hMSCs were seeded in 6-well tissue culture plates (cytochemical staining) or 12-well tissue culture plates (RNA isolation, both Greiner Bio one, Frickenhausen, Germany) at 2 × 104 cells/cm2 for adipogenic or 0.5 × 104 cells/vm2 for chondrogenic and osteogenic differentiation. Cells for adipogenic differentiation were cultivated in α–MEM until confluence and then differentiation was induced with Adipogenic Induction Medium (Lonza, Cologne, Germany). The medium was changed twice a week, altering between Adipogenic Induction Medium and adipogenic maintenance medium (Lonza, Cologne, Germany) for 3 weeks, followed by one week of cultivation in adipogenic maintenance medium. Osteogenic and chondrogenic differentiation was induced using NH Osteo Diff Medium/NH Chrondro Diff Medium (Miltenyi Biotec Bergisch Gladbach, Germany); the medium was changed twice a week. Analysis was performed after 24 days of cell culture.
Before starting the differentiation, all cells were incubated with 300 µg/mL nanoparticles for 24 h and non-treated samples served as a control. All experiments were also performed in α–MEM without differentiation factors (non-differentiated samples).
Before starting the differentiation, all cells were incubated with 300 µg/mL nanoparticles for 24 h and non-treated samples served as a control. All experiments were also performed in α–MEM without differentiation factors (non-differentiated samples).