Fig. 3.
CeONP60/40 and CeONP20/80 pre-treatment to primary hBMSCs reduces IR-induced DNA damage and cellular senescence. [A] Representative confocal micrographs showing DNA damage 3 days after irradiation and following analysis using the Comet Assay®. hBMSCs were pre-treated with either 1 μg/mL or 10 μg/mL of CeONPs for 24 h prior to a single X-ray exposure to 7 Gy. A significant reduction in DNA damage is observed following treatment with both nanozymes and at both concentrations when compared with the untreated control hBMSCs. Images were captured using confocal laser scanning microscopy. [B] Quantification of comet length: ****p < 0.0001. [C] Quantification of tail length: *p < 0.05, ****p < 0.0001. [D] Quantification of tail DNA: *p < 0.05, **p < 0.01. All values are given as the mean ± SD. [E] Representative micrographs of SA‐β‐gal staining for senescent cells (green) following exposure to IR and with or without CeONP pre-treatment. Using an inverted phase microscope, results show fewer IR-induced senescent cells following pre-treatment with CeONPs at both 1 μg/mL and 10 μg/mL concentrations, and after 28-days of culture. The CeONPs were replenished in the media following irradiation and experiments were carried out in triplicate. Black arrows indicate senescent cells. [F] Representative micrographs following staining for ALP (dark blue) after 14 days of culture in osteogenic media. Qualitative analysis indicates increased levels of ALP in cells treated with 1 and 10 μg/mL of CeONP60/40 and CeONP20/80 when compared with the untreated control cell group. [G] Expression of osteogenesis-related genes (ALP, SP7, Col I, and OCN) following X-Ray exposure. *p < 0.05, **p < 0.01, ****p < 0.0001. [H] Western blot analysis of ALP. Endogenous α-Tubulin expression was shown as control. *p < 0.05, ***p < 0.001. [I] The deposition of mineral nodules was qualitatively investigated using Alizarin Red Staining. Areas of red indicate regions of mineralization following 28-days of culture. [J] Mineral deposition by cells was quantified following X-Ray exposure in treated and untreated cells. [K] HIF-1α expression. The level of HIF-1α expression was measured in primary hBMSCs 24h following exposure to 7 Gy. Protein levels were determined using a HIF1α human ELISA kit. The CeONPs were replenished in the media following irradiation. Experiments were carried out in triplicate. All values are given as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.