ObjectiveTo investigate the regulatory effect of simvastatin on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at middle/late stages by p38MAPK pathway under condition of osteoinductive environment. MethodsThe bone marrow of bilateral femur and tibia were harvested from 20 4-week-old female Sprague Dawley rats. BMSCs were isolated and cultured with whole bone marrow culture method; the second generation of cells were randomly divided into 5 groups: control group (complete medium, CM), simvastatin group (simvastatin medium, SIM), osteogenic induction group (osteogenic induction medium, OM), simvastatin and osteogenic induction group (simvastatin+osteogenic induction medium, OM+SIM), and blocker group (SB203580+simvastatin+osteogenic induction medium, OM+SIM+SB). MTT assay was used to detect the cell activity in CM group and SIM group at 2, 3, 4, 5, and 6 days, ELISA method to measure the content of alkaline phosphatase (ALP) in OM group and OM+SIM group at 7 and 14 days. The mRNA and protein expressions of osteocalcin (OCN) were detected by real-time quatitative PCR and Western blot after 1, 12, and 24 hours of osteogenic induction at 21 and 28 days. The protein expressions of phospho-p38 (p-p38) and p38 in OM group, OM+SIM group, and OM+SIM+SB group were detected by Western blot at the best induction time of simvastatin. ResultsMTT assay showed that no significant difference was found in absorbance (A) value between CM group and SIM group at each time point (P > 0.05), indicating no effect of 1×10-7 mol/L simvastatin on cell viability. ELISA results showed that ALP content significantly increased in OM+SIM group when compared with OM group at 7 and 14 days; the ALP content was significantly higher at 7 days than 14 days in OM group and OM+SIM group (P < 0.05). OCN mRNA and protein expressions at 12 hours were significantly higher than those at other time points in each group (P < 0.05), and the expressions of OM+SIM group was significantly higher than those of OM group (P < 0.05). The best induction time of simvastatin was 12 hours. At 12 hours after blocking intervention, the p-p38/p38 in OM+SIM+SB group was significantly lower than that in OM group and OM+SIM group (P < 0.05), and the p-p38/p38 in OM+SIM group was significantly higher than that in OM group (P < 0.05). ConclusionSimvastatin can increase the mRNA and protein expression levels of OCN and the protein of p-p38 in osteogenic differentiation of BMSCs at middle/ late stages, and its best induction time is 12 hours.
ObjectiveTo summarize the research progress of the effects and mechanisms of Hedgehog signaling pathway in regulating bone formation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). MethodsThe related literature concerning the regulations and mechanism of Hedgehog signaling pathway in osteogenic differentiation of BMSCs and bone formation in vivo, in vitro, and ex vivo studies in recent years was analyzed and summarized. ResultsThe in vitro studies indicate that Hedgehog signaling pathway can promote osteogenic differentiation of BMSCs via activation of key molecules Smoothened (Smo) and Gli1 which are downstream of Hedgehog signaling, and Hedgehog signaling can activate mTORC2-Akt signaling by upregulation of insulin-like growth factor which has similar effects. Hedgehog signaling regulates osteoblast differentiation via activation of Hh-Smo-Ptch1-Gli signaling pathway and inhibition of Hh-Gαi-RhoA stress fibre signaling. Hedgehog signaling can regulate key molecules of osteogenesis Runx2 for promoting osteogenic differentiation and matrix mineralization by synergism of bone morphogenetic protein and Wnt signaling, and promotes bone formation and repair and healing for bone defect and bone graft model in vivo. ConclusionHedgehog signaling can regulate bone formation and osteogenic differentiation of BMSCs via activation of Hedgehog signaling and other signaling pathways. Hedgehog signaling pathway may be a potential target for developing treatment for bone related diseases of osteoporosis and fracture healing disorders.