Currently, different techniques to evaluate the biocompatibility of orthopaedic materials, including two-dimensional (2D) cell culture for metal/ceramic wear debris and floating 2D surfaces or three-dimensional (3D) agarose gels for UHMWPE wear debris, are used. Moreover, cell culture systems evaluate the biological responses of cells to a biomaterial as the combined effect of both particles and ions. We have developed a novel cell culture system suitable for testing the all three type of particles and ions, separately. The method was tested by evaluating the biological responses of human peripheral blood mononuclear cells (PBMNCs) to UHMWPE, cobalt-chromium alloy (CoCr), and Ti64 alloy wear particles. 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. 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
Results and Discussion
Total ankle replacement (TAR) is less successful than other joint replacements with a 77% survivorship at 10 years. Predominant indications for revision include: Insert dislocation, soft tissue impingement and pain/stiffness. Insert edge-loading may be both a product and cause of these indications and was reported to affect 22% of patients with the, now withdrawn from market, Ankle Evolutive System (AES) TAR (Transysteme, Nimes, France). Compressive forces up to seven times body weight over a relatively small contact area (∼6.0 to 9.2 cm2), in combination with multi-directional motion potentially causes significant polyethylene wear and deformation in mobile-bearing TAR designs. Direct methods of measuring component volume (e.g. pycnometer) use Archimedes' principle but cannot identify spatial changes in volume or form indicative of wear/deformation. Quantitative methods for surface analysis bridge this limitation and may advance methods for analysing the edge loading phenomena in TAR. Determine the frequency of edge loading in a cohort of explanted total ankle replacements and compare the quantitative surface characteristics using a novel explant analysis method.Introduction
Aim
Wear debris generated by total hip replacements (THRs) may cause mechanical instability, inflammation, osteolysis and ultimately implant loosening, thus limiting the lifetime of such devices [1]. This has led to the development of biocompatible coatings for prostheses. Silicon nitride (SiN) coatings are highly wear resistant and any resultant wear debris are soluble, reducing the possibility of a chronic inflammatory reaction [2]. SiN wear debris produced from coatings have not been characterized 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.Introduction
Methods
Surgical interventions for the treatment of chronic neck pain, which affects 330 million people globally, include fusion and cervical total disc replacement (CTDR). Most of the currently clinically available CTDRs designs include a metal-on-polymer (MoP) bearing. Numerous studies suggest that MoP CTDRs are associated with issues similar to those affecting other MoP joint replacement devices, including excessive wear and wear particle-related inflammation and osteolysis. A standard ISO testing protocol was employed to investigate a device with a metal-on-metal (MoM) bearing. Moreover, with findings in the literature suggesting that the testing protocol specified by ISO-18192-1 may result in overestimated wear rates, additional tests with reduced kinematics were conducted. Six MoM CTDRs made from high carbon cobalt-chromium (CoCr) were tested in a six-axis spine simulator, under the ISO-18192-1 protocol for a duration of 4 million cycles (MC), followed by 2MC of modified testing conditions, which applied the same axial force as specified in ISO-18192-1 (50-150N), but reduced ranges of motion (ROM) i.e. ±3° flexion/extension (reduced from ±7.5°) and ±2° lateral bending (reduced from ±6°) and axial rotation (reduced from ±4°). Foetal bovine serum (25% v/v), used as a lubricant, was changed every 3.3×105 cycles and stored at −20°C for particle analysis. Components were measured after each 1×106 cycles; surface roughness, damage modes and gravimetric wear were assessed. The wear and roughness data was presented as mean ±95% confidence interval and was analysed by one-way analysis of variance (ANOVA) (p=0.05). The mean wear rate of the MoM CTDRs tested under the ISO protocol was 0.246 ± 0.054mm3/MC, with the total volume of wear of 0.977 ± 0.102mm3 lost over the test duration (Fig. 1). The modified testing protocol resulted in a significantly lower mean volumetric wear rate of 0.039 ± 0.015mm3/MC (p=0.002), with a total wear volume of 0.078 ± 0.036mm3lost over the 2MC test duration. Under both test conditions, the volumetric wear was linear; with no significant bedding-in period observed (Fig. 1). The mean pre-test surface roughness decreased from 0.019 ± 0.03µm to 0.012 ± 0.002µm (p=0.001) after 4MC of testing, however surface roughness increased to 0.015 ± 0.002µm (p=0.009) after the additional 2MC of modified test conditions. Following 4MC of testing, polishing marks, observed prior to testing, had been removed. Consistently across all components, surface discolouration and multidirectional, criss-crossing, curvilinear and circular wear tracks, caused by abrasive wear, were observed. Reduced ROMs testing caused similar types of damage, however the circular wear tracks were smaller in size, compared to those produced during testing under the ISO protocol. The wear rates exhibited by MoM CTDRs tested under ISO-18192-1 testing protocol (0.246mm3/MC) were lower, when compared to CTDR designs incorporating MoP bearings, as well as MoM lumbar CTDRs. Wear rates generated under a modified ISO testing protocol were reduced tenfold, similarly to findings that have previously been reported in the literature, and support the hypothesis that the testing protocol specified by ISO-18192-1 may overestimate wear rates. Characterisation of particles generated by MoM CTDRs and biological consequences of those remain to be determined.
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. 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.Introduction
Materials and Methods
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]. 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.Introduction
Materials and Methods
Silicon nitride (SiN) is a recently introduced bearing material for THR that has shown potential in its bulk form and as a coating material on cobalt-chromium (CoCr) substrates. Previous studies have shown that SiN has low friction characteristics, low wear rates and high mechanical strength. Moreover, it has been shown to have osseointegration properties. However, there is limited evidence to support its biocompatibility as an implant material. The aim of this study was to investigate the responses of peripheral blood mononuclear cells (PBMNCs) isolated from healthy human volunteers and U937 human histiocytes (U937s) to SiN nanoparticles and CoCr wear particles. 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.Introduction
Methods
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. 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.Introduction
Methods
Hip replacements are falling short of matching the life expectancy of coxarthritis patients, due to implanting THR in younger patients and due to increasingly active patients. The most frequently implanted hip prostheses use cross linked (XL) polyethylene (PE) on metal bearings in the USA and most of the Western world. Concerns remain in the long term around the potential of wear debris-induced aseptic loosening. Thus exploring lower-wearing alternative bearings remains a major research goal. PEEK (poly-ether-ether-ketone) is a thermoplastic polymer with enhanced mechanical properties. This study compared the wear of PEEK to the wear of cross linked polyethylene, when sliding against cobalt chrome (CoCr) metallic counterfaces, and compared the wear of carbon-fibre reinforced (CFR)-PEEK to cross linked polyethylene when sliding against metallic and ceramic counterfaces under different contact stresses within the hip joint. The following materials were studied: unfilled PEEK (OPTIMA, Invibio) and CFR-PEEK (MOTIS, Invibio) against either high carbon (HC) CoCr or Biolox Delta ceramic plates. The comparative control material was a moderately cross-linked PE (Marathon, DePuy Synthes). A simple geometry wear study was undertaken. A rotational motion of ±30° across a sliding distance of ±28 mm (cross shear of 0.087), and contact pressures of 1.6 or 4 MPa were applied. The lubricant was 25% (v/v) bovine serum and the wear test was conducted for 1 million cycles at 1 Hz. Wear was assessed gravimetrically. A validated soak control method was used to adjust for serum absorption-induced mass changes during the wear test. Surface profilometry was assessed pre and post wear test.Introduction
Methods
Ceramic-on-ceramic hip replacements have generated great interest in recent years due to substantial improvements in manufacturing techniques and material properties1. Microseparation conditions that could occur due to several clinical factors such as head offset deficiency, medialised cup combined with laxity of soft tissue resulting in a translation malalignment, have been shown to cause edge loading, replicate clinically relevant wear mechanisms2,3 and increase the wear of ceramic-on-ceramic bearings3,4. The aim of this study was to investigate the influence of increasing the femoral head size on the wear of ceramic-on-ceramic bearings under several clinically relevant simulator conditions. The wear of size 28mm and 36mm ceramic-on-ceramic bearings (BIOLOX® INTRODUCTION
MATERIALS AND METHODS
Retrieval and clinical studies of metal-on-metal (MoM) bearings have associated increased wear1 and elevated patient ion levels2 with steep cup inclination angles and edge loading conditions. The University of Leeds have previously developed a hip simulator method that has been validated against retrievals and shown to replicate clinically relevant wear rates and wear mechanisms3,4. This method involves introducing lateral microseparation to represent adverse joint laxity and offset deficiency. This study aimed to investigate the effect of microseparation representing translational malpostion, and increased cup inclination angle, representing rotational malposition, in isolation and combined on the wear of different sizes (28 and 36mm) MoM bearing in total hip replacement (THRs). The wear of size 28mm and 36mm MoM THRs bearings was determined under different INTRODUCTION
MATERIALS AND METHODS
