Fig. 5

Selective inhibition of NTPDase3 activity with the pharmacological inhibitor, PSB 06126 (3 µM; panels A–C), or with the monoclonal antibody, hN3-B3_S (0.5 µg/ml; panels D–F), facilitates osteogenic differentiation and mineralization of cultured BM-MSCs from Pm women via the activation of P2X7 and/or P2Y₆ receptors. Each inhibitor was added to osteogenic-inducing media at culture day 0 and remained throughout the assay, i.e. until day 35. Panels A and D show the per cent variation of the ALP activity of Pm BM-MSC cultures on days 7 and 21 corrected for cells growth/viability (MTT assay) determined in the presence of NTPDase3 inhibitors versus the control situation with no added drugs (100%; 9.6 ± 2.6 and 18.3 ± 4.0 nmol/min/MTT at days 7 and 21, respectively). Panels B and E show the per cent variation of the total mineralized area of Pm BM-MSC at culture day 35 in the presence of NTPDase3 inhibitors compared to the control situation with no added drugs (100%; 10,079.2 ± 4210.5 µm²). ^§P < 0.05 (Wilcoxon signed rank test for comparing medians with a hypothetical null variation, 100%) represent significant differences. Involvement of P2X7 and P2Y₆ purinoceptors activation in extracellular matrix mineralization was confirmed using selective antagonists, A438079 (3 µM) and MRS 2578 (100 nM), respectively, which were added to culture media synchronously to NTPDase3 inhibitors. Panels C and F show Alizarin Red staining indicating extracellular matrix mineralization and bone nodule formation of BM-MSC cultures (red-brownish spots) in two Pm women; the scale bar is 100 µm. Boxes and whiskers represent pooled data from four to six Pm women (70 ± 3 years old); three to eight replicas were made per individual experiment. *P < 0.05 and **P < 0.01 (one-way ANOVA with Sidák’s multiple comparison test, single pooled variance) represent significant differences