The Impact Of Extracorporeal Shock Wave On Bone Mesenchymal Stem Cells Differentiated Towards Osteoclast Around Femoral Head Necrosis Area And Relative Clinical Trials

Osteonecrosis of the femoral head of (ONFH) has a high incidence onclinical and showed a progressive development of the disease.ONFH ischaracterized by necrosis of bone cells due to local blood supply insufficiency.The tissue of femoral head is composed of osteoblasts, osteoclasts, osteocytesand other components. When necrosis occurs on femoral head, dynamicbalancing state of bone formation of osteoblasts and bone resorption ofosteoclasts (OC) is broken, osteogenic activities of osteoblast weakened,whileresorption activity of osteoclast enhanced.MSC is a kind of stem cell which has multi-differention potential,it canbe differentiated into osteoclast or osteoblast in certain microenvironment,andthe direction of the differentiation depends on the local environment. MSCcan be induced into osteoclast under the environment of receptor activator ofnuclear factor kappa-B of ligand(RANKL), which causes increased boneresorption activity. RANKL and other cytokines present in themicroenvironment of the femoral head necrosis,some cells around necrosiszone can be differentiated into osteoclast induced by RANKL.Extracorporeal Shock Wave (ESW) is a mechanical wave generated bythe electronic hydraulic devices or electromagnetic devices,which plays a rolein the solid and liquid substances, as well as human tissues.It can promoteproliferation of bone cells in fracture fragments, bone defects and bonenecrotic area,and promote MSC to the bone progenitor cells,which has theexact osteogenic effects. There is no report on whether ESW has the ability toinhibit transplanted MSC differentiated into osteoclast.The effect of ESW onMSC transplantation therapy to treat ONFH is not clear yet.We use ESW treating the process of MSC around necrosis zone differentiated into osteoclast to see the effect.And combined with clinicaltrials,to provide better evidence for the therapy of treating ONFH.Part1The impact of extracorporeal shock wave on bone mesenchymalstem cells differentiated towards osteoclast around femoral head necrosisareaObjective:We using HE staining, phalloidin staining and TRAP staining to observemorphology and quantity changes of osteoclast. We observe the differencesof specific gene expression during differentiation by the technology ofRT-PCR. We use scanning electron microscope (SEM) and Corning Osteobone cell culture surfaces to analyse bone resorption ability quantitatively. Sowe can explore the effects of MSC differentiated into osteoclast interferedwith ESW.Methods:1The obtainment and grouping of experimental cells:We selected10patients aged18to30,who had received MSC transplantation to treat ONFHin Armied Police General Hospital. We collected15mL blood from necrosiszone when drilling the femoral in the case of the informed consent ofpatients.Then subculture the cell in the laboratory, add cytokine of RANKLand M-CSF, to induce osteoclast. ESW intervention on the process of MSCinducted to osteoclast as the experimental group, while no ESW interventionas the control group.2The effect of ESW intervention the differentiation of osteoclastinduced by RANKL: On days1,3,5,7and9of the induction of osteoclast,the cells were given2000frequency,0.25mJ/mm~2ESW intervention.Cellmorphology and the cell number was observed under light microscope.AfterHE staining, TRAP staining and phalloidin staining, multinucleated cells werecounted, and multinucleation rate was calculated to analyze the impact ofESW on the differentiation of MSC to osteoclast.Observe the osteoclast-specific gene expression level changes by RT-PCR.Using SEM scanning toobserve bone resorption lacunae, and analyze area and the relative depth of the lacunae. Using Corning Osteo bone cell culture surface quantitative to analyzebone resorption ability. The experimental results were analyzed statistically byt test, and setting p <0.05as statistically significant.Results:1MSC inducted under50ng/ml RANKL and25ng/ml M-CSF, from theseventh day,we can see the emergence of TRAP staining positive(TRAP+)multinucleated osteoclasts, on the14th day,a large number of osteoclast wereobserved. After HE staining, phalloidin staining and TRAP staining,multinucleated cells were counted, and multinucleation rate was calculated.Compared with control group,the number of multinucleated osteoclast as wellas multinucleation rate of experimental group were decreased. RT-PCRdetection of osteoclast-specific expression of the genes,TRAP and Cat Klevels in the ESW group was lower than the control group.2After14days MSC cultured on bone slice with induction medium,using SEM to scanning the resorption pit.Image J software was used toanalyze area and the relative depth of resorption.The experimental group were:12266.7±573.06μm~2,123.2±6.12respectively while control groupwere19557.9±711.39μm~2,155.4±5.93.The difference between the twogroups were statistically significant (p <0.05).While MSC seeded on CorningOsteo bone cell culture surface for14days, the absorption area analyzed withImage J software, the experimental group for9124.5±915.85μm~2while thecontrol group15800.7±713.12μm~2. the difference was statistically significant(p <0.01).The bone resorption capacity of osteoclast is reduced after ESWintervention.Conclusion:1ESW can inhibit the process of MSC differentiated to osteoclast, thenumber of differentiated multinucleated osteoclasts from MSC were significantly reducedm, And reduced theosteoclast-specific gene expression level.2ESW can effectively inhibit bone resorption ability of osteoclastdifferentiated from MSC, and reduce bone resorption activity. Part2Clinical studie of effect of extracorporeal shock wave on bonemarrow mesenchymal stem cell transplantation for the treatment of earlyosteonecrosis of femoral headObjective：We use ESW to treat ONFH patients after MSC transplantation therapy and assess treatment effects by harris hip score,necrotic areachanges on MRI before and after the treatment.Methods:1The selection and intervention of the experiment subject in thesection of clinical trials According to certain inclusion and exclusioncriteria,we choose36patients(21males and15females) aged18to45yearsold with early femoral head necrosis from General Hospital of People's ArmedPolice between March2010and March2011, which were all unilateral.24cases randomly divided into two groups, the MSC transplantation group andthe ESW+MSC group(n=12),respectively,in accordance with the randomnumber table in advance.Patients enter into the groups according torandomized serial number. The control group was also12cases,which werethe outpatients with ONFH who refuse clinical treatment but receivefollow-up. The ESW group processed ESW treatment using energy fluxdensity with0.25-0.30mJ/mm~2, impact frequency with60-100/min, andimpact number with2000,once every other day,5times for a course oftreatment, patients were treated with2-3course,and the interval was threemonths.MSC transplantation group receiving transplantation surgery in theoperating room.The ESW+MSC group treated with ESW after MSCtransplantation.The follow-up time were all1year after treatment.2Evaluation of treatment of ONFH after the intervention One yearafter the intervention in each group received Harris hip score evalution, andthe data were statistically analyzed. Using Image J software to analyzenecrotic area of femoral head on coronal MRI with the same level before andafter the intervention.And paired t-test and analysis of variance were used toprocess the data.Results：After one year of follow-up, the Harris hip score of the MSC transplantation group, ESW+MSC group and control group were86±3.49,94.67±3.63,47.33±4.33, respectively.While the score before treatment were56.58±4.94,55.58±4.54,59.08±2.99, respectively. The differences betweenbefore and after treatment in each group were statistically significant.Aftertreatment,MSC group and the ESW+MSC group compared with the controlgroup was statistically significant, and the differences of ESW+MSC groupwith MSC group were statistically significant (p <0.05).2MRI coronal necrosis area on the same level before treatment were1987.01±88.55μm2,1976.13±98.99μm2and1991.37±97.43μm2, whileafter one year of treatment the area changed to269.63±58.50μm246.02±21.07μm2and2256.95±143.34μm~2.The differences were the same as HarrisHip Score by statistical analysis.Conclusion:1MSC transplantation in the treatment of early ONFH have certaintherapeutic effect, and ESW intervention on MSC transplantation in thetreatment of early ONFH can obtain better therapeutic effect.2ESW can inhibit MSC differentiated towards osteoclast may beanother mechanism to enhance the effects of MSC transplantation in thetreatment of early osteonecrosis of femoral head.