Figure 5
Nanostructured Zirconia substrates prevent β-cells death through modulation of hypoxia and NF-κB pathways. (A) Representative immunofluorescence images of islets triple stained with TUNEL (green), anti-insulin antibody (red) and DAPI (blue). Bar = 30 μm. (B) Quantification of β-cell apoptosis by TUNEL assay in human islets grown on different substrates for 2, 10 and 20 days. β-cell apoptosis represents the percentage of TUNEL- and insulin-double positive cells over total insulin-positive cells; non β-cell apoptosis represents the percentage of TUNEL-positive cells over total DAPI-positive and insulin-negative cells. The experiment was performed in triplicate, with three different islet preparations. A minimum of 100 cells for islet preparation was counted (*p < 0.05 vs gelatin; °p < 0.05 vs flat-ZrO2). (C) Western-blotting analysis of hypoxia and NF-κB pathways selected proteins in islets grown on the indicated substrates for 20 days (15 μg protein/sample). On the right, the proteins molecular weight in kDa is reported. (D) Quantitative analysis of protein expression shows that flat-ZrO2 and ns-ZrOx substrates downregulate the hypoxia and NF-κB pathways. Data (mean values ± S.E.; n = 5 independent experiments) are expressed as fold change over gelatin (dashed line). (*p < 0.05, **p < 0.01, ***p < 0.005, ns-ZrOx vs gelatin. °p < 0.05, °°p < 0.01, ns-ZrOx vs flat-ZrO2).
