Receptor activator of nuclear factor kappa-B ligand induces osteoclast precursor culture and differentiation / 中国组织工程研究

ABSTRACT

BACKGROUND:Previous studies have applied long-bone mechanical separation method to obtain osteoclasts, which are terminal y differentiated cells and cannot further proliferate. Therefore a large number of osteoclasts can be harvested with bone marrow cells inducing culture method and RAW264.7 cells inducing culture method to meet clinical requirements. OBJECTIVE:To investigate the optimal method of receptor activator of nuclear factor kappa-B ligand (RANKL) induced osteoclast precursors to differentiate into mature osteoclasts. METHODS:After bone marrow cells were isolated from mouse, RANKL and macrophage colony stimulating factor were added into the medium together, or RAW264.7 cells were cultured with RANKL to induce osteoclasts. The osteoclast precursors were treated with different concentrations of RANKL to observe the number of generated osteoclasts and evaluate the dose-effect relationship between RANKL and osteoclastogenesis. Annexin V-FITC and propidium iodide staining were used for flow cytometry to analyze the mononuclear-macrophage apoptosis during osteoclastogenesis. RESULTS AND CONCLUSION:When 10μg/L RANKL was used, the peak of osteoclastogenesis appeared at days 6-7;when the concentration of RANKL was up to 100μg/L, the peak appeared at days 4-5. The number of new osteoclasts was dose-dependent on the RANKL concentration. 50μg/L of RANKL was the turning point in the fitted curve from osteoclastogenesis and RANKL concentration. After the RANKL concentration was more than 150μg/L, the number of osteoclasts slowed down obviously. RANKL can induce monocyte-macrophage to form osteoclasts and promote monocyte-macrophage apoptosis. The highest number of osteoclasts would be obtained to each unit of RANKL when monocyte-macrophage cells were cultured with 30μg/L of RANKL in the same vaccination density (104/cm2). Experimental findings indicate that, RAW264.7 cells or bone marrow cells inducing culture methods are simple and feasible, the optimum cellseeding density was 104/cm2;the appropriate stimulation concentration of RANKL was 30-50μg/L.