In vitro Osteoclast Differentiation

Human monocytes were seeded on OsteoAssay bone plates (Lonza) at a density of 1 × 10⁴ cells per well. The cells were cultured for three weeks in conditioned mediums from THLE-2 cells in the presence of 25 ng mL⁻¹ recombinant human M-CSF (R&D Systems). The medium was changed every other day. On day 21, bone slices were ultra-sonicated in 1 M NH₄OH to remove adherent cells and stained with 0.1% toluidine blue solution. Bone slice images were captured using electron microscopy. Three fields were randomly selected for each bone slice for further analysis. Pit areas were quantified using Image Pro Plus 6.2 software (Media Cybernetics Inc.)^{57 (link)}.