Establish A Culture Method Of Osteoclasts, And Erythromycin Inhibits Bone Resorption By Osteoclasts

【Objective】To establish a effective culture method of osteoclasts induced from murine bone marrow hematopoietic stem cells.To explore the effects of Erythromycin(EM)on bone resorption by osteoclasts.【Methods】Murine bone marrow cells were incubated with M199 containing macrophage colony stimulating factor(M-CSF)and receptor activator of NF-κB ligand(RANKL).Through comparing the numbers of osteoclasts when the concentration of RANKL is 25,50,100ng/ml respectively and cells density is 10~4,10~5,10~6/ml respectively,we established a effective culture method of osteoclasts induced from murine bone marrow hematopoietic stem cells.The osteoclasts induced from murine bone marrow hematopoietic stem cells were cocultured with bone slices and coverslips,and were divided into 6 groups:Group A:control group; Group B:ultrahigh molecular weight polyethylene(UHMWPE)particles; Group C:UHMWPE particles+Erythromycin(EM)(10μg/ml);Group D:UHMWPE particles+EM(20μg/ml);Group E:UHMWPE particles +EM(40μg/ml);Group F:UHMWPE particles+EM(80μg/ml).Finally The tartrate-resistant acid phosphatase(TRAP)staining was done to observe the quantity of the osteoclasts,and the areas of absorption lacuna were calculated using the JD801 image analysis soft system.【Results】Through comparing the numbers of osteoclasts when the concentration of RANKL is 25,50,100ng/ml respectively and cells density is 10~4,10~5,10~6/ml respectively,we found that the number of osteoclasts is the most when the concentration of RANKL is 100ng/ml and cells density is 10~5/ml.Since that,we established a effective culture method of osteoclasts induced from murine bone marrow hematopoietic stem cells in this culture condition(RANKL:100ng/ml;cells density:10~5/ml).Then we cocultured osteoclasts with Erythromycin(EM) and polyethylene particles.We found that,the areas in Group C,D,E,F were all lower than group B(P＜0.05),it indicated that each concentration of Erythromycin(EM)could inhibit polyethylene particles-induced bone resorption by osteoclasts;The areas in Group D were more than group C,the areas in Group E were more than group C,D,the areas in Group F were more than group C,D(P＜0.05),it suggested that higher concentration of Erythromycin(EM)could inhibit polyethylene particles-induced bone resorption by osteoclasts more effectively;Group E and group F has no statistical significance(P＞0.05), it shows there could be useless to increase concentration when the concentration of EM reach to the high level.TRAP-positive cells localized in coverslips indicated the existence of the osteoclasts.The quantity of osteoclasts on each coverslip suggested that each concentration of Erythromycin(EM)could not effect the quantity of osteoclasts(P＞0.05).【Conclusion】We established a effective culture method of osteoclasts induced from murine bone marrow hematopoietic stem cells.Erythromycin(EM) can inhibits polyethylene particles-induced bone resorption by osteoclasts,which are induced from murine bone marrow hematopoietic stem cells.Erythromycin(EM)could be a potential therapeutic candidate for the treatment and prevention of aseptic loosening.