Bovine cortical bone slices

To assess bone resorption activity, monocytes were seeded on bovine cortical bone slices (ImmunoDiagnostic Systems) and differentiated into MF or OC. Following complete cell removal by several washes with water, bone slices were stained with toluidin blue (Sigma-Aldrich) to detect resorption pits under a light microscope (Leica DMIRB, Leica Microsystems). Surface of bone degradation areas were quantified manually with ImageJ software. Cross-linked C-telopeptide collagen I (CTX) concentrations were measured using betaCrosslaps assay (ImmunoDiagnostic System) in the culture medium of OC grown on bone slices [25 (link)].

250000 bone marrow derived monocytes were seeded in a 24-well plate Corning OsteoAssay surface plate. Osteoclast differentiation protocol was performed as described above. At the end of differentiation time, cells were washed with 1X PBS for 3 times and incubated with 5% solution of sodium hypochlorite for 10 min at RT to remove the cells. Next, plates were air dried at RT and resorption areas for each well were acquired automatically using Leica DMI6000B. Resorbed areas were quantified with ImageJ and plotted using GraphPad Prism 8.
Alternatively, osteoclasts resorptive capacities were done by a pit assay on bovine cortical bone slices (Immunodiagnostic Systems). 250000 bone marrow-derived cells/well were seeded in a 24-well plate containing the cortical bone slices. At the end of differentiation time, media was removed carefully from the wells by aspiration and bone slices were incubated with ammonium hydroxide solution overnight. Next, cells were removed from bone slices by ultrasonication for 1 min (Transsonic T460, Elma) and slices were stained with 0.5% Toluidine Blue. To visualize resorption pits, bone slices were imaged with Olympus BX41 equipped with Olympus DP72 camera at ×10 magnification. Osteoclast number and total area were quantified with the OsteoMeasure software (Osteometrics, Decatur) and plotted using GraphPad Prism 8.

Bovine cortical bone slices (Immunodiagnostic Systems) were sterilized with 70% ethanol for 5 min, followed by 10% penicillin–streptomycin for 5 min. The bones were rinsed twice with complete DMEM and incubated overnight at 37 °C. In a 24 well plate, Raw264.7 cells were seeded on the bone slices at a concentration of 2 × 10⁴ cells/well with 35 ng/mL RANK-L. The medium was replaced every two days. On day 6, cells on the bone slices were removed using a cotton swab. Bone slices were stained using toluidine blue solution (1% toluidine, 0.5% tetraborate in water) for 5 min and rinsed in PBS three times. The resorption pits were quantified and imaged using Leica DM4000 B LED microscope. Resorption pit assay was performed in three independent experiments.