Fig. 3
Effects of HA nanoparticles on osteoblasts DNA damage after 1 day of incubation with particles. (A) COMET assay of HOBIT cells after 1 day of incubation with particles and with H2O2 as a positive control. The scale bar is 100 μm. (B) Immunofluorescence analyses of γH2A.X foci (red). Nuclei were visualized with DAPI (blue). The scale bar is 20 μm. (C) Confocal microscopy determined the number of olive tail moments per cell of randomly selected HOBIT cells after incubation with particles and GSH for 24 h and with H2O2 300 µM for 15 min. Individual values are shown as mean ± SD. The resulting p-value is indicated (*p < 0.05; **p < 0.01; ****p < 0.0001). (D) The number of γH2AX dots per cell of MG63 cells determined by confocal microscopy after incubation with particles and with H2O2 1 mM for 30 min. The resulting p-value is indicated (**p < 0.01; ****p < 0.0001). γH2AX determined by Western blot of HOBIT (E) and MG63 (F). Tubulin was used to normalize γH2AX levels. Densitometric analysis of γH2AX protein expression of HOBIT (G) and MG63 (H) normalized to tubulin. Fold change values relative to untreated cells are shown. Values are mean ± SD. The resulting p-value is indicated (*p < 0.05; **p < 0.01; ****p < 0.0001) (n = 3). (I) γH2AX determined by Western blot of HOBIT after incubation with FeHA/CH particles for different timeframes (1, 2, 4, 8, 16, and 24 h) and with H2O2 1 mM for 10 min