Calcineurin/NFAT pathway mediates wear particle-induced TNF-α release and osteoclastogenesis from mice bone marrow macrophages in vitro

Aim: To investigate the roles of the calcineurin/nuclear factor of activated T cells (NFAT) pathway in regulation of wear particles-induced cytokine release and osteoclastogenesis from mouse bone marrow macrophages in vitro. Methods: Osteoclasts were induced from mouse bone marrow macrophages (BMMs) in the presence of 100 ng/mL receptor activator of NF-kappa B ligand (RANKL). Acridine orange staining and MTT assay were used to detect the cell viability. Osteoclastogenesis was determined using TRAP staining and RT-PCR. Bone pit resorption assay was used to examine osteoclast phenotype. The expression and cellular localization of NFATc1 were examined using RT-PCR and immunofluorescent staining. The production of TNF alpha was analyzed with ELISA. Results: Titanium (Ti) or polymethylmethacrylate (PMMA) particles (0.1 mg/mL) did not significantly change the viability of BMMs, but twice increased the differentiation of BMMs into mature osteoclasts, and markedly increased TNF-alpha production. The TNF-alpha level in the PMMA group was significantly higher than in the Ti group (96 h). The expression of NFATc1 was found in BMMs in the presence of the wear particles and RANKL. In bone pit resorption assay, the wear particles significantly increased the resorption area and total number of resorption pits in BMMs-seeded ivory slices. Addition of 11R-VIVIT peptide (a specific inhibitor of calcineurin-mediated NFAT activation, 2.0 mu mol/L) did not significantly affect the viability of BMMs, but abolished almost all the wear particle-induced alterations in BMMs. Furthermore, VIVIT reduced TNF-alpha production much more efficiently in the PMMA group than in the Ti group (96 h). Conclusion: Calcineurin/NFAT pathway mediates wear particles-induced TNF-alpha release and osteoclastogenesis from BMMs. Blockade of this signaling pathway with VIVIT may provide a promising therapeutic modality for the treatment of periprosthetic osteolysis.