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Objective @#To investigate the activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway molecules during the process by which kaempferol (Kae) promotes osteogenic differentiation of mouse bone marrow mesenchymal cells (BMMCs) under cyclic and uniaxial tension.@*Methods @#BMMCs isolated and cultured in vitro were subjected to uniaxial dynamic tension with a 10% shape variable. The appropriate concentration of Kae was selected by cytotoxicity testing. The endogenous mTOR signal was inhibited by pp242. Four hours after traction, alkaline phosphatase (ALP) and osteocalcin (OCN) were detected by chemical colorimetry and ELISA, and the relative concentration of intracellular calcium was detected by flow cytometry. Phosphorylation of mTOR, 4E/BP1, and ribosomal protein S6 kinases (S6K), which are the main molecules of the endogenous mTORC1 signaling pathway, and expression of osteogenic transcription factors (Runx2 and Osterix) were detected by western blotting (WB), and mRNA expression levels of the above factors were detected by qRT-PCR.@*Results @# The cytotoxicity test showed that 10 μmol/L Kae had little inhibitory effect on cell proliferation but had the strongest osteogenic ability. Four hours after stretching, Kae effectively promoted the osteogenic differentiation of BMMCs. The expression of ALP was （153.04 ± 18.72） U/mg, the expression of OCN was （1.64 ± 0.25） U. The mRNA and protein levels of Runx2 and Osterix were upregulated, and the intracellular calcium content was decreased. The mRNA and protein phosphorylation of mTOR and S6K was upregulated, and the opposite effect was observed with 4E/BP1. After pp242 was added to inhibit mTOR signaling, mTOR and S6K mRNA and protein phosphorylation were downregulated, but 4E/BP1 mRNA and protein phosphorylation was upregulated. The osteogenic differentiation of BMMCs was also significantly inhibited, mRNA and protein expression of Runx2 and Osterix were significantly downregulated, ALP and OCN expression were downregulated, and intracellular calcium content was increased. @* Conclusion@#Kae promotes osteogenic differentiation of mouse BMMCs under uniaxial dynamic tension through the mTORC1 signaling pathway.
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Objective@# To investigate the expression of the mTORC1 signaling pathway during the osteogenic differentiation of mouse bone marrow mesenchymal cells (BMMSCs) under cyclic uniaxial tension and explore its possible role.@*Methods @# The BMMSCs of mice were affected by uniaxial dynamic tensile force. Western blot was used to detect the expression changes of major molecules (mTOR, Raptor, S6K) in the endogenous mTORC1 signaling pathway at 0, 1, 2, 4, and 8 hours after stretching. Chemical colorimetry, ELISA and PCR were used to detect alkaline phosphatase (ALP), osteocalcin (OCN) and Runx2 mRNA, respectively. Then, inhibition, activation and control groups were established by administration of the drugs PP242, MHY1485 and PBS, respectively. Two hours after the stress, the expression of S6K was detected by western blot, and the expression of the osteogenic signal was continuously detected by the above methods.@*Results @#Western blot analysis showed that the main molecules of the mTORC1 signaling pathway were all expressed within 8 hours after traction, and the highest expression was 2 hours after the stress. Compared with those in the control group, the ALP activity and OCN expression decreased and the Runx2 mRNA levels increased after the mTORC1 signal pathway was inhibited (P ＜ 0.001); ALP activity and OCN expression increased after the mTORC1 signal pathway was activated, while the Runx2 mRNA levels decreased (P ＜ 0.001). @*Conclusion @#The mTORC1 signaling pathway participates in the osteogenic differentiation of mouse BMMSCs under tension. The osteogenesis of BMMSCs under cyclic uniaxial tension would be enhanced if the mTORC1 signaling pathway was activated.
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Objective @#To explore the promoting effect of periostin on rapid distraction osteogenesis of the rabbit mandible and provide experimental evidence for the clinical use of periostin to promote osteogenesis.@*Methods@#Twenty-four New Zealand male white rabbits underwent distraction osteogenesis, and after 3 days of retention, they were rapidly stretched at a stretch rate of 2 mm/d (total 5 d). The animals were randomly divided into group A and group B (12 per group). On the last day of the stretch, 0.5 mL of normal saline containing 40 μg of recombinant periostin was given to group B or an equal volume of normal saline was added to the control group (group A) for 8 days. At 4 weeks and 8 weeks post-stretch, 8 animals were randomly selected from each group to undergo a CT scan under general anesthesia. The bone mineral density and bone mineral content were detected by dual energy X-ray absorptiometry. Eight weeks post-stretch, all of the experimental animals were sacrificed. Six animals were randomly selected from each group for micro-CT and a histological examination, and the remaining animals were subjected to biomechanical tests. @*Results @#CT images showed that the new bone formation in the distraction space of group B was significantly better than that of group A at 4 and 8 weeks post-stretch. At 4 weeks and 8 weeks post-stretch, the bone mineral density in group B was (0.157 ± 0.016) g/cm 2 and (0.234 ± 0.023) g/cm 2, respectively, and the bone mineral content was (0.096 ± 0.010) g and (0.204 ± 0.017) g, respectively. The above four means were significantly higher in group B than in group A (P < 0.001). The micro-CT images and data suggest that the stretch gap microstructure of group B has more mature features. Histological experiments showed that the trabecular bone of group B was thick and mature, with few chondrocytes. The biomechanical test results showed that the biomechanical strength of the distraction gap in group B was (228.47 ± 39.98) N, which was 1.24 times that of group A (P = 0.045).@*Conclusion@# Interstitial use of periosteal protein in the distraction space of the mandible in rabbits can promote local new bone formation and mineralization.