Chondrogenic Differentiation of BM-MSCs in ECM-Supplemented Media

2.5 ​× ​10⁵ BM-MSCs were cultured in a micromass pellet system as previously described [40 (link),41 (link)]. Pellets were cultured either in a growth factor free chondrogenic differentiation medium (CDM-) which consisted of high glucose DMEM supplemented with 1% penicillin-streptomycin, 100 ​μg/ml sodium pyruvate (Sigma), 40 ​μg/ml l-Proline (Sigma), 50 ​μg/ml l-ascorbic acid-2-phosphate (Sigma), 1.5 ​mg/ml bovine serum albumin (BSA-Sigma), 1× insulin transferrin selenium (ITS- Gibco), 100 ​nM dexamethasone (Sigma) for 28 days. This media was supplemented with 0.05 ​mg/ml of solubilised AC-ECM or LIG-ECM during twice weekly media changes. As a positive control, pellets were also cultured in CDM ​+ ​that consists of the CDM formulation above plus 10 ​ng/ml transforming growth factor beta-3 (TGF-β3 –PeproTech). sGAG quantification was performed using a 1, 9 dimethylmethylene blue (DMMB) assay according to the manufactures protocol (Blyscan sulfated sGAG assay kit, Biocolor).