Technetium-99 conjugated with methylene diphosphonate inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis

In the present study, we investigated the effects of technetium-99 conjugated with methylene diphosphonate (Tc-99-MDP), an agent used in radionuclide therapy, on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and explored the underlying mechanisms. The murine macrophage cell line RAW264.7 and bone marrow-derived-macrophages from C57BL/6 mice (BMM) were used as models for osteoclastogenesis in vitro. The expression of some key factors in RANKL (50 ng/mL)-induced osteoclastogenesis in RAW264.7 cells was investigated by flow cytometry and real-time reverse transcriptionpolymerase chain reaction (RT-PCR). To detect multinucleated osteoclast formation, RAW264.7 cells were induced with RANKL for 4 days, whereas BMM were induced by 50 ng/mL RANKL and 20 ng/mL macrophage colony-stimulating factor for 7 days, before being stained with tartrate-resistant acid phosphatase. Osteoclastogenesis was evaluated using the osteoclast markers CD51, matrix metalloproteinase (MMP)-9 and cathepsin K. At 0.01 mu g/mL, Tc-99-MDP significantly inhibited RANKL-induced osteoclastogenesis without any cytotoxicity. In addition, Tc-99-MDP abolished the appearance of multinucleated osteoclasts. Real-time RT-PCR analysis of transcription factor expression revealed that Tc-99-MDP inhibited the expression of c-Fos and nuclear factor of activated T cells. In addition, Tc-99-MDP inhibited the expression of the inflammatory factors interleukin (IL)-6, tumour necrosis factor-alpha and IL-1 beta. Finally, Tc-99-MDP inhibited the activation of mitogen-activated protein kinases in RAW264.7 cells following RANKL stimulation. In conclusion, Tc-99-MDP possesses anti-osteoclastogenic activity against RANKL-induced osteoclast formation.