Dynamic observation of the culture and bone resorption function of osteoclast-like cells induced by M-CSF and RANKL in vitro

Objective Mouse bone marrow stem cells were induced to osteoclasts using both macrophage colony-stimulating factor(M-CSF)and receptor activator of NF-κB ligand(RANKL) in vitro.The bone absorption function was observed dynamicly.Methods Bone marrow stem cells obtained from mouse extremities were isolated and cultured with M-CSF and RANKL.The coverslips and bone slices were put in the cultures.The tartrate-resistant acid phosphatase(TRAP) staining was performed to observe cell morphology and staining.The absorption lacuna in bone slices were observed meanwhile.Results TRAP-positive cells with light stained mononucleus or double nucleus appeared after a 3-day culture.TRAP-positive cells became more after 6-day culturing than after 3-day culturing,and the cells were mainly double nucleated.TRAP-positive multinuclear cells appeared after 9-day cuturing,and the cells had more than three nuclei.The absorption lacuna in bone slices were not found after 3-day and 6-day culturing.The blue to purple absorption lacuna appeared with different shape afer a 9-day culturing.Conclusion Utilizing M-CSF and RANKL to induce bone marrow stem cells to osteoclasts was an effective culture method.The osteoclast-like cells were found to have bone resorption fuction after a 9-day induced culturing in this experiment.