Abstract
Introduction
Currently, different techniques to evaluate biocompatibility of orthopaedic materials, including two-dimensional (2D) cell culture for metal and ceramic wear debris and floating 2D surfaces or three-dimensional (3D) agarose gels for UHMWPE wear debris, are used. We have developed a single method using 3D agarose gels that is suitable to test the biocompatibility of all three types of wear debris simultaneously. Moreover, stimulation of the cells by wear particles embedded in a 3D gel better mimics the in vivo environment.
Materials and Methods
Clinically relevant sterile UHMWPE and CoCr wear particles were generated using methodologies described previously [1,2]. Commercially available nanoscale and micron-sized silicon nitride (Si3N4) particles (<50 nm and <1 μm, Sigma UK) were sterilised by heat treatment for 4h at 180°C. Agarose-particle suspensions were prepared by mixing warm 2% (w/v) low-melting-point agarose solution with the particles dispersed by sonication in DMEM culture media. The suspensions were then allowed to set at room temperature for 10 min in 96 well culture plates. Sub-confluent L929 murine fibroblasts were cultured on the prepared gels for up to 6 days in 5% (v/v) CO2 at 37°C. After incubation, the viability of cells was measured using the ATP-lite assay. The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc analysis.
Results and Discussion
The gels were observed to ensure uniform distribution of particles and migration of cells into the gel. No significant reduction in viability was observed for nanoscale and micron-sized Si3N4 particles at low doses (0.5 μm3 per cell) and high doses (50 μm3 per cell), or for UHMWPE wear debris at high doses (100 μm3 per cell) [Figure1]. Moreover, the viability was significantly reduced for high doses of CoCr wear debris (50 μm3 per cell) and the positive control, camptothecin (2 μg.ml−1) at day 6 [Figure1]. These results are consistent with the literature [2,3] and therefore validate our 3D agarose cell culture method for comparing cytotoxicity of polymer, metal and ceramic particles in a single assay, simultaneously.
Conclusion
Biocompatibility ofpolymer, metal and ceramic wear debris can be tested simultaneously by using 3D particle embedded agarose gels.
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.
