RANKL pretreatment plays an important role in the differentiation of pit-forming osteoclasts induced by TNF-α on murine bone marrow macrophages.

Background: Osteoclasts differentiated from bone marrow macrophages (BMMs) induced by TNF-α alone do not have resorbing activity. When BMMs are stimulated with receptor activator of NF-κB ligand (RANKL) before TNF-α stimulation, pit-forming osteoclasts are differentiated. However, the details of the effect of RANKL pretreatment on the pit-forming osteoclast differentiation by TNF-α have not been established. The aim of this study is to examine the condition of RANKL pretreatment for differentiation of pit-forming osteoclasts induced by TNF-α. Murine BMMs were stimulated with various concentrations of RANKL for 24h in the presence of M-CSF, then the medium was changed and TNF-α was added. Osteoclasts and pits formation were examined. Osteoprotegerin (OPG), decoy receptor of RANKL, was added to the culture to examine the necessity of co-existing RANKL with TNF-α on the formation of pit-forming osteoclasts. To investigate the influence of RANKL of sufficient concentration as pretreatment for pit-forming osteoclast formation by TNF-α, dose- and time-dependent changes of osteoclast formation were checked.

Results: The pit formation by osteoclasts in response to TNF-α required 10ng/mL RANKL pretreatment. Stimulation with this concentration of RANKL led to the differentiation of mature osteoclasts in the 72h culture. The pit formation was not inhibited by the OPG.

Conclusions: These results suggested that the concentration of RANKL pretreatment, which also alone can differentiate BMMs into osteoclasts, may be important in the differentiation of pit-forming osteoclasts by TNF-α. In addition, the effects of TNF-α after RANKL treatment might be independent of RANKL.