Chondrogenic Differentiation of MSCs

For chondrogenic differentiation, 2 × 10⁵ MSCs were suspended in 500 µl of chondrogenic differentiation medium composed of high glucose Dulbecco’s Modified Eagle Medium supplemented with 1.5 µg/ml fungizone, 50 µg/ml gentamicin, 1 mM sodium pyruvate (All Thermo Fisher Scientific), 1% v/v Insulin-Transferrin-Selenous acid (ITS™+ Premix, Corning, Bedford, MA, USA), 40 µg/ml proline (Sigma-Aldrich), 25 µg/ml L-ascorbic acid 2-phosphate, 100 nM Dexamethasone (Sigma-Aldrich), and 10 ng/ml transforming growth factor-β3 (R&D systems, Minneapolis, MN, USA). The cell suspension was added to 15 ml conical polypropylene tubes (VWR, Radnor, PA, USA) and centrifuged at 300 g for 8 min to facilitate pellet formation. Chondrogenic MSC pellets were cultured at 37°C and 5% carbon dioxide in a humidified atmosphere, and culture medium was refreshed every 3–4 days for 21 days. Chondrogenic differentiation of MSCs in vitro following 21 days of culture was confirmed histologically by thionine staining ( Supplementary Figure S1 ).