Influence of particle size and reactive oxygen species on cobalt chrome nanoparticle-mediated genotoxicity

Abstract

Patients with cobalt chrome (CoCr) metal-on-metal (MOM) implants may be exposed to a wide size range of metallic nanoparticles as a result of wear. In this study we have characterised the biological responses of human fibroblasts to two types of synthetically derived CoCr particles [(a) from a tribometer (30 nm) and (b) thermal plasma technology (20, 35, and 80 nm)] in vitro, testing their dependence on nanoparticle size or the generation of oxygen free radicals, or both. Metal ions were released from the surface of nanoparticles, particularly from larger (80 nm) particles generated by thermal plasma technology. Exposure of fibroblasts to these nanoparticles triggered rapid (2 h) generation of reactive oxygen species (ROS) that could be eliminated by inhibition of NADPH oxidase, suggesting that it was mediated by phagocytosis of the particles. The exposure also caused a more prolonged, MitoQ sensitive production of ROS (24 h), suggesting involvement of mitochondria. Consequently, we recorded elevated levels of aneuploidy, chromosome clumping, fragmentation of mitochondria and damage to the cytoskeleton particularly to the microtubule network. Exposure to the nanoparticles resulted in misshapen nuclei, disruption of mature lamin B1 and increased nucleoplasmic bridges, which could be prevented by MitoQ. In addition, increased numbers of micronuclei were observed and these were only partly prevented by MitoQ, and the incidence of micronuclei and ion release from the nanoparticles were positively correlated with nanoparticle size, although the cytogenetic changes, modifications in nuclear shape and the amount of ROS were not. These results suggest that cells exhibit diverse mitochondrial ROS-dependent and independent responses to CoCr particles, and that nanoparticle size and the amount of metal ion released are influential.

Introduction

Orthopaedic patients with metal-on-metal (MoM) hip replacements are exposed to CoCr nanoparticles as a result of wear of the implant [1], [2], [3], [4]. Approximately 6.7 × 10¹²–2.5 × 10¹⁴ particles (generally <50 nm) are generated by articulating CoCr surfaces in each patient every year [5], [6]. Calls have been made for the establishment and validation of material characterisation protocols and biological testing methodologies to understand the potential toxicity of these nanoparticles. There is also an increase of circulating metal ions in the blood of these patients [5], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], who may be exposed to metal for up to 60 years after surgery. This internal surgical exposure to nanoparticles is different from the external exposure (such as environmental pollution/inhalation) as it bypasses many of the body's natural defences, for example by macrophage uptake or contact with fluids in the airways before entering the body.

While it is not known whether the release of Co and Cr ions is essentially from the whole implant, or by corrosion of wear debris and/or both; the mechanisms of Co²⁺ [18], [19], [20], [21], and Cr ions (trivalent and hexavalent) [22], [23], [24], [25], [26], [27] toxicity in vitro is well understood with oxidative stress mediated cyto-and genotoxicity playing a major role. We have previously observed increases in aneuploidy in peripheral blood lymphocytes of patients with CoCr-on-CoCr wear debris [28]. We also demonstrated that CoCr nanoparticles (30 nm) which were generated by a pin-on-plate tribometer were significantly more toxic than commercially available micron sized (2.9 μm) CoCr particles in vitro [29], demonstrating that CoCr particle size may be a key factor governing toxicity. Importantly, the CoCr wear debris generated in situ from MoM implants is often a heterogeneous mix of nanoscale particles.

Size dependent but concentration independent toxicity of nanoparticles has been demonstrated previously for other metal oxides such as TiO₂, CuO, ZnO [30], [31]. There is very little known about the mechanisms of toxicity of differentially sized CoCr particles within the nano scale range. The toxicity of metal oxides may also be dependent on the extent of oxidative stress [32], [33], [34]. In this study, we have investigated whether there is a relationship between the size of the CoCr nanoparticle and the amount of genotoxicity. We have used a variety of assays to explore this. We have also explored the role of oxidative stress in the genotoxic response.