Objective For studying effects of exogenous factors on bone metabolism,different in vitro osteoclast (OC) culture systems were compared in this study.
Methods Bone marrow mononuclear cells co-cultured with bone marrow stromal cells (BMSC) in 3-dimensioal culture system,bone marrow mononuclear cells cultured with bone marrow stromal cells conditional medium,bone marrow mononuclear cells and RAW264.7 cell line cultured in the presence of mouse recombinant receptor activator of nuclear factorkappaB ligand (RANKL) for OC differentiation.RAW264.7 cells were labeled with cross-linkable membrane dye CM-DiI to examine the fu sing process of OC differentiation by Laser Scanning Confocal Microscope.OC was stained by Tartrate Resistant Acid Phosphatase (TRAP).
Results All of the procedures successfully developed OC which was indicated by TRAP positive and multinuclear cells fu sing from OC precursors.The OCs induced by exogenous RANKL were easy acquired,whose number and size were more and bigger than those induced by the normal BMSCs.The method of OC differentiation induced by normal BMSCs was complicated,but Was useful for studying the indirect effect of intervention factor on OC differentiation post treated BMSC.Some TRAP positive cells characterized by cell membrane not fusing and TRAP staining only focusing around nucleus were observed in all groups,and contained 1-3 nucleuses.This kind of TRAP positive cells might be OC precursors.
Conclusion Each OC differentiation method shows various advantages and which specific procedure is selected should accord to study objectives.
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