| Objective:Healthy bone is in a state of dynamic equilibrium of bone formation by osteoblasts and bone resorption by osteoclasts. Disruption of balance can lead to osteoporosis, osteopetrosis and other bone metabolic diseases. Previous studies have shown that mechanical stimulation can effectively promote bone formation had an important role in the regulation of bone remodeling and maintenance of bone mass. Osteoclast is the only cell which has the function of resorption, it comes from monocyte/macrophage lineage, and a multinucleated giant cell which consist by a number of mononuclear progenitor cells. The studies on mechanical stimulation of osteoclastic resorption are rare.Extracorporeal Shock Wave (ESW) is a mechanical wave which generated by transient changing of the pressure, the energy it produces has the therapeutic effects when high-energy shock waves were focued by the therapeutic equipment. Extracorporeal shock wave has many advantages, such as quantitative effect on the treatment site, non-invasive, fewer complications, shorter treatment period and low risk. Some studies have shown that the shock wave have ability of increasing activity of osteoblast, thus contributing to fracture healing. The shock waves as an stimulation of energy , at present there are few related studies about the effects on the osteoclast differentiation and the bone resorption by ESWThe experiment will simulate the environment of osteoblasts and osteoclasts in vivo via creating a co-culture system which including osteoblasts and osteoclasts. We will study the effect of ESW on osteoclasts formation and the bone resorption via investigate the morphological changes of osteoclasts, the expression of regulatory genes by RT-PCR, osteoclast specific protein secretion by ELISA and quantitative analysis of the bone resorption ability by electron microscopy.Methods:1 hMSCs were isolated from the bone marrow and induced osteogenic differentiation: The bone marrow got from each of the volunteers(excluding metabolic diseases) was 20ml, hMSCs were got by density gradient centrifugation, there will be numerous hMSCs in culture after 1 week in vitro. We use the classic osteogenic medium(10nM dexamethasone, 50uM ascorbic acid, 10mMβ-glycerophosphate) and get the osteoblasts after induction of for 4 weeks, the activity of osteoblast was verifed by ALP staining and alizarin red staining.2 Establish the co-culture system including Osteoblasts and osteoclasts: The bone marrow was got from each of the 10 volunteers again. Bone marrow mononuclear cells( BMMC) were got by density gradient centrifugation. BMMCs were added to the hanging dish which place a piece of bone slice at the bottom and 1,25 (OH)2VD3(0.1μM) in the culture solution. Thus, we established the co-culture system which osteoblasts at the bottom, osteoclasts on the upper and they can interact by macromolecules.3 The effect of extracorporeal shock wave on osteoclass in the co-culture system: BMMCs were intervened the by the ESW(500 pulses, 0.12mJ/mm2) 12 hours later after adherence, cell morphological changes were observed continuously in the microscopy; The content of cathepsin K, tartrate-resistant acid phosphatase 5b, IL-1βand tumor necrosis factorαwas detected by enzyme-linked immuno sorbent assay; RANK, NFATc1 and c-Fos gene expressed by osteoclast was analysised by RT-PCR after 1 week; Fusion condition of osteoclasts was observed by TRAP staining; The number and size of bone lacuna of the bone slice was calculated by the electron microscopy. The control group including co-culture without ESW, Mononuclear cells cultured alone with ESW and without ESW group. All data was expressed in terms of mean±SD. ANOVA was performed to analyse the data and A p value of less than 0.05 was considered as significant. Results:1 Bone marrow mesenchymal stem cells transform into osteoblasts after 28 days induced culture.Calcium nodules can be seen by ALP and alizarin red staining, show that we get the high activity osteoblasts. After the co-culture has established, mononuclear cells exist aggregate trends in 1-3 day, aggregation density ranging from 2-10, but at this time can still see the cell membrane integrity, fusion did not occur. At 4-6 day shows the fusion of cells, ranging from the formation of more than 2-6 nuclear nuclear giant cells. Intervention group fusion was later and have less uncleus than the control group.2 Specific protein secreted by osteoclast was detected by ELISA. Cathepsin K 2.23±0.35ng/ml, TRAP 5b 1.53±0.29ng/ml, IL1β53.28±7.45 ng/ml, TNFα5.87±0.80ng/ml in ESW and co-culture group. The statistical analysis showed that the intervention group was significantly lower than non-ESW(P<0.05), but higher than the BMMC cultured alone with ESW. Related factors secreted by osteoblast promote the secretion of osteoclast-specific protein in the co-culture system, but the ESW will inhibited the secretion of specific protein. Detect RANK, NFATc1 and c-Fos gene expression by RT-PCR. Fluorescence intensity of intervention group was lower than the group without ESW. It is indicated that ESW inhibit the expression of the key gene in the formation of osteoclasts.3 Mononuclear cells were stained claret by TRAP staining. which prove that tartrate-resistant acid phosphatase specific has expressed in all groups and the cells is osteoblast. Fusion index of osteoblast is 0.46±0.10 in co-culture with ESW group while 0.73±0.14 in the group without ESW. The difference has shown that ESW inhibit the fusion of BMMC in co-culture system(P<0.05). Analysis area of bone lacuna, 12963±952μm2 in co-culture with ESW group and 16257±1423μm2 without ESW(P<0.05). The difference shown that ESW inhibit the bone resorption of osteoclast.Conclusion:1 Sufficient osteoclasts can be obtained to study in osteoblast and BMMCs induced culture by establish a osteoblast and osteoclast co-culture system in vitro.2 Osteoblast has the function of promote fuse of BMMCs to generate mature osteoclasts.3 ESW can inhibit the expression of the related genes and specific proteins during the osteoclast formation process, and inhibit capacity of bone resorption by osteoclast. ESW have obvious inhibitory action of osteoclast formation.
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