| Experiment One The effect of Osthol on fracture healing in MiceObjective To study the positive impact on the open tibial fracture healing in mice with the Osthol,and provide the clinical treatment with in vivo experiment.Method 100 2-month-old C57BL/6 wild-type male mice were randomly assigned to Othole group and control group,and buliding up open tibia fracture model according to postoperative 7th,10th,14th,21th,28th day a total of 5 time points evenly distributed in each time point,10 rats each group,the fracture specimens were collected to do X-ray micro-CT,histomorphometric analysis,RT-PCR detection of Callus Cartilage and bone related gene expression.Results ①Postoperative X-ray shows that the fracture line of the Othole group was obviously fuzzy when compared with the control group,28 days aftte the operation the Osthol group has healed basically,while the control group could still vaguely see the fracture line.② Micro-CT quantitative showed that the callus and the bony callus volume(BV)of Osthol group is higher than the control group after 7,10,14 and 21days of operation,in which after 10,14 days has statistically significant(p<0.5)③Histomorphometric analysis showed that,the Callus Cartilage area of Osthol group increased significantly compared with the control group after 7th day of the operation,and could see more chondrocyte when compared with the control group;the callus bone area of the Osthol group increased significantly.④The expression level of Sox9,Col2al of the Osthol group mice were significantly higher than the control group on the 7th postoperative day;and the expression level of Runx2,Osteocalcin of the Osthol group mice was significantly higher than control group after 21th and 28th days;Osthol group CollOal expression level on the 21 postoperative day were significantly higher than those of control group.Conclusion Osthol could promote Cartilage cell proliferation and differentiation in the fracture position and put the time window forward,so Osthol has positive effect on the open tibial fracture healing of the mice.Experiment TWO The study of osteoblast-or chondrocyte-specific Bmp2 conditional knockout mice mediated by Osthol in the intervention of fracture healing.Purpose The specific Bmp2 conditional knockout mice model from osteoblasts and chondrocytes that used to study the biological functions and its major target cells and phase of Osthol in the process of fracture healing.Methods Construct the osteoblast-specific Bmp2 knockout mice(Collal-Cre;Bmp2fx/fx)and chondrocyte-specific Bmp2 knockout mice(Col2al-Cre;Bmp2fx/fx)which was 2-month-old C57BL/6 male mice,divide them into control group,Cre-negative Osthol group,Col1a1-Cre;Bmp2fx/fx Osthol group and Co12al-Cre;Bmp2fx/fx Osthol group,obtain the fracture specimens according to postoperative7th,10th,14th,21th,28th day and do X-ray,micro-CT,histomorphometric analysis,analysis of biological torsional mechanics,RT-PCR detection of the related gene expression of bone.Results ① Postoperative X-ray showed fracture line was more fuzzy in the 7th,10th,14th,21th day of Osthol mice group compared with the control group,the fracture lines of the chondrocyte-specific Bmp2 knockout mice Osthol group and osteoblast-specific Bmp2 knockout mice Osthol group was clear,The latter two compared the chondrocyte-specific Bmp2 knockout mice Osthol group was more clearly.The fracture of the Osthol group has healed after 28 days of opertation,and the fracture line is also very fuzzy in the the control group,and the fracture line of the chondrocyte-specific Bmp2 knockout mice Osthol group and osteoblast-specific Bmp2 knockout mice Osthol group was Faintly visible,shaping is not yet completed.②The bony callus volume quantitative of Osthol mice is higher than the other three groups through analysis of Micro-CT(BV)in 7th,10th,14,th 21th day after operation,in Which,the 10th,14th day after operation had significant differences.While the chondrocyte-specific Bmp2 knockout mice Osthol group in the 7th,10 th,21th day was lower than the control group,showed statistical significance.And the osteoblast-specific Bmp2 knockout mice Osthol group after operation was also lower than that in control group,in which,21th day,14th day has statistical significance.③Histomorphometric analysis showed that,the Callus Cartilage area of the Osthol group increased significantly when compared with the control group after 7th days of operation,and the amount of cartilage cells in the Osthol group was higher than the control group;callus bone area of increased significantly after 10 days,14 days of operation compared with the control group.The Callus Cartilage area of osteoblast-specific Bmp2 knockout Osthol group mice was lower than that in control group after 7th days of the operation,while On the contrary in the 14th,21th day.and in the 7th,10th,14th day there existed abundant cartilage cells in callus.The Callus Cartilage area of the chondrocyte-specific Bmp2 knockout mice Osthol group was lower than that in control group in the 7th,10th days after the operation,while in the 21th,28th day after operation was higher,in the 7th day after the operation,chondrocytes in callus was significantly less than the other three groups,and could still see chondrocytes in callus after 10th,14th,21th days of the operation.④The analysis of biological torsional mechanics showed that the maximum torque value and the hardness of the chondrocyte-specific Bmp2 knockout mice Osthol group and osteoblast-specific Bmp2 knockout mice Osthol group was significantly less than that of the control group in the 10th,14th,21th,28th days after operation,while the control group of mice was lower than the Osthol group in the 21th,28th day after operation.⑤The expression level of Sox9,Col2a1 in Osthol mice was significantly higher than the other three groups of mice on the 7th postoperative day,the expression of Sox9 of the chondrocyte-specific Bmp2 knockout mice Osthol group was higher than other three groups after 14th day after operation;the expression level of Runx2,Osteocalcin was significantly increased when compared with the control group in the 21th and 28th day after operation,and in the chondrocyte-specific Bmp2 knockout mice Osthol group and osteoblast specific Bmp2 knockout mice Osthol group,the expression level of Runx2 was lower than that in control group after 21th,28th day operation,the expression level of Osteocalcin was lower than control group after 21 days of operation;expression of Col10a1 in the Osthol group was significantly higher than other three groups after 21 days of operation,while the chondrocyte-specific Bmp2 knockout mice Osthol group and osteoblast-specific Bmp2 knockout mice Osthol group was lower than that in control group after 21 days of the operation.Conclusion ①The promoting effect of Osthol on open tibial fracture healing of bone fracture mice model is mainly through the Bmp2 pathway,for Bmp2 is one of the most important downstream mediators in fracture healing.②The expression of endogenous Bmp2 plays a decisive role in the process of fracture healing for it promote the fracture mailnly in the way of endochondral ossification and play a key role in the differentiation of chondrocytes. |
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