CYTOTOXICITY OF CLINICALLY RELEVANT METAL NANOPARTICLES ON MACROPHAGES IN VITRO

Abstract

Despite the satisfactory short-term implant survivor-ship, there is an increasing concern that the metal-on-metal hip resurfacing arthroplasty (MoMHRA) release large amount of very small wear particles and metal ions. The periprosthetic soft-tissue masses such as pseudotumours are being increasingly reported. These were found be locally destructive, requiring revision surgery in most patients. It has been suggested that either an immune reaction or cytotoxic effect of chromium(Cr) or cobalt(Co) may play a role in its aetiology. However, the effect of the phagocytosis of implant-associated metal nanoparticles on macrophages has not been elucidated. The aim of this study was to investigate the in vitro viability and proliferative response of murine macrophages to clinically relevant metal nanoparticles and ions.

Materials and Methods: The RAW 264.7 murine macrophage cell line was cultured in MEM at a seeding density of 10E5 cells/cm2. Culture was set up in the presence of either:(1) negative control: medium alone;(2)Cobalt sulphate heptahyrate and chromium chloride hexahydrate (Sigma) at concentrations of 1uM, 10uM, 100uM;(3)Metal nanoparticles sized 30–35nm (American Elements) of cobalt, chromium and titanium at concentrations from 10E7 to 10E14 particles/ml.

At the end of day 1 and 4, two methods were used to quantify cell proliferation and viability. The AlamarBlue assay(Invitrogen) incorporates a fluorimetric growth indicator and the fluorescence signal correlates with metabolic activity of the cells. LIVE/DEAD stain kit(Molecular Probes) contains two fluorescent dyes to stain living cells green and dead cells red. The viability was calculated by the number of live cells divided by total cell numbers. Inter-group comparisons were performed using one-way ANOVA with Tukey post hoc test. Differences at p< 0.05 were considered to be significant.

Results: Compared with control, Alamar blue assay showed inhibition of cell proliferation in all three metal particles (p< 0.05). The Live/Dead staining showed Co nanoparticles were cytotoxic to most of cells Day 1 and Day 4 at 10E11/mL. At 10E13/mL, the Cr group showed cytotoxicity at day 4 (p< 0.05). There was no difference between Ti and control group. The Co2+ and Cr3+ ions led to inhibition to cell proliferation. At 10uM concentration, Co2+ caused a dramatic decrease in cell number. Live/Dead staining showed that Co2+ were toxic to cells (p< 0.05). Cr3+ group showed cytotoxicity at Day 4 (p< 0.05).

Discussion: This study demonstrates that Co and Cr nanoparticles and ions have dose-dependent proliferation and cytotoxic effects on the macrophages in vitro. The cytotoxicity occurred at the high concentration range that is found in the hip aspirates of MoMHRA patients with pseudotumours. This suggests the formation of pseudotumour may be the local sequelae of cytotoxicity due to increased production of metal wear nanoparticles.