2.3. Macrophage Differentiation and Particle/LPS Stimulation

For experiments, cells were seeded in 24-well plates at a density of 1 × 10⁵ cells/cm² in a final volume of 1 mL. To differentiate monocytes toward macrophage phenotype, cells were cultivated for 3 days in complete media supplemented with 100 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA), followed by 24 h cultivation under PMA-free conditions. For the cell viability, THP-1 cells were treated with particle number concentrations of 1 × 10⁷, 1 × 10⁸ and 1 × 10⁹, representing mass concentrations of 0.876, 8.58 and 89.9 μg/mL for TiO₂ and 0.912, 9.11 and 92.6 μg/mL for ZrO₂ particles in complete culture medium for 12, 24 and 48 h. Therefore, for the cell viability, the particles were analyzed at concentrations between 0 and 100 μg/mL according to the corresponding particle mass concentration applied during experimentation. Concentration-dependent analysis revealed that 1 × 10⁸ and 1 × 10⁹ particles/mL confirmed adverse toxicity effects on macrophages’ viability. Consequently, the particle number concentration of 1 × 10⁷ particles/mL was finally sublethal, and for this reason, was selected for the gene expression assays. In order to assess particle-induced pro-inflammatory gene expression in THP-1 after 12, 24 and 48 h of stimulation by the different particles TiO₂, ZrO₂ and glass (at particle number concentration of 10⁷ particles/mL), cell culture lysates were collected, immediately centrifuged, transferred to a fresh tube and stored at −80 °C until used for RT-PCR assays. Lipopolysaccharides from Porphyromonas gingivalis (P. gingivalis LPS) were purchased from Sigma-Aldrich. Cells were cultured at 37 °C under 5% CO₂ with 1 μg/mL of P. gingivalis LPS for 24 h. The growth medium was then replaced with 100 μL of RPMI 1640 medium containing the TiO₂, ZrO₂ and glass particles at the 10⁷ particles/mL concentrations. Cells were exposed to these agents for a further 48 h to finalize the experiment. Macrophages cultured under particle-free/LPS conditions were used as negative controls.