| Objectives Bone morphogenetic protein 2/7 heterodimer(BMP2/7)can induce osteogenic differentiation of cells.All-trans retinoic acid(ATRA)has different effects on osteogenic differentiation of cells,the effect varies depending on the concentration used.By reducing the application dose of ATRA,using BMP2/7 heterodimer alone or in combination in mouse embryo osteoblast precursor cell(MC3T3-E1)to detect their individual or combined osteogenic effects and to explore the optimal combined concentration of the two cytokines,the results will provide experimental evidence for the combined application of multiple cytokines to repair bone defects.Methods The concentrations of BMP2/7 heterodimer were 5ng/m L and 50ng/m L,and the concentrations of ATRA were 50ng/m L,100ng/m L,200ng/m L and 300ng/m L.In MC3T3-E1 cell culture medium,different concentrations of BMP2/7 and ATRA were added individually or in combination.After stimulation,cell proliferation activity,alkaline phosphatase(ALP)staining,ALP activity,osteocalcin(OCN)expression,osteogenic related gene expression and alizarin red staining were detected.Results 1 Detection of cell proliferation activity: on the first day of the stimulating culture,the combined effects of the BMP2/7 heterodimer and ATRA groups were higher than that of the blank control group and the BMP2/7 alone group(P<0.05).On the 4th and 7th day,regardless of the presence or absence of ATRA,BMP2/7 promoted cell proliferation activity in a concentration dependent manner.The simple effect of each concentration of ATRA inhibited cell proliferation activity.50ng/m L ATRA promoted cell proliferation activity induced by 5ng/m L and 50ng/m L BMP2/7,and the 50 ng /m L BMP2/7+50ng/m L ATRA combined effect group had significantly higher cell proliferation activity than other groups,the cell proliferation effect of the 5ng/m L BMP2/7+50ng/m L ATRA group was similar to that of the 50ng/m L BMP2/7 alone group,and there was no statistical difference between the two groups.2 Detection of ALP staining and ALP activity: on the 4th and 7th day of the stimulation culture,the ALP staining area and ALP activity of the 5ng/m L and 50ng/m L BMP2/7 single-action groups were significantly higher than that of the blank control group,and regardless of the presence or absence of ATRA,the ALP expression of the BMP2/7 group treated with50ng/m L was higher than that of the BMP2/7 group treated with 5ng/m L,the ALP expression induced by BMP2/7 was positively correlated with the concentration of BMP2/7.The ALP expression level of each concentration of ATRA alone was similar to that of the blank control group.The ALP expression effect of 5ng/m L,50ng/m L BMP2/7combined with 50ng/m L ATRA was higher than that of the same concentration of the BMP2/7 single-action group at the same time point.The 50ng/m L BMP2/7+50ng/m L ATRA group had the highest ALP expression level among all the groups,and there were statistical differences with other groups.50ng/m L ATRA could promote ALP expression induced by 5ng/m L BMP2/7,reaching the same effect as 50ng/m L BMP2/7.3 Detection of OCN expression: at any time point,BMP2/7 alone significantly increased the secretion of osteocalcin,and regardless of the presence or absence of ATRA,the content of osteocalcin of the 50ng/m L BMP2/7 action group was higher than that of the 5ng/m L BMP2/7 action group.Compared with the blank control group,ATRA acting on the cells alone could significantly reduce the content of osteocalcin,ATRA inhibited the secretion of OCN.When ATRA was present at a concentration of 50ng/m L,it could promote5ng/m L and 50ng/m L BMP2/7 to induce OCN secretion,on the 4th and 7th day,the expression level of OCN in the 50ng/m L BMP2/7+50ng/m L ATRA group was 1.12 and1.2 times that of the 50ng/m L BMP2/7 alone group,50ng/m L BMP2/7+50 ng/m L ATRA group had the highest expression of osteocalcin,and it was statistically different from other groups.On the 7th day,the expression level of OCN of the 5ng/m L BMP2/7+50ng/m L ATRA group was not significantly different from that of the 50ng/m L BMP2/7 single-action group.4 Detection of osteogenic related genes: 5ng/m L and50ng/m L BMP2/7 alone could increase the expression level of ALP,OCN,Col1 and Runx2 genes,and under the action of the same concentration of ATRA,the expression levels of all osteogenesis related genes of the 50ng/m L BMP2/7 action group were significantly higher than the 5ng/m L BMP2/7 treatment group.50ng/m L and 100ng/m L ATRA alone had no significant effect on the expression levels of ALP and Runx2 genes,but inhibited the expression of OCN and Col1 genes.On the 7th day of the stimulation culture,the combined application of 50ng/m L BMP2/7 and 50ng/m L ATRA significantly promoted the expression of ALP,OCN,Col1 and Runx2 genes,and the expression levels of ALP and Col1 genes of the 5ng/m L BMP2/7+50ng/m L ATRA group were similar to those of the 50ng/m L BMP2/7 alone group.5 Detection of matrix mineralization: on the28 th day of the stimulation culture,the staining area of alizarin red of the 5ng/m L and50ng/m L BMP2/7 single groups was larger than that of the blank control group,and the staining area of the 50ng/m L BMP2/7 alone group was larger than that of the 5ng/m L BMP2/7 alone group,and the staining area of 50ng/m L BMP2/7 was also greater than that of 5ng/m L BMP2/7 when the same concentration of ATRA was present,BMP2/7could promote extracellular matrix mineralization in a concentration dependent manner.The matrix mineralization area of all the combined groups was higher than that of the corresponding equal concentration of the BMP2/7 single application group,it could be seen that BMP2/7 and ATRA could synergistically promote the formation of matrix mineralization,and the 50ng/m L BMP2/7+50ng/m L ATRA group had the largest mineralization area of all the groups.There was no statistical difference in mineralized area between the 5ng/m L BMP2/7+50ng/m L ATRA group and the 50ng/m L BMP2/7alone group(P>0.05).Conclusions 1 Regardless of the presence or absence of ATRA,BMP2/7 heterodimer promotes the osteogenic differentiation of MC3T3-E1 in a concentration dependent manner.2 ATRA alone can inhibit the osteogenic differentiation of cells.3 When the ATRA concentration is 50ng/m L,the combined application with BMP2/7 can synergistically promote the osteogenic differentiation of MC3T3-E1,the combined application of 50ng/m L ATRA and 50ng/m L BMP2/7 has the most significant osteogenic effect,the combined application of 50ng/m L ATRA and 5ng/m L BMP2/7 can achieve the similar effect as that of 50ng/m L BMP2/7 alone.Figure12;Table6;Reference 98... |
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