Cells were directly seeded on prepared Co–Cr alloy discs (15.0 mm diameter, 1.0 mm thickness) or indirectly treated with Co–Cr alloy located at the bottom of an insert well and incubated for 3 days. Cytotoxicity and reactive oxygen species (ROS) production was evaluated by MTS assay and flow cytometry, respectively. Protein and mRNA levels were determined by Western blotting and RT-PCR analysis, respectively.
Cell viability and flow cytometric assay demonstrated that the Co–Cr alloy was cytotoxic to HGFs and osteoblasts, and significantly increased ROS production. In addition, the Co–Cr alloys upregulated pro-inflamamtory cytokines (TNF-α, IL-1β, IL-6, and IL-8) and increased levels of various inflammatory mediators (iNOS derived nitrite oxide, and COX-2-derived PGE2) in both cells. A mechanistic study showed that Co–Cr alloys activates the NRF2 pathway and up-regulate antioxidant enzymes including heme oxygenase-1 (HO-1). Co–Cr alloys activated JAK2/STAT3, p38/ERK/JNK MAPKs and NF-κB signaling pathways. Furthermore, antioxidants (resveratrol and NAC) and HO-1 inhibitor (SnPP) significantly inhibited the production of ROS and inflammatory mediators, as well as the activation of NF-κB signaling in Co–Cr alloy stimulated HGFs and osteoblasts.
This study is the first to show that Co–Cr alloys exert cytotoxic and inflammatory effects via activation of Nrf2/ARE signaling and up-regulation of downstream HO-1, which could represent candidate targets for the regulation of inflammatory responses to Co–Cr alloys.