F actin

To examine the effects of the ZOL/β-TCP for osteoclast formation and activation, mBMSCs were cultured on each ZOL/β-TCP disc in accordance with the schedule of Figure 6. In brief, isolated mBMSCs (5.0 × 10⁵ cells/well) were cultured on ZOL/β-TCP discs in differentiation inducer medium, α-MEM with 10% FBS, 0.1% antibiotics, 30 ng/mL M-CSF and 50 ng/mL receptor for nuclear factor kappa B ligand (RANKL, Peprotech Inc., Cranbury, NJ, USA), for 5, 10 days at 37 °C. In this examination, we used the ZOL/β-TCP disc, immersing in culture medium for 24 h before seeding mBMSCs. The medium was exchanged every 2, 3 days. After culturing, to confirm osteoclast formation, cells were stained with Alexa Fluor^® 488-labeled phalloidin (Invitrogen, Carlsbad, CA, USA) for F-actin and DAPI (Dojindo, Kamimashiki, Kumamoto, Japan) for nuclei. Cells were washed with PBS (pH 7.3) and examined by observation with fluorescence microscopy (BZ-X710, Keyence, Osaka, Osaka, Japan). To confirm osteoclast activation, cells were performed with tartrate-resident acid phosphate (TRAP) staining using TRAP/ALP Stain Kit (Wako Pure Chemical, Osaka, Japan) and observed by phase microscopy (BZ-X710, Keyence, Osaka, Osaka, Japan). We analyzed three indexes from the results of observation: the osteoclasts area, cells size, and TRAP-positive area by BZ-X Analyzer (BZ-H3A (ver. 1.31), Keyence, Osaka, Osaka, Japan).