Etude in vitro de la réponse des macrophages à des particules de matériaux utilisés dans les prothèses totales de hanche : céramiques vs polyéthylène

Mécanismes conduisant à l'ostéolyse périprosthétique -- Débris d'usure et ostéolyse périprosthétique : importance de la taille, la concentration et la composition -- Lien entre médiateurs inflammatoires et ostéolyse péryprosthétique -- Importance du type de mortalité cellulaire -- Biocompatibilité des céramiques : des années 70 à nos jours -- Réponse biologique aux céramiques à l'état massif -- Réponse biologique aux céramiques sous forme particulaire -- Méthodologies utilisées pour l'étude de la réponse cellulaire aux particules -- Particules -- Lignée cellulaire -- Médiateurs inflammatoires -- Type de mortalité -- Étude de la réponse in vitro des macrophages à des particules de céramiques et de polyéthylène -- Étude de la cytotoxicité et des médiateurs inflammatoires -- Étude de la mortalité des macrophages par apoptose -- La cytométrie : performances et limites -- Résultats de phagocytose, cytotoxicité et médiateurs inflammatoires : lien avec le mécanisme de descellement des PTHS. -- Les cytokines : maqueurs de l'activité des macrophages -- L'apoptose : un moyen de traitement thérapeutique de l'ostéolyse périprosthétique?

Characterization and biological effects of wear particles from metal-metal hip implants

Polyethylene wear particles have been the main culprit in initiating osteolysis and aseptic failure of conventional metal-polyethylene total hip arthroplasties. There has been a revived interest in metal-metal (MM) bearings, made of CoCrMo alloys; because of their very high wear resistance compared with conventional bearings. The work that forms the basis of this thesis attempts to examine MM bearings as one potential solution to the problem of osteolysis. The results provide important new information on MM particle characteristics and their effects on tissue, as well as the effects of their corrosion products (Co2+ and Cr3+) on macrophage response in vitro.An enzymatic protocol for isolation and characterization of metal particles was first developed in order to minimize particle changes due to the reagents. Metal particles isolated from a hip simulator and from human tissues around MM implants using this protocol were found to be mainly round to oval and more occasionally needle-shaped, extremely small (∼50 nm), and surprisingly, mostly chromium oxides. In vitro and in vivo run-in wear periods did not exactly correlate, but MM wear particles from both sources were quite similar at specific periods of comparison. Alloy composition and number of running cycles in vitro influenced the overall characteristics of the particles, although the observed differences were relatively small and would probably not induce a difference in the biological response.Tissue response to metal wear particles revealed the presence of IL-1beta and IL-6, two major inflammatory mediators implicated in periprosthetic osteolysis. There was also a high correlation between the amount of these cytokines and the quantity of metal particles. The amounts of cytokines decreased when the quantities of particles increased, most likely due to the high toxicity of the metal particles when present in larger quantities. There was more IL-6 than IL-1beta and TNF-alpha remained at low levels. Apoptosis was clearly observed in some tissues from the patients analyzed, but results were highly variable depending on the tissues analyzed. More patients would be necessary to complete this study on apoptosis.In vitro, Co2+ and Cr3+ ions induced the secretion of TNF-alpha, another major inflammatory mediator implicated in periprosthetic osteolysis, and macrophage mortality in a dose- and time-dependent manner. Co2+ was more toxic than Cr 3+. Macrophage apoptosis was predominant at 24h with both ions, and with the lowest concentrations at 48h. Necrosis was more prevalent with the highest concentrations at 48h. The implication of caspase-3 pathway was also identified in macrophage apoptosis. These results suggest how specific therapeutic treatments could modulate macrophage response to these ions

Effects of metal ions on caspase-1 activation and interleukin-1β release in murine bone marrow-derived macrophages.

Ions released from metal implants have been associated with adverse tissue reactions and are therefore a major concern. Studies with macrophages have shown that cobalt, chromium, and nickel ions can activate the NLRP3 inflammasome, a multiprotein complex responsible for the activation of caspase-1 (a proteolytic enzyme converting pro-interleukin [IL]-1β to mature IL-1β). However, the mechanism(s) of inflammasome activation by metal ions remain largely unknown. The objectives of the present study were to determine if, in macrophages: 1. caspase-1 activation and IL-1β release induced by metal ions are oxidative stress-dependent; and 2. IL-1β release induced by metal ions is NF-κB signaling pathway-dependent. Lipopolysaccharide (LPS)-primed murine bone marrow-derived macrophages (BMDM) were exposed to Co2+ (6-48 ppm), Cr3+ (100-500 ppm), or Ni2+ (12-96 ppm), in the presence or absence of a caspase-1 inhibitor (Z-WEHD-FMK), an antioxidant (L-ascorbic acid [L-AA]), or an NF-κB inhibitor (JSH-23). Culture supernatants were analyzed for caspase-1 by western blotting and/or IL-1β release by ELISA. Immunoblotting revealed the presence of caspase-1 (p20 subunit) in supernatants of BMDM incubated with Cr3+, but not with Ni2+ or Co2+. When L-AA (2 mM) was present with Cr3+, the caspase-1 p20 subunit was undetectable and IL-1β release decreased down to the level of the negative control, thereby demonstrating that caspase-1 activation and IL-1β release induced by Cr3+ was oxidative stress-dependent. ELISA demonstrated that Cr3+ induced the highest release of IL-1β, while Co2+ had no or limited effects. In the presence of Ni2+, the addition of L-AA (2 mM) also decreased IL-1β release, below the level of the negative control, suggesting that IL-1β release induced by Ni2+ was also oxidative stress-dependent. Finally, when present during both priming with LPS and activation with Cr3+, JSH-23 blocked IL-1β release, demonstrating NF-κB involvement. Overall, this study showed that while both Cr3+ and Ni2+ may be inducing inflammasome activation, Cr3+ is likely a more potent activator, acting through oxidative stress and the NF-κB signaling pathway

Effects of metal ions on caspase-1 activation and interleukin-1β release in murine bone marrow-derived macrophages - Fig 1

Interleukin-1β (IL-1β) release by bone marrow-derived macrophages (BMDM) after exposure to (A) Cr3+, (B) Ni2+, and (C) Co2+, with or without Z-WEHD-FMK. Cells were incubated under cell culture conditions with the indicated concentrations of ions in the presence or absence of Z-WEHD-FMK (20 μM), a caspase-1 inhibitor, for 18 to 24h following a 6h priming incubation with lipopolysaccharide (LPS; 500 ng/mL). IL-1β release was analyzed by ELISA and is expressed as a percentage of the release in the negative control (cells with no ions and no Z-WEHD-FMK). An asterisk (*) indicates a significant difference (pp<0.05) between the results obtained with and without Z-WEHD-FMK at a given ion concentration. A double dagger (‡) indicates that the measurement was below the detection threshold. Data are presented as means ± SEM of 3–5 independent experiments performed in triplicate.</p

Caspase-1 activation in bone marrow-derived macrophages (BMDM) after exposure to Cr³⁺, Ni²⁺, and Co²⁺, with or without L-ascorbic acid (L-AA).

Cells were incubated under cell culture conditions with the indicated concentrations of ions in the presence or absence of L-AA (2 mM), an antioxidant, for 18 to 24h following a 6h priming incubation with lipopolysaccharide (LPS; 500 ng/mL). Culture supernatants were analyzed for the presence of caspase-1 (as indicated by the detection of its p20 subunit) by western blotting. The immunoblot presented is representative of three independent experiments.</p