A Preliminary Study Of Osteoclast Exosomes Promote The Osteogenic Differentiation Of Osteoblastic Precursor Cells

Background The pathogenesis of osteoporosis(OP) is that the rate of resorption of bone of osteoclast(OC) more than the relative ability of bone formation of osteoblast(OB), resulting in negative balance. The bone constantly update and reconstruction, is to replace the old and new bone and maintain bone strength balance. This bone reconstruction process relies on the precise coordination of bone resorption of OC and bone formation of OB. In recent years, a large number of studies have showed the wonderful relationship between OC and OB from the cellular level, they through the intracellular pathways and multi-way signals of precision control, involving the positive and negative feedback loops and double-sided mechanisms, to maintain bone homeostasis. Exosomes are a diameter of about 60-100 nm membranous vesicles which released into the extracellular matrix, made up of cells multivesicular bodies(MVB) and membrane after fusion, participate in the exchange of information between cells and a variety of physiological and pathological processes. It can be secreted by lymphocytes, ectomesenchymal stem cells, tumor cells and other cells. Previous studies have shown that exosomes play an important role in myeloma, osteosarcoma, Parkinson's disease, alzheimer's disease, colorectal cancer and other diseases. And now, there are less study reports about osteoclast exosomes at home and abroad. The research of biological characteristics and function mechanism of osteogenic differentiation in osteoclast exosomes can provide new ideas and directions for the prevention and treatment of osteoporosis, osteosclerosis and other metabolic bone diseases.ObjectiveSeparate and identify osteoclast exosomes, observe the effect of osteoclast exosomes in the osteogenic differentiation of kusao cells, to insure whether the osteoclast exosomes promote the osteogenic differentiation of kusao cells, looking for the key factor of the osteogenic differentiation of kusao cells in osteoclast exosomes.Methods 1.Using RANKL to induce Raw cell 264.7 to osteoclast, TRAP staining was used to identify the cells after induction; 2.Isolate osteoclast exosomes from the supernatant of osteoclasts by ultrafiltration centrifugation; 3.Detect the expression of CD9 and CD63 protein of the characteristics of exosomes, and analysis the particle size by Nanosight; 4.Observe the PKH67 labeled exosomes target the receptor of kusao cells; 5.Kusao cells can be divided into three groups, complete medium group(CM group), osteogenic medium group(OM Group), osteogenic medium and osteoclast exosomes group(OME group), change medium the next day. To insure the role of osteoclast exosomes in the osteogenic differentiation of kusao cells by western blot, alizarin red staining, Von kossa silver staining, and q-PCR detection after cultivate 14 days; 6.Analyze the osteoclast exosomes by proteomics analysis, find out the key protein and have further validation by western blot and q-PCR.Results 1.It can see a lot of red multicore, large size and irregular shape of the TRAP positive giant cells, those are osteoclasts; 2.Uniform size and a diameter of about 50-200 nm of membranous vesicles was detected in the supernatant of osteoclasts. It can identify as osteoclast exosomes(OC-exosomes) that express CD9 and CD63 protein of the characteristics of exosomes by western blot; 3.The PKH67 labeled of OC-exosomes can target the receptor of kusao cells; 4.Western blot analysis show that the expression of the RUNX2 protein in OM group are 1.25 times of the CM group, 2.72 times of the OME group; 5.Alizarin red staining results show that the ratio of calcium salt deposition area in total area of CM group, OM group, OME group are as follows: 0.21%, 3.47%, 20.74%; 6.Von kossa silver staining results show that the ratio of calcium salt deposition area in total area of CM group, OM group, OME group are as follows: 0.066%, 2.64%, 20.89%; 7.q-PCR analysis results show that:(1)The expression of RUNX2 on OME group are significantly less than CM and OM group, and the difference have statistically significant(P<0.05);(2)The expression of SPP1 on OME group are less than CM and OM group, and the difference have statistically significant(P<0.05); 8.Proteomics analysis found that the expression of MMP9 in osteoclast exosomes are 13.552 times of Raw cell 264.7 exosomes; 9.Western blot results show that the expression of MMP9 protein in the OC-exosome group are obviously higher than the RC-protein and the OC-protein group(P<0.01); 10.q-PCR analysis results show that after 14 days of osteogenic induction, compared with the OM group, the MMP9 gene expression was significantly increased after adding osteoclast exosomes, have significant difference(P<0.01).Conclusion 1.Exosomes were successfully separated from the supernatant of osteoclasts by ultrafiltration centrifugation; 2.Osteoclast exosomes can promote the osteogenic differentiation of kusao cells; 3. MMP9 are highly expressed in osteoclast exosomes, which may be the key factor to promote osteogenic differentiation of kusao cells.