Osteoclast Differentiation and Resorption Assay

Mature OCs were prepared from the co-culture of BMCs and primary osteoblasts (OBs) as described previously [26 (link)]. Briefly, BMCs (1 × 10⁷ cells) and primary OBs (1 × 10⁶ cells) were incubated in collagen gel-coated culture dishes in the presence of 10⁻⁸ M VitD₃ and 10⁻⁶ M PGE₂ for 10–12 days. Mature OCs were detached using 0.1% collagenase and re-seeded in dentin slices. After 1 h, the cells were transfected with the indicated retrovirus or siRNA as described above and further cultured in the presence of RANKL (100 ng/mL). The cells re-seeded in dentin slices were completely removed using 10% sodium hypochlorite after 48 h. Dentin slices were stained with hematoxylin to detect resorption pits. The total area of resorbing pits was determined under a microscope and were quantified using Image-Pro Plus version 4.0 (Media Cybernetics, Silver Spring, MD, USA). To confirm the survival of mature OC by retrovirus and siRNA transfection, mature OC replanted in 48-well plates in the same manner as in dentin slices, and after 48 h, stained with TRAP solution.