The Effect And Mechanism Of The Combination Of Treadmill Exercise And Low-intensity Pulsed Ultrasound On The Healing Of Bone Injury

Purpose:In recent years,the incidence of osteoporosis and fractures is gragually increased,some of which even developed into delayed union or nonunion,which seriously affected the quality of life.It is very important to find an effective method to prevent and cure bone disease and promote bone healing.Appropriate exercise and low intensity pulsed ultrasound have a positive effect on bone tissue,but there is little research on the role of combination of the two measures on bone tissue,and the specific molecular mechanism is still unclear.In this study,the rat femur and osteoblast were taken as the research object,and the effects of low intensity pulsed ultrasound combined with exercise on bone injury were studied.Method:Animal experiment selected healthy female SD rats as the research object,and they were randomly divided into 10 in each group of 5 groups:normal control group(Normal control group,NC),treadmill exercise group(Treadmill Exercise,TE),drilling group(Drilling Control,DC),training drilling group(training drilling,TED),training drilling and low intensity ultrasound in the intervention group(Treadmill drilling +ultrasound,TEUD).Normal group without training;medium-intensity exercise group rats have the slope treadmill training,each training time was 60 min,28m/min,the experimental time are 6 weeks of training,every week the rats trading 6 days and 1 day of rest;The treatment group was treated with low intensity pulsed ultrasound.The treatment was 20 min per day,and the treatment lasted for 7 days,lasting for about 3 weeks.The ultrasonic intensity is 30 mW/cm2,the duty cycle is 1:4,the ultrasonic frequency is 1 MHz,and the pulse frequency is 100 Hz.The control group was not given any intervention.After the intervention,measured and recorded the grip and weight of rats and then the rats were sacrificed.Collected serum and interception of rat femur and femoral quadriceps,then used biochemical kit,micro-CT scan,H&E staining,three point bending experiment and computer simulation analysis of related parameters of bone metabolism,femoral ultrastructure,the change of bone biomechanical characteristics.Cell experiments in this study using MC3T3-E1 cells as the research object,they were randomly divided into control group(Control group),recombinant protein group(Myostatin group),recombinant protein+antibody group(Myostatin+MsAb group),ultrasound group(group LIPUS)and ultrasound+recombinant protein group(LIPUS+Myostatin group).After the intervention,extracted the RNA and protein of osteoblast,then analized the effect of low intensity pulsed ultrasound and Myostatin recombinant proteins on the proliferation of osteobalast and the biological mechanisms using CCK-8 kit,Western Blot and RT-qPCR technology.Result:1.The weight,holding power and bone weight of rats:compared with NC group,the weight of TE group was lower than that of group NC(P<0.05).The holding power of TE group were higher than NC group(P<0.05);compared with the DC group,the holding power of TE group were higher than DC group(P<0.01);The holding power of DC group was significantly lower than that of TEUD group(P<0.05);compared with TED group,TED group,the holding power is less than TE group,and there was a remarkable difference(P<0.05).There was no remarkable difference in weight of femur and femoral weight between the five groups.2.The rat serum index:There were no significant differences in serum calcium and phosphorus values between the five groups.Compared with DC group,the activity of alkaline phosphatase of TEUD group was higher than that in group DC group,and there was a remarkable difference(P<0.05);compared with DC group,The tartrate resistant acid phosphatase activity of NC group was remarkably lower than that in group DC(P<0.01);The tartrate resistant acid phosphatase activity of TEUD group(P<0.01)and TED group(P<0.05)was significantly lower than that of DC group.3.The rat cancellous bone micro-CT quantitative analysis:The Tb.N(P<0.05),BMD(P<0.05)and BV/TV(P<0.05)in TE group were significantly higher than those in NC group.Compared with the NC group,the Tb.Th(P<0.05)and BMD(P<0.05)of the DC group were significantly lower than those in the NC group,while the Tb.Th(P<0.05),BMD(P<0.05)and BV/TV(P<0.05)in the TED group were remarkably lower than those in the TE group.Compared with DC group,Tb.N(P<0.05)and BMD(P<0.05)in TED group were significantly higher than those in group DC.Compared with DC group,Tb.N(P<0.05),Tb.Th(P<0.05),BV/TV(P<0.05)and BMD(P<0.05)in TEUD group were significantly higher than those in DC group.Compared with TED group,Tb.N,Tb.Th,BV/TV and BMD in TEUD group were higher than those in group TED,the BMD(P<0.05)in group TEUD was significantly higher than that in group TED.4.The rat cancellous bone micro-CT quantitative analysis:Compared with NC group,the BMD of cortical bone in the DC group was significantly lower than group NC(P<0.05);Compared with TE group,the BMD of cortical bone of TED group(P<0.05)and DC group(P<0.05)was significantly lower than that of TE group.Compared with TEUD group,the BMD of cortical bone of DC group(P<0.01)and TED group(P<0.05)was significantly lower than the TEUD group.5.3D reconstruction of femoral injury:The 3D reconstruction results show that compared with the DC group,the bone healing area of TED group and TEUD group was larger than DC group;the bone volume fraction results drilling site showed that compared with the DC group,the BV/TV of drilling site of TED group(P<0.05)and TEUD group(P<0.01)were significantly higher than the DC group;compared with group TED,the BV/TV of drilling site of TEUD group was significantly higher than the TED group(P<0.05).6.Three point bending test results:Compared with NC group,the maximum load of DC group was remarkably lower than NC group(P<0.01);compared with TE group,the maximum load of DC group(P<0.01),TED group(P<0.01)and TEUD group(P<0.05)were significantly lower than TE group.Stiffness test results show that compared with NC group,the stiffness values of TE group was remarkably higher than NC group(P<0.05);compared with NC group,DC group(P<0.05)and the maximum load was lower than NC group;compared with TE group,the stiffness values of DC group(P<0.01)and TED group(P<0.01)and TEUD group(P<0.05)were remarkably lower than TE group;compared with DC group,the maximum load of TEUD group was higher than that in DC group(P<0.05).Energy absorption test results show that,compared with the NC group,the energy absorption of DC group(P<0.01),TED group(P<0.05)were significantly lower than NC group;compared with TE group,the energy absorption of DC group(P<0.01),TED group(P<0.01)were significantly lower than TE group,suggesting that the injury of bone tissue will make bone tissue energy absorption value decreased.Compared with TEUD group,the energy absorption of DC group was remarkably lower than that in TEUD group(P<0.05).Elastic modulus test results showed that compared with the TE group,the elastic modulus of DC group(P<0.01),TED group(P<0.01)were significantly lower than TE group;compared with NC group,the elastic modulus of DC group was significantly lower than that of NC group(P<0.05).7.CCK-8 experiments assay:The proliferation of osteoblasts in the ultrasound group was higher than that in the control group at third day(P<0.01),fifth day(P<0.05)and seventh day(P<0.01).Compared with the Control group,the cell proliferation level of Myostatin group was remarkably lower than that in Control group(P<0.05);compared with Myostatin group,the cell proliferation level was higher in Myostatin+MsAb group than in Myostatin group(P<0.01);there was no significant difference between the cell proliferation level of Control group and Myostatin+MsAb group;compared with Control group,the proliferation of LIPUS the group was significantly higher than that of Control group(P<0.01);compared with group LIPUS,the proliferation level of LIPUS+Myostatin cells were remarkably lower than that of LIPUS group(P<0.05);and the cell proliferation level of Control group and LIPUS+Myostatin group had no significant difference.8.RT-qPCR,Western Blot and immunofluorescence results:low intensity pulsed ultrasound can make the mRNA and proteins expression of the downstream signal molecule of Myostatin including AcvrIIB,Smad2,Smad3 decreased;low intensity pulsed ultrasound will raise the mRNA and protein expression level of Wntl and?-catenin in Wnt/?-catenin signaling pathway and down-regulated the mRNA and protein expression level of GSK-3?.Recombinant Myostatin protein may be through the release of low intensity pulsed ultrasound on inhibition of osteoblasts of Smad3 to down-regulate the(3-catenin expression,thereby blocking the positive effects of the low intensity pulsed ultrasound on promoting the proliferation of osteoblasts.Conclusion:1.Medium-intensity slope treadmill exercise make the femoral microstructure and biomechanical parameters has been optimized,and improve bone strength and bone stiffness;the bone healing ability of TED group is superior to DC,no matter from bone tissue microstructure and its biomechanical properties,suggesting that movement plays a positive role in preventing bone loss and accelerate bone healing.2.Treadmill exercise and LIPUS interventions could improve the bone metabolism,optimize of the femur microstructure and biomechanical characteristics,shorten the time of bone healing,accelerate and promote the healing of bone tissue.The effect of treadmill exercise and LIPUS joint intervention in promoting bone healing is superior to single exercise intervention,indicating that LIPUS as a non-invasive physical therapy curative effect is distinct in terms of accelerating healing bone injury.3.The potential mechanism of low intensity pulsed ultrasound accelerating bone healing may be through inhibiting the expression of the AcvrIIB,Smad2,Smad3 in MSTN signaling pathway,and then regulated the expression of ?-catenin,GSK-3? to activate the Wnt/?-catenin signaling pathway,thereby promoting osteoblast proliferation.