Characterization of Wear Particles Generated from CoCrMo Alloy under Sliding Wear Conditions.

Biological effects of wear products (particles and metal ions) generated by metal-on-metal (MoM) hip replacements made of CoCrMo alloy remain a major cause of concern. Periprosthetic osteolysis, potential hypersensitivity response and pseudotumour formation are possible reactions that can lead to early revisions. To accurately analyse the biological response to wear particles from MoM implants, the exact nature of these particles needs to be characterized. Most previous studies used energy-dispersive X-ray spectroscopy (EDS) analysis for characterization. The present study used energy filtered transmission electron microscopy (TEM) and electron diffraction pattern analysis to allow for a more precise determination of the chemical composition and to gain knowledge of the crystalline structure of the wear particles.Particles were retrieved from two different test rigs: a reciprocating sliding wear tribometer (CoCrMo cylinder vs. bar) and a hip simulator according to ISO 14242-1 (CoCrMo head vs. CoCrMo cup). All tests were conducted in bovine serum. Particles were retrieved from the test medium using a previously published enzymatic digestion protocol.Particles isolated from tribometer samples had a size of 100 - 500 nm. Diffraction pattern analysis clearly revealed the lattice structure of strain induced hcp ε-martensite. Hip simulator samples revealed numerous particles of 15 - 30 nm and 30 - 80 nm size. Most of the larger particles appeared to be only partially oxidized and exhibited cobalt locally. The smallest particles were Cr(2)O(3) with no trace of cobalt. It optically appeared that these Cr(2)O(3) particles were flaking off the surface of larger particles that depicted a very high intensity of oxygen, as well as chromium, and only background noise of cobalt. The particle size difference between the two test rigs is likely related to the conditions of the two tribosystems, in particular the difference in the sample geometry and in the type of sliding (reciprocating vs. multidirectional).Results suggest that there may be a critical particle size at which chromium oxidation and cobalt ionization is accelerated. Since earlier studies have shown that wear particles are covered by organic residue which may act as a passive layer inhibiting further oxidation, it would suggest that this organic layer may be removed during the particle isolation process, resulting in a change of the particle chemical composition due to their pyrophoric properties. However, prior to being isolated from the serum lubricant, particles remain within the contact area of head and cup as a third-body. It is therefore possible that during that time, particles may undergo significant transformation and changes in chemical composition in the contact area of the head and cup within the tribological interface due to mechanical interaction with surface asperities.

[Implant wear and aseptic loosening. An overview]. / Verschleiss und aseptische Prothesenlockerung. Eine Bestandsaufnahme.

Wear of total joint implants is multifactorial in nature. Even for identical materials and geometries, the interaction of those parameters can generate different numbers of particles as well as different particle sizes and shapes. These different wear-particle characteristics will directly influence the biological response to an implant and thereby its clinical success. The long-term success of a total joint replacement requires an optimized compromise among implant material, design, surgical procedure, and biological performance.

Wear particles from metal-on-metal total hip replacements: effects of implant design and implantation time.

Detailed characterization of wear particles is necessary to understand better the implant wear mechanisms and the periprosthetic tissue response. The purposes of the present study were to compare particle characteristics of current with older designs of metal-on-metal (MM) total hip replacements (THRs), and to determine the effect of implantation time on wear particle characteristics. Metal wear particles isolated from periprosthetic tissues from 19 patients with MM THRs of current and older designs and at different implantation times (very short, longer, and very long) were studied using transmission electron microscopy and energy dispersive X-ray analysis. The particles from the current design implants with implantation times of not more than 15 months (very short-term) were almost exclusively round to oval chromium oxide particles. In all other cases, although the predominance was still round to oval chromium oxide particles, greater proportions of cobalt-chromium-molybdenum (Co-Cr-Mo) particles, mainly needle-shaped, were detected. Very long-term THRs implanted for more than 20 years had the highest percentage of needle-shaped Co-Cr-Mo particles. Particle lengths were not markedly different between the different designs and implantation times except for the current design implants of not more than 15 months, which had a significantly smaller mean length of 39 nm. In conclusion, the implant design did not seem to have a significant influence on particle characteristics whereas the implantation time appeared to have the most effect on the particles. It should be noted that, because of the limited number of tissue retrievals available, some uncertainty remains regarding the generality of these findings.

Effects of digestion protocols on the isolation and characterization of metal-metal wear particles. I. Analysis of particle size and shape.

Isolation of metal wear particles from hip simulator lubricants or tissues surrounding implants is a challenging problem because of small particle size, their tendency to agglomerate, and their potential for chemical degradation by digestion reagents. To provide realistic measurements of size, shape, and composition of metal wear particles, it is important to optimize particle isolation and minimize particle changes due to the effects of the reagents. In this study (Part I of II), transmission electron microscopy (TEM) was used to examine and compare the effects of different isolation protocols, using enzymes or alkaline solutions, on the size and shape of three different types of cobalt-based alloy particles produced from metal-metal bearings. The effect on particle composition was examined in a subsequent study (Part II). Large particles (<1200 nm) were generated by dry abrasion of CoCrMo alloy against itself and small particles (<300 nm) were generated by hip simulator testing of a metal-metal implant pair in the presence of either distilled-deionized water or a 95% bovine serum solution. The reagents changed particle size and to a lesser extent particle shape. For both large particles and small particles generated in water, the changes in size were more extensive after alkaline than after enzymatic protocols and increased with alkaline concentration and time in solution, up to twofold at 2 h and threefold at 48 h. However, when isolating particles from 95% serum, an initial protective effect of serum proteins and/or lipids was observed. Because of this protective effect, there was no significant difference in particle size and shape for both oval and needle-shaped particles after 2 h in 2N KOH and after enzymatic treatments. However, round particles were significantly smaller after 2 h in 2N KOH than after enzymatic treatments. Particle composition may also have been affected by the 2N KOH treatment, as suggested by a difference in particle contrast under TEM, an issue examined in detail in Part II.

Effects of digestion protocols on the isolation and characterization of metal-metal wear particles. II. Analysis of ion release and particle composition.

The isolation of metal wear particles from hip simulator lubricants is important for understanding wear mechanisms and the tissue response to particulate material. Part I of this study demonstrated that isolation protocols involving digestion reagents can chemically attack metal-metal wear particles, reducing their size and changing their shape. In part II of this study, Co and Cr ion concentrations in solution after each digestion protocol were measured by flame atomic absorption spectrometry, and wear particle composition was determined by X-ray analysis spectra. The exposure of wear particles in water to alkaline solutions caused an increasing release of Cr ions in solution with alkaline concentration and time, and a corresponding decrease in particle Cr peak intensity on X-ray spectra. As a result, particles exposed to 12N KOH for 48 h displayed Co peaks and no Cr. In contrast, enzymatic protocols caused a release of Co ions in solution and a corresponding decrease in particle Co peak intensity on X-ray spectra, especially with sodium phosphate as a buffer. However, when isolating particles from 95% serum, there was an initial protective effect of serum proteins, presumably because of their binding to Co and Cr. As a result, the extent of Cr ion release from metal wear particles in 95% serum after alkaline treatments was diminished, although still present, whereas both enzymatic protocols resulted in a negligible release of Co and Cr ions into solution. Particle composition analysis after enzymatic treatments revealed the presence of chromium oxide particles and CoCrMo particles with variable Co/Cr ratios. After alkaline treatments, the chromium oxide particles increasingly disappeared with time and alkaline concentration, demonstrating a change in particle composition after these treatments. This study demonstrated that digestion reagents can induce chemical changes that affect particle composition. Of all the protocols tested, the enzymatic protocols were the least damaging to the particles and appeared to be the best compromise for isolation and characterization of metal particles, especially in 95% serum. Special care on the choice of buffers should be taken when isolating particles from a lower concentration of serum.

Cytotoxic and apoptotic effects of cobalt and chromium ions on J774 macrophages - Implication of caspase-3 in the apoptotic pathway.

The aim of this study was to evaluate the cytotoxic and apoptotic effects of cobalt and chromium ions on macrophages in vitro, and analyze the implication of caspase-3 in the apoptotic pathway. J774 mouse macrophages (5 x 10(5) cells/ml) were exposed for up to 24 h to 0-10 ppm Co2+ and 0-500 ppm Cr3+. The cytotoxic effect of ions was measured by Trypan blue exclusion. DNA analysis on agarose gel was used as a specific test for detection of DNA fragmentation into oligonucleosomes that occurs in apoptotic cells. The proteolytic cleavage of poly(ADP-ribose)polymerase (PARP), closely associated with the induction of apoptosis, was also analyzed along with the appearance of the active fragment of caspase-3, implicated in several apoptosis pathways. Results demonstrated that both Co2+ and Cr3+ ions induce macrophage mortality in a dose-dependent manner. However, Co2+ is more toxic inducing a cell mortality up to 28% with only 10 ppm vs. 37% with 500 ppm of Cr3+. DNA analysis demonstrated that both Co2+ and Cr3+ ions induce DNA fragmentation, between 6-10 ppm Co2+ and 250-500 ppm Cr3+ after 24 h incubation. PARP cleavage and the appearance of caspase-3 active fragment were observed after 6 h with both Co+ and Cr3+ ions, with a stronger signal after 24 h and 10 ppm of Co2+ or 500 ppm of Cr3+. In conclusion, this study demonstrates that after 24 h incubation, both Co2+ and Cr3+ ions can induce macrophage mortality, and more specifically apoptosis. The results also suggest that apoptosis occurs via a caspase-3 pathway. However, the relative importance of necrosis and apoptosis and the effects of longer exposure times on the induction of macrophage death by these metal ions remain to be investigated.

Induction of macrophage apoptosis by ceramic and polyethylene particles in vitro.

The purpose of this study was to investigate in vitro the presence of apoptotic cell death after macrophage stimulation with different ceramic (Al2O3 and ZrO2) and high density polyethylene (HDP) particles. We also analyzed the effects of particle size, concentration, and composition. The J774 mouse macrophage cell line was exposed to commercial particles of different sizes (up to 4.5 microm) and concentrations (up to 500 particles per macrophage). Fluorescence microscopy and DNA laddering were used to investigate the presence of apoptosis in cell cultures after 24 h of incubation. Fluorescence microscopy of propidium iodide stained cells showed two characteristic morphological features that occur in apoptotic cells, namely nuclear condensation and heterogeneity of stain uptake. The effect of ceramic particles on apoptotic nuclear morphology was size- and concentration-dependent and reached a plateau above 150 particles per macrophage at 1.3 microm. With regards to composition, we did not find any difference in cell morphology between Al2O3 and ZrO2. Ceramic and HDP particles induced DNA fragmentation into oligonucleosomes as evidenced by DNA laddering, another characteristic of apoptosis. The induction of DNA laddering was size- and concentration-dependent whereas particle composition (Al2O3 vs. ZrO2 and Al2O3 vs. HDP) had no effect. In conclusion, our results demonstrated that ceramic and HDP particles induce macrophage apoptotic cell death in vitro and open doors for possible modulation of debris-induced periprosthetic osteolysis.

Cytotoxicity and macrophage cytokine release induced by ceramic and polyethylene particles in vitro.

Although the response of macrophages to polyethylene debris has been widely studied, it has never been compared with the cellular response to ceramic debris. Our aim was to investigate the cytotoxicity of ceramic particles (Al2O3 and ZrO2) and to analyse their ability to stimulate the release of inflammatory mediators compared with that of high-density polyethylene particles (HDP). We analysed the effects of particle size, concentration and composition using an in vitro model. The J774 mouse macrophage cell line was exposed to commercial particles in the phagocytosable range (up to 4.5 microns). Al2O3 was compared with ZrO2 at 0.6 micron and with HDP at 4.5 microns. Cytotoxicity tests were performed using flow cytometry and macrophage cytokine release was measured by ELISA. Cell mortality increased with the size and concentration of Al2O3 particles. When comparing Al2O3 and ZrO2 at 0.6 micron, we did not detect any significant difference at the concentrations analysed (up to 2500 particles per macrophage), and mortality remained very low (less than 10%). Release of TNF-alpha also increased with the size and concentration of Al2O3 particles, reaching 195% of control (165 pg/ml v 84 pg/ml) at 2.4 microns and 350 particles per cell (p < 0.05). Release of TNF-alpha was higher with HDP than with Al2O3 particles at 4.5 microns. However, we did not detect any significant difference in the release of TNF-alpha between Al2O3 and ZrO2 at 0.6 micron (p > 0.05). We saw no evidence of release of interleukin-1 alpha or interleukin-1 beta after exposure to ceramic or HDP particles.

Flow cytometric analysis of macrophage response to ceramic and polyethylene particles: effects of size, concentration, and composition.

Using the J774 macrophage cell line, we designed an in vitro model to analyze by flow cytometry the effects of size, concentration, and composition of ceramic (Al2O3 and ZrO2) and high density polyethylene (HDP) particles on phagocytosis and cell mortality. Inflammatory mediator (TNF-alpha) also was measured by ELISA. Kinetic studies revealed that phagocytosis of the particles begins very early after cell exposure, increasing with time and particle concentration and reaching a plateau after 15 h. This implies that the optimum period to evaluate cellular response to particulate debris is between 15 and 24 h of incubation. Results also showed that phagocytosis increases with concentration for particles up to 2 microns. For larger particles (up to 4.5 microns), phagocytosis seems to reach a plateau independent of size and concentration, which suggests a saturation of phagocytosis that is most likely dependent on overall particle volume ingested. We did not detect any significant difference in phagocytosis between Al2O3 and ZrO2 at 0.6 microns. Al2O3 seems to be more easily phagocytosed than HDP at the same size (4.5 microns) and concentrations. Cytotoxicity studies revealed that macrophage mortality increases with particle size and concentration for sizes greater than 2 microns. Smaller particles (0.6 microns) cause cell mortality only at higher concentrations (from 1,250 particles per cell), but the mortality is still very low (10%). No significant difference in cell mortality and TNF-alpha release was found between Al2O3 and ZrO2. Effects of Al2O3 and HDP at 4.5 microns were compared by measuring TNF-alpha release. Results showed that TNF-alpha release increases with particle concentrations and is higher with HDP than with Al2O3.