| Objective: 1.The objective of this study is to compare the osteogenic differentiation of MC3T3-E1 cells induced by classical chemical composition(dexamethasone,ascorbic acid and ?-glycerophosphate),BMP2 and BMP7 to provide a basis for the selection of sustained-release material loading the factor in the next experiment.2.Bone morphogenetic protein(BMP2)is used to promote bone formation in many clinical applications.However,BMP2 are inherently unstable in vivo,so they need to be combined with carriers for controlled delivery.In this study,a novel and efficient fibrin glue/fibronectin/heparin(FG/Fn/Hep)sustained-release system was developed for bone defect repair.Methods: 1.In the experiment of comparing the osteogenic induction effect of classical chemical composition with BMP2 and BMP7,the osteogenic induction effect was evaluated by alizarin red staining calcium nodule,quantitative detection of ALP,immunohistochemistry and ELISA detection of runt related transcription factor-2(Runx-2),OPN and OCN.2.In the preparation of a novel FG/Fn/Hep-BMP2 sustained-release system,FG/Fn-BMP2 hydrogel was used to induce osteogenesis in vitro.Fibrin glue was designed as a temporary scaffold to repair the infiltration of cells.Fibronectin provided adhesion sites for cells and served as a connector between fibrin and heparin.Heparin was also designed as a storage for controlling the release of BMP2 and preventing its degradation by protease in vivo.The incorporation of fibronectin and heparin can significantly slow down the release of BMP2,but basically does not affect the structure and hardness of fibrin glue.The release of BMP2 from hydrogel was detected by in vitro release experiment.The osteogenic induction ability of hydrogel was detected by MC3T3-E1 cell induction experiment.The characteristic indexes were calcium nodule,ALP,Runx-2,OPN,OCN and type I collagen(CoL-I).Rat craniums implant experiment was conducted to evaluate the ability of hydrogel to repair bone defects in vivo.Results: 1.The results of alizarin red staining,quantitative detection of ALP,immunohistochemistry and ELISA showed that on the 7th day,a small amount of calcium nodules appeared in MC3T3-E1 cells induced by BMP 2,BMP 7,BMP 2 + BMP 7 and chemical group and there was no significant difference in the expression of ALP between the groups.The expression of Runx-2 in BMP2 group was significantly increased compared with other groups,while OPN expression in chemical group was significantly increased compared with other groups.At this time,there was no significant difference in OCN expression among the groups.On the 14 th day,there were a large number of calcium nodules in each induction group,and the staining areas of calcium nodules in chemical group and BMP2 group were large.ALP expression in BMP 2 group and BMP 2 + BMP 7 group was significantly higher than that in other groups.The expression of Runx-2 in chemical group and BMP2 group was significantly higher than that in other groups.At this time,the expression of OPN in the chemical group was still the highest,and the expression of OCN in each induction group was significantly different from that in the control group.On the 21 st day,the pigmented area of calcium nodules in the chemical group,BMP 2 group and BMP2+BMP7 group increased significantly,while the pigmented area of calcium nodules in BMP 7 group increased less than that on the 14 th day.ALP expression in BMP 2 group was significantly higher than that in other groups.At this time,the expression of Runx-2 in the BMP2 group decreased,and the expression of Runx2 in the chemical group was still significantly different from that in the other groups,and the expression of OPN in each induction group decreased.At this time,only the chemical group and the BMP2 group had significant differences compared with the control group,and the expression of OPN in the chemical group was still the highest.The expression of OCN in each induction group was significantly different from that in the control group.In the process of inducing osteogenic differentiation of MC3T3-E1 cells,the synergistic effect of BMP2 and BMP7 was not significant.The experimental results showed that the induction effect of the chemical composition and BMP2 was more prominent,but because some ingredients in the chemical composition,such as dexamethasone,have certain side effects on bone healing,we chose BMP2 as the load factor of the sustained release material.2.In the preparation of FG/Fn/Hep-BMP2 sustained-release system,the incorporation of fibronectin and heparin could significantly slow down the release of BMP2,but basically did not affect the structure and hardness of fibrin glue.The release of BMP2 from FG/Fn/Hep-BMP2 hydrogel was mainly hydrogel degradation rather than simple diffusion.In vitro release experiments and MC3T3-E1 cell induction experiments showed that hydrogel could significantly reduce the initial burst of BMP2,so that BMP2 could release smoothly and induce MC3T3-E1 cells to differentiate into osteoblasts.Its characteristic is calcium nodule,ALP,and Runx-2,OPN,OCN and collagen I(CoL-I)are significantly increased compared with the control group.In vivo evaluation showed that when the FG/Fn/Hep-BMP2 hydrogel was filled,the healing of bone defect area increased rapidly.After three months of healing,more than 80% of the original defect area was covered by calcified tissue radiograph.Conclusion: 1.BMP2 had a significant ability to induce osteogenic differentiation,which was similar to the chemical composition and had stronger osteogenic ability than BMP7.2.During osteogenesis,ALP and Runx-2 were highly expressed in BMP-2 group,while ALP and Runx-2 were both early markers of osteogenesis,indicating that BMP2 had a more significant induction effect in early osteogenesis.3.According to the expression of osteogenic indexes,the combined induction effect of BMP2 and BMP7 was not good.4.FG/Fn/Hep-BMP2 hydrogel significantly promoted bone regeneration in rat calvarial critical-size defect model and was expected to be used to repair bone defects. |
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