferation by inhibiting BKα expression. • EXD-containing serum promotes osteoblasts osteogenic differentiation by increasing BKα expression. • EXD-containing serum activates PTEN/Akt/Foxo1 signaling pathway through BK channel to accelerate bone formation. [ABSTRACT FROM AUTHOR]

Background and Objective Follicular dendritic cell secreted protein ( FDC- SP), has been identified in human periodontal ligament ( PDL) in a recent study. It is suggested that the expression of FDC- SP might be associated with the osteogenic differentiation and mineralization of human periodontal ligament cells (h PDLCs). However, the intrinsic mechanism regarding this is still unclear. The aim of this study was to establish h PDLCs with safe and efficient overexpression of FDC- SP and to elucidate the influence of FDC- SP transfection on h PDLC osteogenesis in periodontal regeneration. Material and Methods We first applied a recombinant lentiviral vector containing FDC- SP to transfect h PDLCs via different multiplicity of infection ( MOI) levels (1, 10, 20, 50 and 100). Western blot was performed to confirm the expression of FDC- SP. MTT assay was employed to evaluate the proliferation status of transfected cells. Then, the extent of osteogenic differentiation was investigated by simultaneous monitoring of alkaline phosphatase ( ALP) activity assessment, immunofluorescent staining, the expression patterns of osteoblastic markers and mineralization staining. Results We found that h PDLCs transfected via MOI 20, 50 and 100 exhibited expression of FDC- SP protein compared with MOI 1 and 10. There was no significant effect of FDC- SP transfection (at different MOI levels of 1, 10 and 20) on the proliferation of h PDLCs, whereas higher MOI levels (50 and 100) inhibited cell proliferation ability. In addition, ALP activity decreased significantly in FDC- SP-transfected h PDLCs at day 7. When stained with alizarin red, cells overexpressing FDC- SP formed less mineralized nodules at 21 d post-induction of differentiation, compared with the control cultures. Osteogenic inhibition was also confirmed by ALP immunostaining. Moreover, m RNA expression levels of osteoblastic markers decreased after FDC- SP transfection, which were in accordance with western blot results. Conclusion Our data suggest that MOI 20 is optimal to transfect h PDLCs, which achieves safe and efficient overexpression of FDC- SP in transfected cells. Moreover, FDC- SP overexpression inhibits osteogenic differentiation of h PDLCs. The present study contributes to a better understanding of the biological functions governing FDC- SP-induced h PDLC differentiation. [ABSTRACT FROM AUTHOR]

An S, Ling J, Gao Y, Xiao Y. Effects of varied ionic calcium and phosphate on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells in vitro . J Periodont Res 2012; 47: 374-382. © 2011 John Wiley & Sons A/S Background and Objective: A number of bone-filling materials containing calcium (Ca2+) and phosphate (P) ions have been used in the repair of periodontal bone defects; however, the effects that local release of Ca2+ and P ions has on biological reactions are not fully understood. In this study, we investigated the effects of various levels of Ca2+ and P ions on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells (hPDLCs). Material and Methods: The hPDLCs were obtained using an explant culture method. Defined concentrations and ratios of ionic Ca2+ to inorganic P were added to standard culture and osteogenic induction media. The ability of hPDLCs to proliferate in these growth media was assayed using the Cell Counting Kit-8. Cell apoptosis was evaluated by the fluorescein isothiocyanate-annexin V/propidium iodide double-staining method. Osteogenic differentiation and mineralization were investigated by morphological observations, alkaline phosphatase activity and Alizarin Red S/von Kossa staining. The mRNA expression of osteogenic related markers was analysed using RT-PCR. Results: Within the ranges of Ca2+ and P ion concentrations tested, we observed that increased concentrations of Ca2+ and P ions enhanced cell proliferation and formation of mineralized matrix nodules, whereas alkaline phosphatase activity was reduced. The RT-PCR results showed that elevated concentrations of Ca2+ and P ions led to a general increase of Runx2 mRNA expression and decreased alkaline phosphatase mRNA expression, but gave no clear trend on osteocalcin mRNA levels. Conclusion: The concentrations and ratios of Ca2+ and P ions could significantly influence proliferation, differentiation and mineralization of hPDLCs. Within the range of concentrations tested, we found that the combination of 9.0 m m Ca2+ ions and 4.5 m m P ions were the optimal concentrations for proliferation, differentiation and mineralization in hPDLCs. [ABSTRACT FROM AUTHOR]

Abstract: Aim of the study: Panax notoginseng saponins (PNS) is the main effective component of Panax notoginseng, have various pharmacologic activities such as antioxidant, anti-inflammatory, and estrogen-like bioactivities, have been shown to be an effective agent on anti-osteoporosis. Bone marrow stromal cells (BMSCs) play a crucial homeostatic role in skeletal modeling and remodeling due to their capability to differentiate into osteooblasts. Whether PNS has effect on osteogenic differentiation of BMSCs are unknown. This study was designed to investigate the effects of PNS on the proliferation and osteogenic differentiation of BMSCs in vitro. Materials and methods: When BMSCs cultivated in the basal medium or the osteogenic induction medium (OS with or without PNS), cell proliferation was analyzed using an MTT assay, the mineralization was assessed using Alizarin red S staining, the alkaline phosphatase activity was measured using a commercial kit, the mRNA level of osteogenic gene and PPARγ2 gene were determined using RT-PCR, the protein level of PPARγ2 was analyzed by Western blotting. Results: BMSCs cultured in the basal medium with PNS caused a significant increase in proliferation. PNS treatment increased ALP activity, Alizarin red S staining and mRNA level of ALP, Cbfa 1, OC, and BSP, whereas decreased the mRNA level and protein expression of PPARγ2 during osteogenic induction. In addition, the effects of PNS treatment were dose-dependent relationship. Conclusion: PNS could stimulate BMSCs proliferation and promote their osteogenic differentiation by up-regulation expression of osteogenic marker gene and down-regulation expression of adipogenic marker gene in a dose-dependent manner. Thus, PNS may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs. [Copyright &y& Elsevier]

Ossification of the posterior longitudinal ligament (OPLL) is a spinal disorder characterized by progressive ectopic bone formation in the PLL of the spine. Dickkopf-1 (Dkk1) is a secreted inhibitor of the Wnt pathway that negatively regulates bone formation during skeletal development. However, whether Dkk1 impacts the pathogenesis of OPLL has not been reported. This study is to investigate the role of Dkk1 in the development of OPLL. Our results show that the serum levels of Dkk1 are decreased in OPLL patients compared with non-OPLL controls. The expression of Dkk1 is also reduced in OPLL ligament cells. Downregulation of Dkk1 in ligament cells is associated with activation of the Wnt/β-catenin signaling, as indicated by stabilized β-catenin and increased T-cell factor-dependent transcriptional activity. Functionally, Dkk1 exerts a growth-inhibitory effect by repressing proliferation but promoting apoptosis of ligament cells. Dkk1 also suppresses bone morphogenetic protein 2-induced entire osteogenic differentiation of ligament cells, and this suppression is mediated via its inhibition of the Wnt pathway. Our results demonstrate for the first time that Dkk1 acts as an important negative regulator in the ossification of the PLL. Targeting the Wnt pathway using Dkk1 may represent a potential therapeutic strategy for the treatment of OPLL., (© 2020 International Federation for Cell Biology.)