Co-Cr dental alloys induces cytotoxicity and inflammatory responses via activation of Nrf2/antioxidant signaling pathways in human gingival fibroblasts and osteoblasts.

Objective: Although cobalt-chromium (Co-Cr) dental alloys are routinely used in prosthodontics, the biocompatibility of Co-Cr alloys is controversial. The aims of the present study were to investigate the effects of Co-Cr alloys on human gingival fibroblasts (HGF) and osteoblasts in an in vitro model as well as their potential molecular mechanisms, focusing on NF-E2-related factor 2 (Nrf2) pathways.

Methods: Cells were directly seeded on prepared Co-Cr alloy discs (15.0mm diameter, 1.0mm 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.

Results: 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.

Significance: 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.