Sml1594

Serum starvation was performed overnight (18 h) and recovered by resupplementation of media containing 10% FCS for 10 min. Where indicated, amino acid starvation was performed for 1 h (after one wash with PBS) in RPMI 1640 without amino acids (US Biologicals), and recovery was achieved by addition of 1× RPMI 1640 amino acid solution (R7131; Sigma-Aldrich) with or without 10% FCS, pH 7.2, for 10 min. Where indicated, cells were incubated for the last 2 h before harvest/fixation with the ROCKi Y-27632 (50 µM; SCM075; Sigma-Aldrich) or the integrin-binding peptide cilengitide (5 µM; SML1594; Sigma-Aldrich). Where indicated, cells were preincubated with 100 nM rapamycin for 2 h.

To test if we could abrogate the effect of the accelerated mineralisation we observed in our cell culture model, we purchased the ITGAV/B3 and ITGAV/B5 integrin inhibitor molecule cilengitide from Sigma Aldrich (SML1594). Using a concentration of 10 μM, that has been previously reported to enhance adipogenesis in the presence of AM without significantly comprising cell proliferation [55 ], cilengitide was added to the OM of shock wave exposed DP and BM-MSCs, as well as the GM of cells not exposed to the shock wave. As a positive control cells were also cultured in OM and GM without the presence of cilengitide. After 14 days in culture, staining for the presence of mineral deposits was performed using Alizarin Red S which is described in detail in the Quantification of osteogenesis section. Viability of BM-MSCs cultured in OM with the addition of 10 μM, or a range of concentrations, including 1 nM, 10 nM, 100 nM, 1 μM and 10 μM were assessed using the previously described method in section Assessment of cell viability and proliferation. Lastly, after 21 days in culture, using the same concentrations of cilengitide as in the viability test, BM-MSCs were assessed for mineral deposition using the same method as described in Quantification of osteogenesis.