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

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.

The genotoxicity of physiological concentrations of chromium (Cr(III) and Cr(VI)) and cobalt (Co(II)): an in vitro study.

Humans are exposed to chromium and cobalt in industry, from the environment and after joint replacement surgery from the CoCr alloy in the implant. In this study we have investigated whether Cr(III), Cr(VI), Co(II) and Cr in combination with Co could induce chromosome aberrations in human fibroblasts in vitro at the same concentrations that have been found in the peripheral blood of exposed humans. We used 24 colour M-FISH as a sensitive way to detect translocations and aneuploidy and examined the effects of a 24-h exposure and its consequences up to 30 days after the exposure in order to record genomic instability and/or repair. At these physiological doses the metals induced predominantly numerical rather than structural aberrations. Co was the least reactive and Cr(VI) especially in combination with Co the most. All metals at the highest physiological doses caused simple (gain or loss of 3 or less chromosomes) and complex (more than 49 chromosomes) aneuploidy. All metals at the lowest physiological dose caused a significant increase of total aberrations. Cr(VI) was much more effective than Cr(III) in causing chromosome fragments, which were only induced at the highest doses. There was a slow resolution of aneuploidy with time after exposure. This involved a reduction in the proportion of aneuploid cells and a reduction of the number of chromosomes within cells showing complex aneuploidy. We conclude that these metal ions can cause chromosome aberrations at physiological concentrations and that their main effect is aneugenic.

Thresholds for indirect DNA damage across cellular barriers for orthopaedic biomaterials.

Cobalt-chromium particles and ions can induce indirect DNA damage and chromosome aberrations in human cells on the other side of a cellular barrier in tissue culture. This occurs by intercellular signalling across the barrier. We now show that the threshold for this effect depends on the metal form and the particle composition. Ionic cobalt and chromium induced single strand breaks at concentrations equivalent to those found in the blood of patients with well functioning metal on metal hip prostheses. However, they only caused double strand breaks if the chromium was present as chromium (VI), and did not induce chromosome aberrations. Nanoparticles of cobalt-chromium alloy caused DNA double strand breaks and chromosome aberrations, of which the majority were tetraploidy. Ceramic nanoparticles induced only single strand breaks and/or alkaline labile sites when indirectly exposed to human fibroblasts. The assessment of reproductive risk from maternal exposure to biomaterials is not yet possible with epidemiology. Whilst the barrier model used here differs from the in vivo situation in several respects, it may be useful as a framework to evaluate biomaterial induced damage across physiological barriers.

The in vitro genotoxicity of orthopaedic ceramic (Al2O3) and metal (CoCr alloy) particles.

One of the biggest problems with orthopaedic joint replacements has been the tendency for metal-on-polyethylene implants to produce particulate wear debris. These particles stimulated adjacent macrophage infiltration, which caused destruction of bone and soft tissue, resulting in aseptic loosening of the implant. This problem led to the development of new implants with articulating surfaces that produce less volumetric wear (metal-on-metal, MOM, and ceramic-on-ceramic, COC). To determine whether there could be adverse biological effects from exposure to particulate wear debris after total hip replacement (THR), we investigated the in vitro genotoxic effects of alumina ceramic (Al(2)O(3)) particles in comparison with cobalt-chrome metal (CoCr alloy) particles. Primary human fibroblasts were exposed to Al(2)O(3) nanoparticles or CoCr alloy particles (0.1-10mg/T-75 flask) for 5 days. There were no significant differences in cell viability between control and ceramic-treated cells, at all doses and time-points studied. Cells exposed to CoCr alloy particles showed both dose- and time-dependent cytotoxicity. There was a small but significant increase in micronucleated binucleate cells after 24h of treatment with >1mg/T-75 flask of alumina particulates compared with controls, although no clear dose-response was observed. The induction of micronuclei was unaffected by the size or shape of the ceramic particles. The increase in micronucleated binucleate cells was much greater after exposure to CoCr particles for 24h, showing a clear dose-response curve. No increase in gamma-H2AX foci was noted in cells exposed to ceramic particles, in contrast with a significant increase of these foci in cells exposed to CoCr particles at comparable mass/surface doses. Cytogenetic analysis showed that both types of particle caused mainly numerical rather than structural chromosomal aberrations, with a greater number and variation of lesions induced by CoCr particles. In conclusion, our results show that alumina (Al(2)O(3)) ceramic particles are only weakly genotoxic to human cells in vitro when compared with metal (CoCr alloy) particles.