Inhibitory effects of AM630 on inflammatory osteolysis induced by titanium particles

Abstract

Objective To explore the effect of cannabinoid receptor 2 (CB2) selective antagonist (AM630) on the regulation of osteoclast differentiation from a murine macrophage cell line (RAW264.7)stimulated with titanium(Ti) particles and the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL).Methods The effect of AM630 and Ti particles on RAW264.7 cell viability was examined by using the methyl thiazol tetrazolium (MTT) assay.Osteoclast formation was measured by tartrate resistant acid phosphatase (TRAP) staining using a commercial kit.The mRNA levels of CB2,creatine phosphokinase(CPK) and NF-κB receptor activation factor(RANK) were detected by using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR).Enzyme-linked immunosorbent assay (ELISA)was performed to determine interleukin (IL)-1β and tumor necrosis factor (TNF)-t.Results AM630 (0-200 nmol/L) did not affect viability of RAW264.7 cells cultured with Ti particles.The mature osteoclasts were obtained from RAW264.7 cells stimulated with RANKL and Ti particles for 6 days and detected by TRAP staining.AM630 at a doses of ≥ 100 nmol/L significantly reduced the number of TRAP-positive cells as compared with controls.After culture for 6 days,mRNA levels of CB2,RANK and CPK were significantly higher in the media with RANKL and Ti particles than in those without RANKL and Ti particles.When 100 nmol/L AM630 in combination with RANKL and Ti particles was added to the culture system,mRNA levels of CB2,RANK and CPK were significantly decreased.The concentrations of IL-1β and TNF-α in RAW264.7 cell cultures were measured by using ELISA.After culture for 24 h,IL-13 and TNF-α concentrations were significantly increased in the media with Ti particles as compared with those in the media withou Ti particles.The cytokine concentration was further increased with culture time.Conclusion AM630 could significantly inhibit Ti particle-induced inflammatory osteoclastogenesis. Key words: Osteoclasts ; Inflammation ; Titanium particles ; Cannabinoid receptor 2