Fig. 6

Load-induced release of NO, PGE₂ and relevance of NFκB for SOX9 and ECM synthesis. Conditioned medium supernatant (20 h) from AC- and MSC-derived neocartilage compressed on day 21 and from control samples was analyzed for A nitrite concentration by the Griess test (n = 6–9 per group) and B PGE₂ content by ELISA. PGE₂ data were log₂ transformed to cope with the more than 100-fold different levels of AC- and MSC-derived samples (n = 6–9). C, D MSC-derived cartilage was treated with 0.75 µM of the IκBα phosphorylation inhibitor Bay11-7082 (Bay11) or DMSO starting 45 h before beginning of loading on day 21. Changes in GAG synthesis per DNA were determined by ³⁵S-sulfate incorporation and changes in collagen protein synthesis per DNA were determined by ³H-proline incorporation over 24 h following termination of loading (n = 4). E Regulation of SOX9 protein levels in MSC-derived neocartilage treated by the NFκB inhibitor or DMSO and subjected to loading was determined by Western blot analysis using β-actin as a loading control. F MSC-derived cartilage pre-cultured for 21 days was subjected to dynamic loading (3 h) or non-loaded samples were instead treated with 10 ng/ml IL1-β for 3 h before changes in GAG synthesis and collagen synthesis were determined by ³⁵S-sulfate or ³H-proline incorporation, respectively. Values were normalized to the DNA content and data are depicted relative to non-loaded and non-treated samples set to 1 (dashed line, n = 3–4 donors). G PGE₂ content in conditioned medium supernatants (20 h) was determined by ELISA in controls and after 3 h of cyclic compression or stimulation with 10 ng/mL IL1-β (n = 3–4). T-test with Bonferroni correction, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compressed vs. controls; #p ≤ 0.05, ##p ≤ 0.01, ### p ≤ 0.001 AC versus MSC