Glass coverslips

ASCs differentiation potential into adipocytes, osteoblasts, and chondrocytes, were evaluated in third passage with a commercial medium (Lonza, Walkersville, EUA). The medium inducer was changed every 3 days during 3 weeks. To adipogenic and osteogenic differentiation, cells were seeded on glass coverslips (Sarstedt, Newton, NC, USA) in 24-well plates (Sarsted). Briefly, cells were treated with Bouin's fixative (Biotec, Labmaster, Paraná, Brazil) for 10 min, washed twice with 70% ethanol and once with Milliq water. Oil Red O (Sigma-Aldrich) was used to visualize lipid-rich vacuoles and hematoxylin-eosin (HE) (Biotec) was used for nuclear staining. Osteogenic differentiation was evaluated by Alizarin Red S (Fluka Chemie, Buchs, UK) and light green (Sigma-Aldrich) was used to counterstain. For chondrogenic differentiation, cells were grown in micromass culture. Briefly, 5 × 105 cells in 0.5 ml of medium were centrifuged at 300 g for 10 min in a 15 mL polypropylene tube to form a pellet. Without disturbing the pellet, cells were cultured for 21 days with medium inducer. On day 21, cell aggregates were fixed in 10% formaldehyde for 1 h dehydrated in serial ethanol dilutions and embedded in paraffin blocks. Toluidine Blue staining (Sigma-Aldrich) demonstrate the presence of intracellular matrix proteoglycans. Control cells were kept in DMEM-F12 medium with 15% FCS.