Figure 1.
TEM images of MG63 after the uptake of GNPs.
TEM images of MG63 cells after 20 hours of treatment with GNPs at concentrations of 1 ppm (a) and 10 ppm (b). GNPs aggregated within the cytoplamic vesicles shown with arrows.
Figure 2.
Dark-field images of MG63 after uptake of GNPs.
Dark-field (a) scattering and (b) fluorescence images of two MG63 cells undergoing mitosis. Cytoskeletal F-actin (red) are stained with Texas Red-X phalloidin. Cell nuclei (blue) are stained with Hoechst 33258. The bright spots represent endosomes that have enclosed GNPs. Scale bar: 10 µm.
Figure 3.
LSCM fluorescence images of MG63 after uptake of GNPs.
LSCM fluorescence images of (a) MG63 cells treated with GNPs at a concentration of 1 ppm, (b) 10 ppm, and(c) normally cultured cells. Cytoskeletal F-actin (red) are stained with Texas Red-X phalloidin. Cell nuclei (blue) are stained with Hoechst 33258. The green spots in the circled areas are GNPs.
Figure 4.
Effect of GNPs on the differentiation of MG63.
(a) Dark-field hyperspectral image of MG63 treated with GNPs for 21 days; the image shows the specific nodule-like formation. (b) The corresponding scattering spectrum of the marked endosome.
Figure 5.
The effect of GNPs on the viability of MG63.
The viability of MG63 cells treated with GNPs at a concentration of either 1 ppm or 10 ppm for 20 hours and then cultured in fresh medium for 21 days. Data are presented as the mean ± SD (n=9) and were analyzed using the non-parametric Kruskal-Wallis H-test. Differences at p < 0.05 were considered statistically significant.
Figure 6.
Effect of GNPs on the progressive apoptosis of MG63.
The MG63 cells were exposed to H2O2 (control) and GNPs for 20 hours and then cultured for 1 to 8 days: (a) 1 d, (b) 2 d, (c) 4 d and (d) 8 d. Representative dot plots of Annexin V/PI staining are shown. The upper-left quadrant shows the necrotic (Annexin V-/PI+) population. The upper-right quadrant shows the late apoptotic/necrotic (Annexin V+/PI+) population. The lower-left quadrant shows the vital (Annexin V-/PI-) population. The lower-right quadrant shows the early apoptotic (Annexin V+/PI-) population. The result is from one experiment representative of three similar independent experiments. (e) The percentage of viable cells, early apoptotic cells, late apoptotic cells and necrotic cells after being exposed to GNPs for 20 hours and then cultured for up to 8 days. The results were summarized from three separate experiments and are presented as the mean ± SD. Data were analyzed using the non-parametric Kruskal-Wallis H-test. Differences at p < 0.05 were considered statistically significant.
Figure 7.
Real-time PCR analyses of the expressions of three bone-associated genes and alkaline phosphatase activity of MG63 treated with GNPs.
(a) OPN levels, (b) type I collagen levels, and (c) OCN levels. The gene expression levels are normalized against the 18S ribosomal RNA levels. (d) The alkaline phosphatase activity of the cells. Data are presented as the mean ± SD (n=3). Based on statistical analyses, the expression levels of the three specific bone-associated genes and the alkaline phosphatase activity of the MG63 treated with GNPs show no significant difference in comparison with the control group. Data were analyzed using the non-parametric Kruskal-Wallis H-test. Differences at p < 0.05 were considered statistically significant.