Adherent endotoxin mediates biological responses of titanium particles without stimulating their phagocytosis

Abstract

Aseptic loosening of orthopaedic implants is thought to be primarily due to stimulation of cytokine production by wear particles from the implants. The cytokines increase osteoclast differentiation, leading to osteolysis and implant loosening. Accumulating evidence indicates that adherent endotoxin mediates the biological responses induced by the wear particles. One mechanism by which adherent endotoxin may act is by increasing phagocytosis of the wear particles. To test this hypothesis, the effect of adherent endotoxin on phagocytosis of titanium particles was determined. First, we developed reliable confocal and fluorescence microscopy methods to examine both the attachment and internalization steps of phagocytosis. Use of these methods showed that adherent endotoxin does not detectably alter the rate or the extent of phagocytosis of titanium particles by RAW 264.7 cells. Despite this lack of an effect on phagocytosis, adherent endotoxin dramatically increases the ability of RAW 264.7 cells to produce TNF-a and induce osteoclast differentiation. Thus, adherent endotoxin mediates these biological responses by a mechanism that does not rely on increased phagocytosis. These results also demonstrate that phagocytosis is not sufficient to induce cytokine production and osteoclast differentiation but do not rule out the possibility that phagocytosis is required for induction of these responses by titanium particles with adherent endotoxin.

Polymorphisms in the interleukin‐1 receptor antagonist and interleukin‐6 genes affect risk of osteolysis in patients with total hip arthroplasty

“…These included endotoxin-stripped particles (Ti) and commercially pure endotoxin-stripped particles with adherent lipopolysaccharide (LPS) added back (Ti plus LPS). The protocols for preparation of the 2 types of particles are described in detail elsewhere, as are their size characteristics and their ability to induce cytokine production in mononuclear cells (28,29). High Sensitivity Limulus Amebocyte Lysate assay (BioWhittaker, Walkersville, MD) confirmed endotoxin levels of 0 EU/ml and 140 EU/ml for the Ti and Ti plus LPS particles, respectively.…”

Individual susceptibility to periprosthetic osteolysis is associated with altered patterns of innate immune gene expression in response to pro‐inflammatory stimuli

“…Seventy-five percent of the particles had a mean diameter of <6.5 mm, and their ability to induce cytokine production in PBMCs has been described elsewhere, as has the ability of conditioned media taken from these cultures to induce osteoclastogenesis and bone resorption. 16 …”

Effects of AS-cast and wrought cobalt-chrome-molybdenum and titanium-aluminium-vanadium alloys on cytokine gene expression and protein secretion in J774A.1 macrophages

“…Endotoxin is ubiquitous remnants from gram-negative cell walls, with a strong pro-inflammatory capacity. Particles with adherent endotoxin generates a larger proinflammatory response in cell cultures than particles without endotoxin (Bi et al, 2002;Cho et al, 2002).…”

“…These findings can be explained by the fact that macrophages express and secrete cytokines independently of the internalization process (Bi et al, 2002;Fritz et al, 2006;Nakashima et al, 1999). In physiological environment adherent endotoxin is unavoidable (Tatro et al, 2006) and may also contribute to the pro-inflammatory response generated by the foreign objects in the body.…”

“…Fibronectin and vitronectin also adsorb to the implant surface and act as a ligand for macrophages through RGD-integrin receptor domains. These ligand-interactions and several others are the primarily interaction between implant surface and macrophages (Ziats et al, 1988) resulting in cytokine release independently of internalization of wear particles (Bi et al, 2002;Fritz et al, 2006;Nakashima et al, 1999). The release of metal ions take place as a result of the dissolucytes (macrophages) controlling the chemical milieu in the membrane (Danscher, 2002;Larsen et al, 2007) in a process called dissolucytosis.…”