Fig. 2

Effects of HA nanoparticles on osteoblasts metabolism and redox activity. Metabolic activity by PrestoBlue assay of HOBIT (A) and MG63 (B) after 1 and 3 days of incubation with particles. 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 < 0.001; ****p < 0.0001) (n = 3). Cell number determined by cell counting of HOBIT (C) and MG63 (D) after 1 and 3 days of incubation with particles. 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 < 0.001; ****p < 0.0001) (n = 3). ROS production was determined using H2-DCFDA as a substrate of HOBIT (E) and MG63 (F) after 1 day of incubation with particles and/or with reduced glutathione (GSH). 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 < 0.001; ****p < 0.0001) (n = 3). PrxSO3 was determined by Western blot of HOBIT (G) and MG63 (H) after 1 day of incubation with particles and with H₂O₂ 1 mM for different timeframes; specifically, HOBIT cells were incubated with H₂O₂ 1 mM for 2, 4, 8 and 10 min, whereas MG63 for 10 min. Tubulin was used to normalize PrxSO3 levels. (I) PrxSO3 was determined by Western blot analyses 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. Tubulin was used to normalize PrxSO3 levels. In all tests, the particle concentration was adjusted to have the same HA concentration in all samples