Recombinant human il 6rα

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.

hASCs (1 × 10⁴ cells/cm²) were seeded in 24-well plates and cultured in α-MEM containing 1% PSF, 10 IU/mL heparin, and 2% human platelet lysate, at 37°C in 5% CO₂ in air. hASCs were allowed to attach for 24 h before stimulation with cytokines. After cytokine stimulation, the medium was replaced with osteogenic medium (OM), consisting of α-MEM containing 1% PSF, 10 IU/mL heparin, 2% human platelet lysate, 50 μM ascorbic acid-2-phosphate (vitamin C; Sigma, St. Louis, MO, USA), 5 mM β-glycerophosphate (βGP; Sigma), and 10 nM 1,25-(OH)₂ vitamin D₃ (Sigma). Recombinant human TNF-α (R&D Systems, Minneapolis, MN, USA), recombinant human IL-4 (R&D Systems), recombinant human IL-6 (R&D Systems), recombinant human IL-6Rα (R&D Systems), recombinant human IL-8 (R&D Systems), and recombinant human IL-17F (R&D Systems) were added to the OM at 10 ng/mL and incubated for 72 h at 37°C in 5% CO₂ in air. Then, the medium was changed to OM without cytokines and was replaced every 3 days. hASCs were harvested at 6 and 48 h (early time points) and at 4, 7, and 14 days (late time points) to assess proliferation and osteogenic differentiation of hASCs.