Chondrogenic Differentiation of BM-MSCs

Donor-matched IC-BM and VB-BM MSCs (expanded for three passages) were seeded at 2.5x10⁵ per conical tube for the chondrogenic assays. Triplicates were used for quantitative measurements of glycosaminoglycan (GAG) levels and duplicates for the GAG staining. As previously described [32 (link)], cells were cultured in chondrogenic media, consisting of high glucose DMEM (Thermo Fisher Scientific) supplemented with; l-ascorbic acid-2-phosphate, sodium pyruvate, proline, Bovine serum albumin, penicillin/streptomycin, dexamethasone, insulin-transferrin-selenium (all from Sigma-Aldrich) and TGFβ3 (R&D Systems, Abingdon, UK). Following 21 days of culture, cell pellets were digested in papain solution (100mM Sodium Phosphate Buffer supplemented with 5mM Na₂EDTA, 10mM l-cysteine and papain, all from Sigma) and the levels of GAG were measured using a Blyscan™ kit (Biocolor Life Sciences, Co Antrim, Ireland) as per manufacturer instructions. For the GAG staining, the cells were treated with 1% toluidine blue (Sigma-Aldrich) then the images for GAG-stained cells were captured using an Eclipse E1000 light microscope (Nikon, Surrey, UK).