| Objective 1. To explore whether orthosilicic acid promotes rat bone marrow mesenchymal stem cells (BMSCs) to osteogenic differentiation by increasing the expression of bone morphogenetic protein 2 (BMP-2).2. To define whether orthosilicic acid promotes collagen type 1 (COL-1) and osteocalcin through BMP-2/Smadl/5/RUNX2 signaling pathway.Methods The rat BMSCs were incubated with different concentrations of orthosilicic acid (0,5,10,20,50 μM). After 48 h, the expression of BMP-2 was detected by Western blot. The rat BMSCs were incubated with 10 μM orthosilicic acid for different times (0、24、48、72 h). The expression of BMP-2 was detected by Western blot. The cells were first treated with 100ng/ml noggin for 2 h. Then the expression of BMP-2 was detected by Western blot. The rat BMSCs were incubated with different concentrations of orthosilicic acid (0,5,10,20,50 μM). After 72 h, the expression of COL-1 was detected by Western blot. The rat BMSCs were incubated with 10 μM orthosilicic acid for different times (0、48、72、96 h). The expression of COL-1 was detected by Western blot. Colorimetry was used to measure the intracellular ALP activity after the cells were incubated with different concentrations of orthosilicic acid (0,5,10,20,50 μM) for 7 d. The influence of noggin was also used the same way. After the rat BMSCs were incubated with different concentrations of orthosilicic acid (0,10 μM), the formation of mineralized nodules was observed with alizarin red S staining. MG-63 and U2-OS cells were stimulated with different concentrations of orthosilicic acid (0,10,20,50 μM). After 72h, the expression of COL-1, osteocalcin and the relevant proteins of BMP-2/Smadl/5/RUNX2 signaling pathway was detected by Western blot. The cells were first treated with noggin for 2 h. Then the expression of BMP-2、P-Smad1/5 and RUNX2 was detected by Western blot following exposure to noggin. In MG-63 cells, immunofluorescence methods were applied to detect changes in the expression of BMP-2, P-Smad1/5 and RUNX2. Colorimetry was used to measure the intracellular ALP activity after the cells were incubated with different concentrations of orthosilicic acid (0,10,20,50 μM) for 7 d.Results After being incubated with different concentrations of orthosilicic acid for 48 h, the expression of BMP-2 began to increase following orthosilicic acid at 5 uM. Orthosilicic acid promotes the expression of BMP-2 and exposure to orthosilicic acid at 10μM produced the maximum effect compared with the control group, and the difference was statistically significant (P<0.05). But with the increase of the concentration, to 50 μM, the expression of BMP-2 did not significantly increase (P>0.05). The expression of BMP-2 was increased under orthosilicic acid treatment at 10 μM for 24h and 48h compared with the control group (P<0.05). Exposure to orthosilicic acid for 48 h produced the maximum effect. When incubated with orthosilicic acid for 72 h, the level of BMP-2 expression decreased and approached to the control group (P>0.05). Noggin (100 ng/ml) preconditioning inhibited the orthosolicic acid-induced over-expression of BMP-2. After being incubated with different concentrations of orthosilicic acid for 72 h, the expression of COL-1 increased in all experimental groups (P<0.05). Orthosilicic acid promotes the expression of COL-1 and exposure to orthosilicic acid at 10μM produced the maximum effect. But with the increase of the concentration, to 50 μM, the expression of COL-1 did not significantly increase (P>0.05). After being incubated with 10 μM orthosilicic acid for 48 h,72 h and 96 h, the expression of COL-1 increased compared with the control group (P<0.05). Orthosilicic acid promotes the expression of COL-1 and exposure to orthosilicic acid for 72 h produced the maximum effect compared with the 48 h,96 h group. Incubated with 10 μM and 20 μM orthosilicic acid, the activity of ALP increased compared with the control group (P<0.05), and exposure to 10 μM produced the maximum effect. The addition of orthosilicic acid at 50 μM did not significantly increase the activity of ALP (P>0.05). Noggin (100 ng/ml) preconditioning inhibited the activity of ALP. The BMSCs treated with 10 μM Orthosilicic acid for 21 d formed more bone mineralized nodules than the control group. Exposure to 10 μM orthosilicic acid for 72 h markedly increased the expression of BMP-2, P-Smad1/5, RUNX2, COL-1 and osteocalcin in MG-63 and U2-OS cell lines. Enhanced ALP activity was also observed under these conditions. Treatment with 10 μM orthosilicic acid resulted in an increase in the number of BMP-2, P-Smadl/5 and RUNX2 fluorescent cells compared with the control cells. Preconditioning with noggin inhibited the orthosilicic acid-induced up-regulation of P-Smad1/5, RUNX2 and COL-1 expression.Conclusion 1. Orthosilicic acid can promote BMSCs to osteogenic differentiation and the up-regulated expression of BMP-2 may be one of the mechanisms in this process. 2. The BMP-2/Smadl/5/RUNX2 signaling pathway participates in the orthosilicic acid-mediated induction of COL-1 and osteocalcin synthesis. |
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