Complete α mem

BMMs as prepared previously were seeded in droplets of 5 μl (5 × 10³ cells) in 96-multiwell plates (Nunc, Roskilde, Denmark). The cells were left to adhere for 10 min whereafter 200 μl of complete α-MEM supplemented with M-CSF (30 ng/ml) and with or without RANKL (4 ng/ml) (R&D Systems/Biotechne) and CCL5 (1 μg/ml) (R&D Systems/Biotechne) was added. The cells were incubated at 37 °C in 5% CO₂ for various times (designated in Results section), whereafter the culture media were harvested and stored in aliquots at −20 °C until analysis of the active isoform 5b of TRAP5b (Immunodiagnostic systems [IDS], Copenhagen, Denmark). Adherent cells were fixed at the bottom of the 96-well plate and stained for TRAP using the leukocyte acid phosphatase kit (Sigma–Aldrich, St Louis, MO, USA), following the manufacturer's instruction. TRAP-positive cells with three or more nuclei were considered osteoclasts. Images were acquired using an Olympus BX41 light microscope. For gene expression analyses, BMMs were seeded in droplets of 40 μl (4 × 10⁴ cells) in 12-multiwell plates (Nunc) and cultured as mentioned previously.

RAW264.7 cells (mouse macrophage cells) were obtained from America Type Culture Collection (Mannassasa, VA, USA) and maintained in complete α-MEM (α-MEM, 10% heat inactivated FBS, 2 mM l-glutamine and 100 U/mL penicillin/streptomycin). Freshly isolated bone marrow macrophages (BMM) were isolated from C57BL/6 mice by flushing the marrow from the femur and tibia. Primary BMMs were seeded in complete α-MEM supplemented with macrophage-colony stimulating factor (M-CSF), purchased from R&D Systems, (Minnneapolis, MN, USA). All cell cultures were maintained in 5% CO₂ at 37 °C.

All the animal experiments were approved by the Ethics Committee of West China Hospital of Stomatology (WCHSIRB‐D‐2017‐029). C57BL/6 mice were dissected to acquire femurs and tibias. Then, the bone marrow cells were flushed into a culture dish and cultivated for 24 h in complete α‐MEM (HyClone) supplemented with 10% FBS and 1% penicillin–streptomycin (HyClone) at 37°C under 5% CO₂. Then, the cells were cultured for 72 h in complete medium containing 30 ng/ml M‐CSF (Catalog#416‐ML, R&D Systems), whereupon they were regarded as bone marrow‐derived macrophages (BMMs). These macrophages and RAW 264.7 monocytic cells (Shanghai Cell Center) were subsequently seeded onto the PDMS substrates in dishes and cultured in complete α‐MEM supplemented with 30 ng/ml M‐CSF and 100 ng/ml RANKL (Catalog#462‐TEC, R&D Systems). After 7 days of culture, during which the media containing inducing factors were replaced three times, osteoclastogenesis assays were performed to identify TRAP‐positive multinucleated cells (nuclei number ≥3) using an acid phosphatase staining kit (387A, Sigma‐Aldrich).