1 25 dihydroxyvitamin d3

Primary osteoblast isolated from Dicer^{gtRosaCreERT2} mice were seeded as 8,000 cells per well in 96-well plates and treated with 1μM 4-OHT for 3 days as described above. Bone marrow cells from wild-type mice were isolated and 200,000 cells per well were added on top of primary osteoblasts cultivated in alpha-MEM supplemented with 10 nM 1,25-dihydroxyvitamin D3 (Sigma-Aldrich, St. Louis, USA). Medium was changed every 3 days. After 9 days, cells were fixed and stained for TRAP activity.

Samples were sterilized in 70% ethanol for 1 h, followed by four washes with phosphate buffered saline (PBS; Sigma, Steinheim, Germany) and overnight incubation in an expansion medium (minimum essential medium alpha, α-MEM; Gibco, Thermo Fisher Scientific, Paisley, UK) supplemented with 10% FBS (fetal bovine serum; South American Origin, Biowest, Nuaillé, France), 1% PSN (5 units/ml of penicillin, 5 mg/ml of streptomycin, 10 mg/ml neomycin; Sigma, St. Louis, MO, USA) and 1 ng/mL human basic fibroblast growth factor (Sigma, Jerusalem, Israel). Normal human bone marrow derived mesenchymal stem cells (hMSCs) were purchased from Lonza (Walkersville, MD, USA). The cells were isolated from bone marrow of a 40-year-old female. The cells used in the experiments were from passage 4. MSCs were seeded at a density of 2.5 × 10⁵ per scaffold and incubated in expansion for 7 days and in osteogenic medium (α-MEM supplemented with 10% FBS, 1% PSN, 50 µM ascorbic acid phosphate (Sigma, Osaka, Japan), 2 mM β-glycerophosphate (Sigma, St. Louis, MO, USA), 10 nM 1,25-dihydroxy-vitamin D3 (Sigma, Jerusalem, Israel) and 10 nM dexamethasone (Sigma, Shanghai, China) for an additional 7 days.

Cells were induced with osteogenic inducing medium containing 10% FBS, 10 mM b-glycerophosphate (Sigma), 100 nM dexamethasone (Sigma), 50 mg/ml ascorbic acid and 0.01 mM 1,25-dihydroxy-vitamin D3 (Sigma) [18 (link)] for 14 d. Medium was changed every 2 d. After 14 d of culture, induced DFCs were washed 3 times in PBS after being fixed in 4% paraformaldehyde for 10 minutes and then incubated in 0.1% alizarin red solution (Sigma) in Tris-HCl (pH 8.3) at 37°Cfor 30 minutes. Cells were washed and observed using a phase-contrast inverted microscope (Nikon, Japan).

For osteoclast differentiation, bone marrow was flushed out of the femora from 6 to 8 mice at the age of 12 weeks with α-MEM/FBS. Cells were then plated at a density of 5 × 10⁶ cells per ml, and after 24 h the adherent cells were cultured in α-MEM/FBS containing 10 nM 1,25-dihydroxyvitamin-D3 (Sigma-Aldrich). Beginning at day 4 after seeding M-CSF and RANKL (both from Peprotech) were added to a final concentration of 20 ng/ml and 40 ng/ml, respectively, and the cells were cultured for 7 days to generate osteoclasts. For the stimulation experiments, 100 ng/ml IL-6^{47 (link)} and 100 ng/ml hyper-IL-6^{34 (link),48 (link)} were added to the culture medium during the whole period of osteoclast differentiation.
Formation of multinuclear cells was assessed by tartrate-resistant acid phosphatase (TRAP) activity staining as described previously^{49 (link)}. In brief, after removal of the medium and two washing steps with phosphate-buffered saline (PBS), cells were fixed with cold methanol for 5 min. After washing and drying, cells were stained with Naphthol AS-MX-Phosphate (Sigma-Aldrich) for 30 min before the number of TRAP-positive multinuclear cells per well was counted.