ABSTRACT
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.
ABSTRACT
Objective@#To compare the in vitro biocompatibility of bone marrow mesenchymal cells on polyetheretherketone (PEEK) and titanium (Ti) surfaces.@*Methods @#PEEK and Ti foils with thicknesses of 1 mm and diameters of 10 mm were prepared. First, bone marrow mesenchymal cells were separated and purified by the whole bone marrow adherent culture method in vitro. Then, osteogenesis-induced bone marrow mesenchymal cells were cultivated on the surfaces of the PEEK and Ti foils. Scanning electron microscopy (SEM), the Alamar Blue test, an alkaline phosphatase (ALP) kit and Alizarin Red staining were used to analyze calcium nodules and compare the adhesion, proliferation and osteogenic differentiation ability of bone marrow mesenchymal cells on the surfaces of the PEEK and Ti foils.@*Results @# ① The morphology of the bone marrow mesenchymal cells cultured on the PEEK and Ti foils at 1 h, 4 h and 24 h showed no significant differences. ② In the 1 h, 3 h, 1 d and 3 d cultures of the bone marrow mesenchymal cells inoculated on the surfaces of the foils, the number of living cells in the PEEK group was greater than that in the Ti group (P < 0.05). ③ In the 7 d and 14 d osteogenesis-induced cultures of the inoculated bone marrow mesenchymal cells, the ALP activity of the PEEK group cells was significantly greater than that of the Ti group cells (P < 0.05). ④ Semiquantitative analysis after Alizarin Red staining showed that the mineralization degree of the bone marrow mesenchymal cells induced by osteoblasts was greater in the PEEK group than in the Ti group (P < 0.05). @*Conclusion@#PEEK has better in vitro biocompatibility than Ti and can better promote cell adhesion, proliferation and osteogenic differentiation compared with Ti, and so it is expected to become a new dental implant material.
ABSTRACT
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.