| Objectives The treatment of large-scale critical bone defects is still a difficult problem that clinicians often face.Because of its advantages such as safety and low side effects,it has gradually become a hot spot in the research of bone tissue engineering technology at home and abroad.In this study,the optimal concentration of BMP2/7 and BGN cytokines combined to induce osteogenic differentiation of cells was preliminarily screened.And preliminarily explore the internal mechanism of the combination of the two induced osteogenesis.Methods 5ng/ml,50ng/ml BMP2/7 and 2μg/ml,4μg/ml,6μg/ml BGN were used alone or in combination,and the experiment was divided into 12 groups.Various concentrations of BMP2/7 and BGN were used in MC3T3-E1 cells alone or in combination to detect cell proliferation activity,alkaline phosphatase(ALP)staining area,ALP activity,osteocalcin(OCN)detection,and osteogenesis-related genes in each group.(ALP,OCN,Col 1,Runx 2)expression and extracellular matrix mineralization level.Results 1 The cell proliferation activity experiment showed that BMP2/7 promoted the proliferation activity of MC3T3-E1 cells in a concentration-dependent manner regardless of whether BGN was added or not.After 1 day of stimulation,the proliferation activity of cells in the 50ng/ml BMP2/7 group was significantly higher than that in the blank control group.The cell proliferation activity of the combined application of the two factors had no significant difference compared with the single application of each factor at the same concentration.The cell proliferation activity of the +4μg/ml BGN group was significantly higher than that of the control group and higher than that of the two factors alone.Among them,the combined application of 50ng/ml BMP2/7+2μg/ml BGN group had the best cell proliferation activity.2 Alkaline phosphatase(ALP)activity and staining experiments showed that BMP2/7 alone could significantly increase ALP activity and staining area.The detection of ALP activity showed that the ALP activity of the combined application group of BMP2/7 and 2μg/ml BGN of 5ng/ml and 50ng/ml was significantly higher than that of the blank control group and the two alone application after the 4th and 7th day of stimulation culture.In the ALP staining experiment,the ALP staining area in the combined application group of 50ng/ml BMP2/7 and 2μg/ml,4μg/ml and 6μg/ml BGN was significantly higher than that in the control group and BMP2/7 and 6μg/ml after stimulation culture for 4 and 7days.BGN two separate application groups.3 Osteocalcin(OCN)test showed that there was no significant difference in OCN concentration in each group after 4 days of stimulation and culture.After 7 days of stimulation and culture,the OCN concentration in each experimental group was significantly higher than that in the blank control group.The concentration was significantly higher than that of the two factors BMP2/7 and BGN applied alone at the same concentration.4 The detection of osteogenic gene expression showed that BMP2/7 promoted the expression of ALP,OCN,Col 1 and Runx 2 genes in MC3T3-E1 cells in a concentrationdependent manner;7 and 2μg/ml,4μg/ml,6μg/ml BGN two factors combined application group ALP gene expression level was significantly higher than the control group and BMP2/7 and BGN two factors alone application group;in OCN gene expression,stimulation culture After 4 and 7 days,the expression level of OCN gene in the combined application group of 50ng/ml BMP2/7 and 2μg/ml,4μg/ml and 6μg/ml BGN was significantly higher than that in the control group and BMP2/7 and BGN alone.In the expression of Col 1 and Runx 2 genes,the expression levels of Col 1 and Runx 2 genes in the combined application group of BMP2/7 and BGN were significantly higher than those in the control group,and after 7 days of stimulation and culture,50ng/ml The expression levels of Col 1 and Runx 2in BMP2/7 combined with 2μg/ml,4μg/ml and 6μg/ml BGN were significantly higher than those in BMP2/7 and BGN groups at the same concentration.5 In the extracellular matrix mineralization experiment,on the 4th and 7th day,the alizarin red staining area of 5ng/ml,50ng/ml BMP2/7 and 2μg/ml,4μg/ml,6μg/ml BGN alone was the same.Significantly higher than the control group,and the alizarin red staining area in the combined application group of BMP2/7 and BGN was significantly higher than that in the single application group of the two factors at the same concentration.The above differences were statistically significant(P<0.05).Conclusions 1 BMP2/7 heterodimer alone can significantly promote the proliferation activity,ALP activity and staining,OCN activity,osteogenic gene expression and extramatrix mineralization of MC3T3-E1 cells;2 2μg/ml,4μg/ml BGN factor alone When MC3T3-E1 cells were cultured with stimulation,it can improve cell proliferation activity,ALP staining area,OCN activity,Runx 2 osteogenic gene expression and extramatrix mineralization;3 BMP2/7 and BGN can synergistically promote the combination of two factors MC3T3-E1 cell proliferation activity,ALP activity and staining,OCN activity,osteogenic gene expression and extramatrix mineralization,among which 50ng/ml BMP2/7combined with various concentrations of BGN induced osteogenic differentiation significantly.In the cell proliferation activity,ALP activity and staining,OCN activity and extramatrix mineralization experiments.Figure 11;Table 3;Reference 77... |
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