1 25 oh 2 vitamin d3

All hormone injections were performed in the home cage of the mice after random treatment allocation of individual ear‐marked mice. For 1,25(OH)₂‐vitamin D₃ treatment, male C57BL/6N mice aged 13–15 weeks were subcutaneously injected with 2 μg/kg body weight 1,25(OH)₂‐vitamin D₃ (Sigma D1530) in ethanol 1% in NaCl 0.9%. The dose and application route was the same as previously used in our laboratory. Control mice were injected with 1% (v/v) ethanol in NaCl 0.9%. Mice were sacrificed 6 hr after injection.
For PTH treatment, male C57BL/6N mice aged 12–13 weeks were subcutaneously injected with 80 μg/kg body weight human PTH fragment 1–34 (hPTH1‐34) (Sigma P3796) in NaCl 0.9% or NaCl 0.9% alone as vehicle and sacrificed 2 hr after injection. The dose and application route used was determined from the literature (Kramer, Loots, Studer, Keller, & Kneissel, 2010).
For estradiol treatment, male C57BL/6N mice aged 16 weeks received one daily subcutaneous injection of 15 μg 17β‐Estradiol (Sigma E8875) in ethanol 0.1% (v/v) in NaCl 0.9% for five consecutive days and were sacrificed 4 hr after the last injection. The dose per body weight and the drug application route was derived from the literature, as shown Van Abel et al. (2002) to induce Trpv5 expression. Control mice were subcutaneously injected with 0.1% ethanol in NaCl 0.9% for 5 days.

HASCs (10 × 10³ cells/cm²) were seeded into 24‐wells plates and cultured in αMEM containing 1% PSF, 10 IU/ml heparin, and 2% human PL in a humidified atmosphere containing 20% O₂ (normoxia [standard condition]) or 1% O₂ (hypoxia), and allowed to attach for 24 hr at 37°C. The next day, the medium was replaced with osteogenic medium, consisting of αMEM with 1% PSF, 10 IU/ml heparin, 2% human PL, 50 µM ascorbic acid‐2‐phosphate (vitamin C; Sigma‐Aldrich), 5 mM β‐glycerophosphate (Sigma‐Aldrich) and 10 nM 1,25‐(OH)₂vitamin D₃ (Sigma‐Aldrich). Recombinant human IL‐4 (R&D Systems, Minneapolis, MN), and/or recombinant human IL‐6 (R&D Systems) and recombinant human IL‐6Rα (R&D Systems) were added to the osteogenic medium. IL‐4 or IL‐6 were added in a final concentration of 1 and 10 ng/ml, respectively, and IL‐4 in combination with IL‐6 in a final concentration of 10 ng/ml. After addition of osteogenic medium supplemented with the cytokines, hASCs were incubated in 1% O₂ or 20% O₂ at 37°C during 3 days. Then, the medium was replaced by osteogenic medium without cytokines, and refreshed every 3 days during 11 days. hASCs were harvested after 2, 7, and 14 days of culture for analysis of proliferation, osteogenic differentiation, and VEGF expression as described below.

The human monocytic cell line, THP-1, was obtained from ECACC and used for this study between passages 7 and 35. THP-1 cells were cultured in R10 medium composed of RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco, Massachusetts, Grand Island, NY, USA) and 2 mM L-glutamine, in the absence of exogenously added antibiotics. Pro-inflammatory (M1-like) macrophages and anti-inflammatory (M2-like) macrophages were generated by differentiation of these monocytes in the presence of 25 ng/mL phorbol 12-myristate 13-acetate (PMA) for three days or 10 nM 1,25-(OH)₂-Vitamin D₃ (Sigma-Aldrich, Poole, UK), for seven days, respectively [33 (link)]. M1-like macrophages were then washed and allowed to rest in fresh R10 medium without PMA for an additional 24 h [34 (link)]. Macrophages were plated out to a final density of 5 × 10⁵ cells/500 µL/well in R10 medium in 24 flat-bottomed well tissue culture plates.