Osteoclast (OC) precursor culture v1

Osteoclast precursors from human peripheral blood were cultured as previously described. 15 mL of heparin anti-coagulated peripheral blood was obtained by venipuncture from the patient, and a healthy age- and sex-matched control. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque Solution. Cells were washed in PBS twice, and plated on 24-well plates at a density of 1×106/well at 37°C in α-MEM, supplemented with 10% FBS, 1% penicillin/streptomycin and 25 ng/ml of macrophage colony stimulating factor (M-CSF) (R&D Systems, Inc., Minneapolis, USA). 6 days later, osteoclast precursor differentiation was induced with the medium supplemented with both 25ng/ml of M-CSF and 30ng/ml RANKL (R&D Systems, Inc., Minneapolis, USA). PBMCs in three wells from each subject were cultured in the absence of M-CSF and RANKL as a negative control.7 days later, TRAP staining was performed using a kit from Sigma-Aldrich (sigma Chemical Co., St. Louis, MO, USA). TRAP-positive cells containing 3 or more nuclei were counted as OCs. 6mm*6mm bovine cortical bone slices were put into cell culture wells at the beginning of OC differentiation. 7 days after co-cultures, the slices were removed and evaluated for OC morphology and pit formation by scanning electron microscope (INCA PENTAFET X3, Oxford Instruments, Abingdon, Oxfordshire, UK).