Chondrogenic Re-differentiation of Cells

Chondrocyte phenotype was determined under re-differentiation. For chondrogenic re-differentiation, a pellet culture assay with chondrogenic differentiation media (ChM) was performed for 21 days in a 96 deep-well format as described elsewhere [43 (link),44 (link)]. Briefly, 3 × 10⁵ cells per pellet were used, and cultured in ChM composed of high glucose DMEM including L-glutamine (Sigma Aldrich, D6546, USA), dexamethasone (0.1 µM, Sigma Aldrich, D2915, USA), L-ascorbic acid (50 µg/mL, Sigma Aldrich, A8960, USA), L-proline (40 µg/mL, Sigma Aldrich, P5607, St. Louis, MO, USA) sodium pyruvate (0.1 mg/mL, AppliChem, A4859, Darmstadt, Germany) ITS (6.25 µg/mL, Sigma Aldrich, I1884, St. Louis, MO, USA), BSA (1.25 mg/mL, Sigma Aldrich, A9418, St. Louis, MO, USA), linoleic acid (5.35 µg/mL, Sigma Aldrich, L1012, St. Louis, MO, USA), penicillin (10 U/mL)/streptomycin (10 µg/mL) (Biochrom, A2213, Berlin, Germany) and recombinant human TGF-ß1 (10 ng/mL, Peprotech, 100-21-5, Cranbury, NJ, USA). Negative controls were cultured in the same medium but without TGF-ß1. Media change was performed every 3–4 days. Chondrogenic phenotype was determined by proteoglycan assay, type II collagen immunostaining, Alcian blue staining, as well as by expression of type II collagen, type X collagen, MMP13, Sox9 and aggrecan mRNAs.