Application Of "Accordion Technology" To Promote Osteogenic Differentiation In Vitro And In Vivo And Its Mechanism Research

Objective: "Accordion technology" has shown good application in clinic,but its basic research is few and the mechanism of action is unclear,which can not provide theoretical basis for the further application of this technology.Therefore,in this study,alternating stretch-compression "accordion technique" was used in vivo and in vitro to verify that "accordion technique" can effectively promote osteogenic differentiation and bone regeneration in the stretched osteogenic region in vivo and in vitro,and to preliminatively clarify the optimal period and mechanism of its use.Methods: In this study,the signal pathway of "Accordion technique" and the in vivo experiment were studied.1.Rat bone marrow mesenchymal stem cells(BMMSCs)were used as the research object in the in vitro experiment,and the rat BMMSCs physical stimulation model was constructed in vitro.The experiment was divided into three groups,the "Accordion technique" group,the simple stretch group and the control group.The "Accordion technique" group was stretched for 2.5 days with a 1h frequency of 0.5Hz and 2000μstrain per day,and then contracted for 2.5 days with the same force.The simple stretch group was stretched with the same mechanical strength for 5 days.The control group did not need any treatment,and the three groups of cells were cultured under the same conditions.Detection: The effects of three different conditions on the osteogenic differentiation and proliferation of BMMSCs were compared by CCK-8detection,ALP staining,Alizarin red staining and detection of the relative expression of Osteocalcin(OCN).2.The experiment was divided into four groups: "Accordion technique" group,"Accordion technique" + XAV939 group,XAV939 group and control group.q RT-PCR was used to detect the relative expression levels of β-catenin,TCF1 and Axin2 after 5days of different intervention methods.It was proved that the Wnt/β-catenin signaling pathway was activated or inhibited,and its role in osteogenic differentiation was described.3.In vivo experiments using the left tibia bone lengthening of Mongolian short-tailed sheep as a model,local intervention in the distraction zone,including the "accordion technique" in the early,middle and late stages of mineralization,using X-ray,Micro-CT and biomechanical detection Investigate the relative best time to use the "Accordion technique".Results: 1.The cell proliferation and ALP activity of rat BMMSCs in the intermittent mechanical stimulation "Accordion technique" group and the simple stretch group were increased,the number of calcified nodules formed after cell stress increased,and the relative expression of osteogenesis-related gene OCN m RNA Significantly increased(P<0.05).Among them,the ability of promoting cell proliferation in the "Accordion technique" group was stronger than that in the simple stretch group and the control group(P<0.05).From the ALP staining and Alizarin red staining,it can also be seen that the "Accordion technique" group had more ALP activity and calcified nodules than the other two groups.2.In the mechanism study of "accordion technology",q RT-PCR was used to detect the relative m RNA expression levels of β-catenin,TCF1 and Axin2 related genes in the Wnt/β-catenin signaling pathway in cells,and it was found that the "Accordion technology" group was compared with the control group.The expressions of β-catenin,TCF1 and Axin2 were significantly increased(P<0.0001);the relative expressions ofβ-catenin,TCF1 and Axin2 in the "Accordion technology" + XAV939 group were decreased compared with the "Accordion technology" group(P<0.0001)),indicating that "Accordion Technology" and XAV939 activated and inhibited the Wnt/β-catenin signaling pathway,respectively.3.In the in vivo experimental results of the "Accordion technique",X-ray films and Micro-CT 3D reconstruction: the "Accordion technique" group in the mid-mineralization stage had faster callus formation,smaller fracture stump gap,and better continuity of the medullary cavity than the other three groups.BMD and BV/TV:The two indicators of the early "Accordion technology" and the middle "Accordion technology" group were higher than those of the other groups,and the difference was statistically significant(P<0.05).Among them,the middle "Accordion technology" group was higher than the early "Accordion technology" group(P<0.05)..Biomechanical test: The maximum bending force and maximum deflection of the "Accordion technique" in the middle stage were significantly higher than those of the other groups,and the difference was statistically significant(P<0.05),indicating that the "Accordion technique" in the middle stage of mineralization was better than that in the early and late stages of mineralization."Accordion technique" callus shape is stronger.Conclusions: 1.In vivo and in vitro experiments in this study confirmed that the "Accordion technique" can effectively accelerate the proliferation and osteogenic differentiation of BMMSCs.2.The expression levels of β-catenin,TCF1 and Axin2 were specifically enhanced during the osteogenic differentiation of rat BMMSCs stimulated by the "Accordion technique",indicating that β-catenin was positively expressed during this process,which was verified at the molecular level.The Wnt/β-catenin signaling pathway is involved in the process of osteogenic differentiation under the "Accordion technique".3.After using XAV939 inhibitor,it was found that the expression of these genes all decreased,indicating that XAV939 effectively inhibited the Wnt/β-catenin signaling pathway.4.It is confirmed that the sheep distraction osteogenesis model is an effective model to study bone regeneration.And through the "Accordion technique" in different mineralization stages,it was confirmed that the best period of "Accordion technique" in the sheep distraction osteogenesis model may be the middle stage of mineralization,which provides experimental basis for accelerating callus formation for various clinical fractures.