Methods

Clinically relevant sterile UHMWPE, CoCr, and Ti64 wear particles were generated in a pin-on-plate wear simulator. Whole peripheral blood was collected from healthy human donors (ethics approval BIOSCI 10–108, University of Leeds). The PBMNCs were isolated using Lymphoprep (Stemcell, UK) and seeded into the wells of 96-well and 384-well cell culture plates. The plates were then incubated for 24 h in 5% (v/v) CO₂ at 37°C to allow the attachment of mononuclear phagocytes.

Adherent phagocytes were incubated with UHMWPE and CoCr wear debris at volumetric concentrations of 0.5 to 100 µm³ particles per cell for 24 h in 5% (v/v) CO₂ at 37°C. During the incubation of cells with particles, for each assay, two identical plates were set up in two configurations (one upright and one inverted). After incubation, cell viability was measured using the ATPlite assay (Perkin Elmer, UK). Intracellular oxidative stress was measured using the DCFDA-based reactive oxygen species detection assay (Abcam, UK). TNF-α cytokine was measured using sandwich ELISA. DNA damage was measured by alkaline comet 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

Cellular uptake of UHMWPE, CoCr and Ti64 particles was confirmed by optical microscopy. PBMNCs incubated with UHMWPE particles did not show any adverse responses except the release of significant levels of TNF-α cytokine at 100 µm³ particles per cell, when in contact with particles. PBMNCs incubated with CoCr wear particles showed adverse responses at high particle doses (100 µm³ particles per cell) for all the assays. Moreover, cytotoxicity was observed to be a combined effect of both particles and ions, whereas oxidative stress and DNA damage were mostly caused by ions. Ti64 wear particles did not show any adverse responses except cytotoxicity at high particle doses (100 µm³ particles per cell). Moreover, this cytotoxicity was mostly found to be a particle effect. In conclusion, the novel cell culture system is suitable for evaluating the biological impact of orthopaedic wear particles and ions, separately.

Methods

Commercial silicon nitride particles of <50nm (Sigma Aldrich) were incubated with formalin fixed sheep synovium at a volume of 0.01mm³ /g of tissue (n=3). The tissue was digested with papain (1.56mg/ml) for 6h and subsequently proteinase K (1mg/ml) overnight. Proteinase K digestion was repeated for 6h and again overnight, after which samples appeared visibly homogeneous [Figure 1]. Samples were then subjected to density gradient ultracentrifugation using sodium polytungstate (SPT) [3]. The resulting protein band was removed from the pellet of particles. Control tissue samples, to which no particles were added, were also subjected to the procedure. Particles were washed with filtered water to remove residual SPT using ultracentrifugation and filtered onto 15nm polycarbonate filters. The filtered particles were imaged by cold field emission scanning electron microscopy (CFE-SEM) and positively identified by elemental analysis before and after the isolation procedure. To validate whether the isolation method affected particle size or morphology, imaging software (imageJ) was used to determine size distributions and morphological parameters of the particles. A Kolmogorov-Smirnov test was used to statistically analyse the particle morphology.

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.

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.

Methods

SiN nanopowder (<50nm, Sigma UK) and CoCr wear particles (nanoscale, generated in a multidirectional pin-on-plate reciprocator) were heat-treated for 4 h at 180°C and dispersed by sonication for 10 min prior to their use in cell culture experiments. Whole peripheral blood was collected from healthy donors (ethics approval BIOSCI 10–108, University of Leeds). The PBMNCs were isolated using Lymphoprep® as a density gradient medium and incubated for 24 h in 5% (v/v) CO2at 37°C to allow attachment of mononuclear phagocytes. SiN and CoCr particles were then added to the phagocytes at a volume concentration of 50 µm3 particles per cell and cultured for 24 h in RPMI-1640 culture medium in 5% (v/v) CO2 at 37°C. Cells alone were used as a negative control and lipopolysaccharide (LPS; 200ng/ml) was used as a positive control. Cell viability was measured after 24 h by ATPLite assay and tumour necrosis factor alpha (TNF-α) release was measured by sandwich ELISA. U937s were co-cultured with SiN and CoCr particles at doses of 0.05, 0.5, 5 and 50 µm3 particles per cell for 24h in 5% (v/v) CO2 at 37 C. Cells alone were used as a negative control and camptothecin (2 µg/ml) was used as a positive control. Cell viability was measured after 0, 1, 3, 6 and 9 days. Results from cell viability assays and TNF-α response were expressed as mean ±95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc analysis.

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.