Fig. 2

Assay of cell proliferation ATP activity, GAG content and ALP activity after 21 days of chondrogenic differentiation in pellet cultures derived from five different donors. Pellet cell cultures containing mesenchymal stromal cells (MSCs) were incubated in chondrogenic differentiation medium containing 500 ng/ml and 1000 ng/ml of the mutant R57A, 500 ng/ml and 1000 ng/ml of growth and differentiation factor 5 (GDF-5) or transforming growth factor beta (TGF-β)1 for 21 days to induce chondrogenesis. Untreated cultures treated with standard cell culture medium were maintained as negative controls. Adenosine Triphosphate (ATP)-Assays (a) were performed to examine cell proliferation after 3, 7, 14 and 21 days. Glycosaminoglycan (GAG)-Assays (b) were conducted to examine the quantitative synthesis of the chondrogenic marker protein GAG in respective pellets after 3, 7, 14 and 21 days. In addition, alkaline phosphatase (ALP)-Assays (c) were performed to evaluate relative ALP-activity after 3, 7, 14 and 21 days. Asterixis (*) represent significant differences (P < 0.05) compared to negative control samples at this particular time. The respective group of pictured bars refer to MSCs treated as controls, with R57A 500 ng/ml, with R57A 1000 ng/ml, with GDF-5 500 ng/ml, with GDF-5 1000 ng/ml and TGF-ß1 10 ng/ml (from left to right). ATP, Adenosine Triphosphate; ALP, alkaline phosphatase; GAG, Glycosaminoglycan; GDF-5, growth and differentiation factor 5; MSCs, mesenchymal stromal cells; TGF-ß, transforming growth factor beta