| Objective: Osteoporosis is a disease characterized by a decrease in bone mass leading to increased bone fragility and an increased risk of fracture. Osteoporosis results from a negative balance between new bone formation and resorption, and in most cases, is associated with a relative increase in the number and activity of bone-resorbing osteoclasts. Osteoclasts are derived from bone marrow monocyte / macrophage Cell Lineage,the differentiation of which is characterized by the fusion of osteoclast precursors, creating a multinucleated giant cell. Osteoclast is the only multinucleated giant cells which has the ability of bone resorptive function. Bone resorptive function is resulting from the activation of osteoclast. Therefore , osteoclasts play a key role in bone formation and bone mass regulation . In Pathological conditions , many metabolic bone diseases (such as osteoporosis, bone sclerosis, etc.), are due to bone resorption caused by hyperthyroidism. Therefore,the research on osteoclast formation and bone resorption is significant in treating with metabolic bone diseases such as osteoporosis.Extracorporeal Shock Wave Therapy is used to treat urinary calculi physiotherapy in recent 20 years. In 20th century,the early 90s,it was reported that ESWT could treat many diseases of orthopedics. experimental study found that a certain energy intensity of ESW can increase the bone-forming osteoblast activity, thus contributing to fracture healing. But the function of ESW, as a kind of to stimulative energy form,affecting osteoclast formation and bone resorption activity,is not known at present.In this study,we give a certain energy ESW to interfere osteoclasts in vitro,so as to disscuss The effect of Extracorporeal shock wave on osteoclast formation and bone resorption Methods: 1.Isolation and culture MSCs in vitrobone marrow stromal stem cells were isolated in patients with osteoporosis by way of density gradient centrifμgation, then inoculated in 24-well plates which put the coverslip and the bone slices in advance . Using 1,25-(OH)2D3 as the inducer in vitro culture. 24-well plates inoculated with bone marrow stromal stem cells were randomly divided into two groups: ESW intervention group and control group.2. The effect of ESW on osteoclast formationESW intervention group was interfered with ESW(0.08mJ/mm2, 100 times)in 4th day.Two groups were cultured in vitro induced by the first 3rd ,6th ,9th ,12th days.Both groups were extracted in 3rd ,6th 9th 12th days.Using TRAP staining to observe morphology of osteoclasts; count osteoclast cells and osteoclast like cells and so as to analyze the effect of ESW on osteoclast formation ; Using RT-PCR technology combined with the OD value semi-quantitative to analyze NFATc1 and c-Fos gene expression of the two groups at different time points.3. The effect of ESW on the bone resorption activity of osteoclastsESW intervention group was interfered with ESW(0.08mJ/mm2, 100 times)in 12th day ,and the bone chips were extractede in the 15th day, l% toluidine blue stained and observed under the microscope,counting the number of absorption laeuna;scanning with SEM, using Image pro plus5.0 program to anaylyze the absorption laeuna, reflecting the bone resorption activity of osteoclasts .4.The experiments were repeated 3-5 times and the experimental data was expressed with x±s and Using SPSS 11.0 tatistical software to test the difference between the two groups.Repeated measures design ANOVA was used to compare the means of osteoclasts and osteoclast like cells in different times of the 2 groups. Independent-samples t-test was used to compare the means of the number and area of absorption laeuna on bone chip in the 2 group. p <0.05 was considered statistically significant difference.Results: 1. TRAP + cells appeared on the 3rd day in Both of the groups .The number of osteoclasts and osteoclast like cells of the 2 groups shows that there is no significant differences between the two groups, (p >0.05).From the 6th day, there are more TRAP + cells in control group, and the emergence of more dual-core osteoclast like cells and scattered three nuclears osteoclasts could be observed. In ESW intervention group ,we also found less dual-core osteoclast like cells,and three nuclears osteoclasts had not be found .the different between the two groups is significant (p<0.05).In the 9th day, there is a large number of dual-core osteoclast like cells, and much more three nuclear osteoclasts; In ESW intervention group, dual-core osteoclast like cells and three nuclear osteoclasts is less than the control group .the different between the two groups is significant (p<0.05).In the 12th day, there is a large number of typical TRAP + multi-core osteoclasts in control group.The number of dual-core osteoclast like cells and three nuclear osteoclasts is much less than the number of control group, the different between the two groups is significant (p<0.05).PCR showed: ESW group and control group were both detected c-Fos and NFATc1 expression on the 3rd day. The expression gradually increased and compared with control group, except the 3rd day ,on the rest of the time points,the ESW group`s NFATc1 and c-Fos gene expression is much less, the differences were statistically significant (p <0.05).2. By comparing the amount and area of absorption laeuna in ESW group with control group ,we can see that the amount and area of absorption laeuna in ESW group is less than the control group . the differences were statistically significant (p <0.05).The result sμggested that extracorporeal shock wave can inhibit osteoclastic bone resorption.Conclusion: 1.A certain energy intensity of ESW can inhibit osteoclast differentiation and formation. During Osteoclast differentiation, NFATc1 and c-Fos gene expression gradually increased. A certain energy intensity of ESW could inhibitors osteoclast differentiation by inhibiting NFATc1 and c-Fos gene expression.2.ESW can significantly inhibit bone resorption inmature osteoclasts. 3.ESW can be a positive and effective method of physical therapy to the clinical treatment of osteoporosis.
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