Abstract
Introduction
Significant reduction in the wear of current orthopaedic bearing materials has made it challenging to isolate wear debris from simulator lubricants. Ceramics such as silicon nitride (SiN), as well as ceramic-like surface coatings on metal substrates have been explored as potential alternatives to conventional implant materials. Current isolation methods were designed for isolating conventional metal, UHMWPE and ceramic wear debris. The objective of this study was to develop methodology for isolation and characterisation of modern ceramic or ceramic-like coating particles and metal wear particles from serum lubricants under ultra-low wearing conditions. Sodium polytungstate (SPT) was used as a novel density gradient medium due to its properties, such as high water solubility, the fact that it is non-toxic and acts as a protein denaturant, coupled with a large density range of 1.1–3.0 g/cm3 in water.
Methods
SiN nanoparticles (<50nm nanopowder, Sigma-Aldrich) and clinically relevant cobalt-chromium wear debris were added to 25% (v/v) bovine serum lubricant at concentrations of 0.03 and 0.3 mm3/ million cycles. The particles were isolated by a newly developed method using SPT gradients. The sample volume was reduced by centrifuging the lubricant at 160,000 g for 3 h at 20°C. Then, re-suspended pellet was digested twice with 0.5 mg/ml proteinse K for 18 hours at 50°C in the presence of 0.5% (w/v) SDS. Particles were then isolated from partially hydrolysed proteins by density gradient ultracentrifugation at 270,000 g for 4 h using SPT gradients [Figure 1]. At the end of centrifugation, particles were pelleted at the bottom of the centrifuge tube, leaving protein fragments and other impurities suspended higher up the tube. Isolated particles were then washed with pyrogen free water, dispersed by sonication and filtered through 15 nm polycarbonate membrane filters for SEM and EDX analysis.
Results and Discussion
The morphology and size distribution of the SiN and cobalt-chromium particles was not altered after isolation [Figure 2] [Figure 3]. The mode size of the SiN particles was 30–40 nm, while the mode size of cobalt-chromium particles was 10–20 nm [Figure 3]. Unlike current isolation methods, the present study developed a highly sensitive method which uses cost effective commercially available reagents and components. Furthermore, the particles are recovered in solution and can be readily analysed using commercial size analysers, prior to use in cell studies. This study also confirmed the aggregation characteristics of silicon nitride particles in aqueous medium as observed in previous studies. The above method may also be used to isolate wear debris of materials that have density higher than 1.6 g/cm3. This includes the majority of ceramics, metals and ceramic-like coatings used in TJR components such as alumina, zirconia toughened alumina, titanium, chromium nitride coating, titanium nitride coating and chromium carbon nitride coating.
Conclusions
The new isolation method successfully isolated silicon nitride nanoparticles and cobalt-chromium wear debris from serum lubricant at ultra-low concentrations of 0.03 mm3/million cycles.
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.
