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

Abstract

BackgroundSilver 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).Methods and ResultSilver 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.ConclusionThese 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.

Silver Nanoparticles in Orthopedic Applications: New Insights on Their Effects on Osteogenic Cells

“…Previous studies on human fetal osteoblasts have demonstrated an impairment of cell viability with 30–60 µg/mL Ag NP after 24 and 48 h because of the increase of iNOS [14]. Differently from [14], we did not detect any significant modulation of nitric oxide synthesis after exposure to Ag NP from 0.5 to 100 µg/mL for 24 and 48 h (data not shown). It should be noted that discrepancies could be attributed, at least in part, to the different treatment protocols used, to NP composition, and to their different size.…”

Role of betaine in liver injury induced by the exposure to ionizing radiation

“…The results demonstrate that exposure to ionizing radiation generates an excess of free radicals that contribute to the increase of fibrotic factors. In this context, it has been shown that oxidative stress induces the release of mitochondrial cytochrome c into the cytosol, where it can activate caspases and lead to apoptosis In addition, the apoptosis‐related genes ( p53 and TNF‐α ) are activated by oxidative stress . The increase of ALT, AST, ALP, and GGT, verify that irradiation has affected liver function.…”

Effect of exposure of osteoblast-like cells to low-dose silver nanoparticles: uptake, retention and osteogenic activity

“…However, recent studies have revealed potential health risks associated with use of AgNPs for orthopaedic tissue repair. For example, AgNPs were shown to have cytotoxic effects (induction of cell death, cell-cycle arrest) on osteoblast or osteoblast-like cells [12][13][14]. However, the effects of AgNPs on bone cells and bone formation and their underlying mechanisms are not well characterized.…”