Abstract
Introduction
Wear debris and metal ions originating from metal on metal hip replacements have been widely shown to recruit and activate macrophages. These cells secrete chemokines and pro-inflammatory cytokines that lead to an adverse local tissue reaction (ALTR), frequently requiring early revision. The mechanism for this response is still poorly understood. It is well documented that cobalt gives rise to apoptosis, necrosis and reactive oxygen species generation. Additionally, cobalt stimulates T cell migration, although the effect on macrophage motility remains unknown. This study tests the hypothesis that cobalt ions and nanoparticles affect macrophage migration stimulating an ALTR.
Methods
This study used Co2+ ions (200µM) and cobalt nanoparticles (CoNPs, 100µM, 2–60nm diameter). PMA differentiation of the U937 cell line was used as macrophage-like cells. The effect of cobalt on macrophage migration was investigated by live cell imaging. After 12 hours of each treatment, timelapse images of 20 cells were collected over a 6 hour period with images captured every 5 min. Migration of individual cells was tracked in 2D using ImageJ software. The transwell migration assay was also applied to study the effect of cobalt on macrophage directional migration. U937 cells in serum free medium were added to the upper chamber of a 8µm pore size Transwell insert in the presence of cobalt, whilst the lower chamber was filled with medium plus 10% FBS. After 6 hours treatment, cells remaining on the membrane were fixed, stained with crystal violet and counted. Cellular F-actin and podosomes were visualized by labeling with TRITCconjugated phalloidin and anti-vinculin antibody after 12 hours of cobalt exposure (Co2+ and CoNPs).
Results
Cells incubated with cobalt ions and nanoparticles showed a substantial reduction in cell migration compared with control cells. The total migration path length of cells treated with Co2+ (362.4±96.6µm) and CoNPs (217.3±128.1µm) were significantly shorter than those for untreated cells (801.1±198.3µm). The ability of macrophages to migrate through the transwell membrane was significantly impaired by pre-treatment with cobalt, with 16±4 and 18± migrated cells/field for Co2+ and CoNPs respectively with the control at 42±7 migrated cells/field. In addition, cobalt influenced macrophage morphology and actin cytoskeletal organization with a dramatic increase in the presence of intracellular podosome-type adhesions structure.
Discussion
Co2+ ions and nanoparticles dramatically inhibited the migration of U937 macrophages in contrast to the enhanced migration reported for T cells. We propose that macrophages recruited into the area of CoCr implants would lose their responsiveness to migration signals and be retained in situ due to cobalt-induced cytoskeleton rearrangement. This enhanced macrophage accumulation and cobalt-induced formation of podosomes may therefore represent a mechanism through which cobalt wear debris and metal ions from joint prostheses exacerbate the ALTR leading to revision surgery.
