Abstract
Introduction
Particle-induced oxidative stress in cells is a unifying factor that determines toxicity and carcinogenicity potential in biomaterials. A previous study by Bladen et al. showed the production of significant levels of reactive oxygen species (ROS) following the stimulation of phagocytes by UHMWPE and CoCr wear debris [1]. Latest generation bearing materials such as silicon nitride also need to be tested for potential generation of ROS in phagocytic cells. This study aimed to investigate the production of reactive oxygen species in L929 fibroblasts stimulated with clinically relevant doses of nanoscale and micron-sized silicon nitride (Si3N4) particles, silica nanoparticles, and CoCr wear debris. Silica nanoparticles were included as a comparison material for situations where the Si3N4 particle's surface are oxidised to silicon dioxide [2].
Materials and Methods
Si3N4 particles (<50 nm and <1 µm, Sigma), silica nanopowder (<100 nm, Sigma) and clinically relevant CoCr wear particles were heat-treated at 180°C for 4 h to remove endotoxin. Particles were then re-suspended in sterile water by sonication. L929 murine fibroblasts were cultured with low doses (0.5 µm3/cell) and high doses (50 µm3/cell) of Si3N4 particles, and high doses (50 µm3/cell) of silica nanoparticles and CoCr wear debris. Cells were incubated for three and six days at 37°C with 5% (v/v) CO2. tert-Butyl hydroperoxide (TBHP) was used as a positive control for the production of ROS in the cells. Intracellular ROS was measured using Image-IT LIVE kit (Invitrogen). This assay is based on carboxy-2',7'-dichlorodihydro-fluorescein diacetate (carboxy-H2DCFDA), which forms a non-fluorescent derivative by intracellular esterases and then reacts with intracellular ROS to form green fluoroscence producing derivative carboxy- dichlorodihydro-fluorescein. Images were captured using a confocal microscope and analysed using ImageJ for corrected total cell fluorescence (CTCF). The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc tests.
Results and Discussion
Si3N4 nanoparticles significantly reduced the ROS levels in L929 fibroblasts at low doses (0.5 μm3/cell) and high doses (50 μm3/cell) over a period of six days; whereas no significant change in the levels of ROS was observed in cells treated with micron-sized Si3N4 particles [Figure 1]. Only a few cells treated with high doses of CoCr wear particles (50 μm3/cell) survived for up to six days and produced significantly higher levels of ROS [Figure 1, 2]. Interestingly, cells challenged with high doses (50 μm3/cell) of Si3N4 and silica nanoparticles produced statistically similar levels of ROS in cells [Figure 1]. This might be due to the potential surface oxidation of Si3N4 nanoparticles, which makes their surface chemistry and biological identity similar to silica nanoparticles.
Conclusion
Unlike existing implant materials such as UHMWPE and CoCr, silicon nitride has demonstrated the capacity to reduce or maintain normal levels of ROS in macrophages depending on the particle size and dose.
Acknowledgements
The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. GA-310477 LifeLongJoints.
