Osteoclast Formation and Bone Resorption Assay

BMMs (1 × 10⁴ cells/well) were seeded onto 96-well plates in triplicate, containing 30 ng/mL M-CSF and 50 ng/mL RANKL. After 7 days' culture, 4% PFA was used to fix osteoclasts for 20 min at 37°C [27 (link)]. The rhodamine-conjugated phalloidin (Cytoskeleton, Inc., Denver, CO, USA) was used to detect the cytoskeleton (F-actin ring). BMMs were stained at 37°C for 1 h, and then, washed with PBS three times, each time for 10 min. A LSM5 confocal microscope (magnification, ×10; Carl Zeiss AG, Oberkochen, Germany) was used to observe the F-actin ring. The images were analyzed using the Image-Pro Plus 6.0 software. Sterile bone pieces from the 4 groups were placed into 96-well plates, containing BMMs (1 × 10⁴ cells) with 30 ng/mL M-CSF and 50 ng/mL RANKL. After 12 days, the 0.25% (v/v) trypsin was used to digest the cells on the bone pieces, and the cells were then washed 3 times with PBS. A Quanta 250 scanning electron microscope (SEM; FEI; Thermo Fisher Scientific, Inc.) with a magnification of 10 kV was used to obtain on the bone surface. The resorption area was measured using Image-Pro Plus 6.0.