Pretreatment of hFOB 1.19 with L-NIL attenuated AgNPs-induced cell death.

<p><b>A representative dot plot of flow cytometry.</b> A representative dot plot of flow cytometry. AgNPs induced apoptosis and necrosis in hFOB 1.19 cells an effect attenuated by L-NIL. Viable cells are shown in the lower left field (low Annexin V and PI staining; AV- PI-). The lower right field (AV+ PI-) represents the apoptotic cells, and the higher right field (AV+ PI+) indicates late apoptotic/necrotic cells. The higher left field (AV- PI+) shows the dead cells. L-NIL significantly attenuated number of apoptotic and dead cells.</p

Molecular Mechanism of Silver Nanoparticles-Induced Human Osteoblast Cell Death: Protective Effect of Inducible Nitric Oxide Synthase Inhibitor

<div><p>Background</p><p>Silver nanoparticles (AgNPs) show strong antibacterial properties, making them excellent candidates to be used in orthopaedic repair and regeneration. However, there are concerns regarding the cytotoxicity of AgNPs and molecular mechanisms underlying AgNPs-induced bone cells toxicity have not been elucidated. Therefore, the aim of our study was to explore mechanisms of AgNPs-induced osteoblast cell death with particular emphasis on the role of nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS).</p><p>Methods and Result</p><p>Silver nanoparticles used in this study were 18.3±2.6 nm in size, uncoated, spherical, regular shape and their zeta potential was -29.1±2.4 mV as measured by transmission electron microscopy (TEM) and zetasizer. The release of silver (Ag) from AgNPs was measured in cell culture medium by atomic absorption spectroscopy (AAS). The exposure of human osteoblast cells (hFOB 1.19) to AgNPs at concentration of 30 or 60 μg/mL for 24 or 48 hours, respectively resulted in cellular uptake of AgNPs and changes in cell ultrastructure. These changes were associated with apoptosis and necrosis as shown by flow cytometry and lactate dehydrogenase (LDH) assay as well as increased levels of pro-apoptotic Bax and decreased levels of anti-apoptotic Bcl-2 mRNA and protein. Importantly, we have found that AgNPs elevated the levels of nitric oxide (NO) with concomitant upregulation of inducible nitric oxide synthase (iNOS) mRNA and protein. A significant positive correlation was observed between the concentration of AgNPs and iNOS at protein and mRNA level (r = 0.837, r = 0.721, respectively; p<0.001). Finally, preincubation of osteoblast cells with N-iminoethyl-l-lysine (L-NIL), a selective iNOS inhibitor, as well as treating cells with iNOS small interfering RNAs (siRNA) significantly attenuated AgNPs-induced apoptosis and necrosis. Moreover, we have found that AgNPs-induced cells death is not related to Ag dissolution is cell culture medium.</p><p>Conclusion</p><p>These results unambiguously demonstrate that increased expression of iNOS and generation of NO as well as NO-derived reactive species is involved in AgNPs-induced osteoblast cell death. Our findings may help in development of new strategies to protect bone from AgNPs-induced cytotoxicity and increase the safety of orthopaedic tissue repair.</p></div

AgNPs-induced necrosis in hFOB 1.19 cells and protective effect of L-NIL.

<p>Nec-1 failed to protect cells from AgNPs-induced cell death. Data are expressed as means ± SD of 3 independent experiments. *p<0.05; ***p<0.001 exposed cells v/s control or as indicated.</p

AgNPs-induced generation of nitrotyrosine in hFOB 1.19 and its inhibition by L-NIL.

<p>(A) A bar graph showing data as measured by ELISA; (B) Representative immunoblots. Data are expressed as means ± SD of 3 independent experiments. *p<0.05; **p<0.01 exposed cells v/s control or as indicated.</p

The TEM examination of hFOB 1.19.

<p>(A) The ultrastructural features of a control cell; mitochondria (M), nucleus (N), Golgi complex (Ga); (B-C) Cells exposed to AgNPs 30 μg/mL for 24 h. cell. (B) AgNPs are found within the cell (arrows). Micrographs show vacuoles (V) with randomly oriented AgNPs of quite regular morphology (arrows). The well-developed Golgi complex (Ga) is located in the cytoplasm in perinuclear areas. (C) Cells exposed to AgNPs 30 μg/mL for 24 h show swelling of the endoplasmic reticulum. Ribosomes may have dissociated from the endoplasmic reticulum (RER) and the cytoplasm is filled with free ribosomes. The nucleus (N) presents a normal aspect.</p

Evaluation of cytotoxicity of AgNPs and Ag released in cell medium from 30 and 60 μg/mL AgNPs on hFOB 1.19 cells.

<p>Results are expressed as % LDH release by cells into the culture medium and presented as mean ± standard deviation of 3 independent experiment. ***p<0.001 exposed cells v/s control.</p

The summary characteristics of AgNPs.

<p>The summary characteristics of AgNPs.</p

AgNPs-induced apoptosis in hFOB 1.19 cells and protective effect of L-NIL.

<p>(A) AgNPs-induced apoptosis, (B) Bax, Bcl-2 protein and (C) Bax, Bcl-2 mRNA levels. L-NIL significantly attenuated: (A) number of apoptotic cells, (B) Bax protein, (C) mRNA level and significantly increased: (B) Bcl-2 protein, (C) mRNA level. Data are expressed as means ± SD of 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 exposed cells v/s control or as indicated.</p

Characterization of AgNPs using transmission electron microscopy (TEM).

<p>(A) A representative microscopy image shows shape of AgNPs. (B) The histogram illustrates the range of particle size distribution with the mean size of 18.3 nm obtained from TEM measurements of 200 particles.</p

Transfection of hFOB 1.19 with iNOS siRNA prevents AgNPs-induced cell death.

<p>The hFOB 1.19 cells were transfected with either 50 nM iNOS or control siRNA for 24 h, then cells were exposed for 48 h to 60 μg/mL AgNPs (the highest working concentration). (A) A representative immunoblot demonstrates efficient of transfection of iNOS siRNA. iNOS siRNA prevents AgNPs-induced increase of (B) NO, (C) NT, (D) apoptosis level and attenuated AgNPs-induced (E) cell death. Data are expressed as means ± SD of 3 independent experiments. **p<0.01; ***p<0.001 exposed cells v/s control or as indicated.</p