Osteogenic differentiation from mesenchymal progenitor cells (MPCs) are initiated and regulated by a cascade of signaling events. Either Wnt/β-catenin or estrogen signaling pathway has been shown to play an important role in regulating skeletal development and maintaining adult tissue homeostasis. Here, we investigate the potential crosstalk and synergy of these two signaling pathways in regulating osteogenic differentiation of MPCs. We find that the activation of estrogen receptor (ER) signaling by estradiol (E2) or exogenously expressed ERα in MPCs synergistically enhances Wnt3A-induced early and late osteogenic markers, as well as matrix mineralization. The E2 or ERα-mediated synergy can be effectively blocked by ERα antagonist tamoxifen. E2 stimulation can enhance endochondral ossification of Wnt3A-transduced mouse fetal limb explants. Furthermore, exogenously expressed ERα significantly enhances the maturity and mineralization of Wnt3A-induced subcutaneous and intramuscular ectopic bone formation. Mechanistically, we demonstrate that E2 does not exert any detectable effect on β-catenin/Tcf reporter activity. However, ERα expression is up-regulated within the first 48h in AdWnt3A-transduced MPCs, whereas ERβ expression is significantly inhibited within 24h. Moreover, the key enzyme for the biosynthesis of estrogens aromatase is modulated by Wnt3A in a biphasic manner, up-regulated at 24h but reduced after 48h. Our results demonstrate that, while ER signaling acts synergistically with Wnt3A in promoting osteogenic differentiation, Wnt3A may crosstalk with ER signaling by up-regulating ERα expression and down-regulating ERβ expression in MPCs. Thus, the signaling crosstalk and synergy between these two pathways should be further explored as a potential therapeutic approach to combating bone and skeletal disorders, such as fracture healing and osteoporosis.

Background: Mesenchymal stem cells (MSCs) play a crucial role in maintaining the dynamic balance of bone metabolism. Melatonin may have a regulatory effect on bone metabolism by regulating the lineage commitment and differentiation signalling pathways of MSCs. Among the BMP families, the osteogenesis of BMP9 is considered to be one of the strongest in MSCs. Here, we explored whether melatonin and BMP9 act synergistically on MSC osteogenic differentiation. Methods: The C3H10T1/2 osteogenic differentiation function induced by melatonin synergizes with BMP9, as detected by the expression of osteogenic markers at different periods. The result was further confirmed by foetal limb explant culture and in vivo stem cell implantation experiments. The effects of the AMPK/β-catenin pathway on the osteogenic differentiation of C3H10T1/2 cells were evaluated by Western blotting. Results: Melatonin combined with BMP9 significantly enhanced the expression of osteogenic markers at different periods in C3H10T1/2 cells, effectively enhancing BMP9-induced bone formation in cultured foetal explants and ectopic bone formation in vivo in stem cell transplantation experiments. Melatonin increases the expression of BMP9 in C3H10T1/2 cells and induces Smad1/5/8 translocation from the cytoplasm to the nucleus. In addition, melatonin and BMP9 synergistically promote AMPK and β-catenin phosphorylation, which can be largely eliminated by AMPK siRNA pretreatment. Conclusions: Melatonin and BMP9 in C3H10T1/2 cells synergistically promote osteogenic differentiation at least in part by activating the AMPK/β-catenin signalling pathway.

Background. Mesenchymal stem cells (MSCs) differentiate into osteocytes, adipocytes, and chondrocytes. Resveratrol and bone morphogenetic protein 9 (BMP9) are known osteogenic induction factors of MSCs, but the effect of both resveratrol and BMP9 on osteogenesis is unknown. Herein, we explored whether resveratrol cooperates with BMP9 to improve osteogenic induction. Methods. The osteogenic induction of resveratrol and BMP9 on C3H10T1/2 cells was evaluated by detecting the staining and activity of the early osteogenic marker alkaline phosphatase (ALP). In addition, the late osteogenic effect was measured by the mRNA and protein levels of osteogenic markers, such as osteopontin (OPN) and osteocalcin (OCN). To assess the bone formation function of resveratrol plus BMP9 in vivo, we transplanted BMP9-infected C3H10T1/2 cells into nude mice followed by intragastric injection of resveratrol. Western blot (WB) analysis was utilized to elucidate the mechanism of resveratrol plus BMP9. Results. Resveratrol not only enhanced osteogenic induction alone but also improved BMP9-induced ALP at 3, 5, and 7d postinduction. Both the early osteogenic markers (ALP, Runx2, and SP7) and the late osteogenic markers (OPN and OCN) were significantly increased when resveratrol was combined with BMP9. The fetal limb explant culture further verified these results. The in vivo bone formation experiment, which involved transplanting BMP9-overexpressing C3H10T1/2 cells into nude mice, also confirmed that resveratrol synergistically enhanced the BMP9-induced bone formation function. Resveratrol phosphorylated adenosine monophosphate- (AMP-) activated protein kinase (AMPK) and stimulated autophagy, but these effects were abolished by inhibiting AMPK and Beclin1 using an inhibitor or siRNA. Conclusions. Resveratrol combined with BMP9 significantly improves the osteogenic induction of C3H10T1/2 cells by activating AMPK and autophagy.

The osteogenic differentiation capacity of senescent bone marrow mesenchymal stem cells (MSCs) is reduced. p53 not only regulates cellular senescence but also functions as a negative regulator in bone formation. However, the role of p53 in MSCs senescence and differentiation has not been extensively explored. In the present study, we investigated the molecular mechanism of p53 in MSCs senescence and osteogenic differentiation. We found that p53 was upregulated during cellular senescence and osteogenic differentiation of MSCs respectively induced by H₂O₂ and BMP9. Similarly, the expression of p53-induced miR-145a was increased significantly. Furthermore, Overexpression of miR-145a in MSCs promoted cellular senescence and inhibited osteogenic differentiation. Then, we identified that p53-induced miR-145a inhibited osteogenic differentiation by targeting core binding factor beta (Cbfb), and the restoration of Cbfb expression rescued the inhibitory effects of miRNA-145a. In summary, our results indicate that p53/miR-145a axis exert its functions both in promoting senescence and inhibiting osteogenesis of MSCs, and the novel p53/miR-145a/Cbfb axis in osteogenic differentiation of MSCs may represent new targets in the treatment of osteoporosis.

Objective. The purpose of this study was to evaluate the effect of bisphosphonates in improving bone mineral density (BMD) and decreasing the occurrence rate of fractures and adverse events in patients with inflammatory bowel disease (IBD). Methods. Randomized controlled trials (RCTs) which use bisphosphonates in IBD patients were identified in PubMed, MEDLINE database, EMBASE database, Web of Knowledge, and the Cochrane Databases between 1990 and June 2016. People received bisphosphonate or placebos with a follow-up of at least one year were also considered. STATA 12.0 software was used for the meta-analysis. Results. Eleven randomized clinical trials were included in the meta-analysis. The data indicated that the percentage change in the increased BMD in the bisphosphonates groups was superior to that of the control groups at the lumbar spine and total hip. At the femoral neck, there was no significant difference between the two groups. The incidence of new fractures during follow-up showed significant reduction. The adverse event analysis revealed no significant difference between the two groups. Conclusion. Our results demonstrate that bisphosphonates therapy has an effect on bone loss in patients with IBD but show no evident efficiency at increasing the incidence of adverse events.