Elsevier

Biomaterials

Volume 29, Issue 36, December 2008, Pages 4760-4765
Biomaterials

Systemic trafficking of macrophages induced by bone cement particles in nude mice

https://doi.org/10.1016/j.biomaterials.2008.09.004Get rights and content

Abstract

Macrophages play an important role in the biological response to wear particles, which can result in periprosthetic osteolysis and implant loosening. In this study, we demonstrate that polymer particles induce systemic trafficking of macrophages by non-invasive in vivo imaging and immunohistochemistry. The distal femora of nude mice were injected with 10% (w/v) Simplex bone cement (BC) suspensions or saline (PBS). Reporter RAW264.7 macrophages which stably expressed the bioluminescent reporter gene fluc, and the fluorescence reporter gene gfp, were injected intravenously. Bioluminescence imaging was performed immediately and periodically at 2-day intervals until day 14. Compared to the non-operated contralateral femora, the bioluminescent signal of femora injected with BC suspension increased 4.7 ± 1.6 and 7.8 ± 2.9-fold at day 6 and 8, respectively. The same values for PBS group were 1.2 ± 0.2 and 1.4 ± 0.5, respectively. The increase of bioluminescence of the BC group was significantly greater than the PBS group at day 8 (p < 0.05) and day 6 (p < 0.1). Histological study confirmed the presence of reporter macrophages within the medullary canal of mice that received cement particles. Modulation of the signaling mechanisms that regulate systemic macrophage trafficking may provide a new strategy for mitigating the chronic inflammatory response and osteolysis associated with wear debris.

Introduction

Excessive production of wear particles from joint replacements is associated with periprosthetic osteolysis, which can lead to implant loosening [1], [2], [3], [4], [5], [6], [7]. Phagocytic cells engulf particulate debris and become activated; releasing proinflammatory cytokines, chemokines, degradative enzymes, reactive oxygen radicals and other substances which stimulate osteoclasts to undermine the prosthetic bed [8], [9], [10], [11], [12], [13], [14]. The key cell in the foreign body and chronic inflammatory response to wear particles is the macrophage [15], [16], [17]. Cells of the monocyte/macrophage lineage differentiate and maturate into phagocytic macrophages, foreign body giant cells and osteoclast precursors. These cells (in communication with stromal cells and other cell types) are primarily responsible for the cascade of events culminating in periprosthetic osteolysis. Despite ongoing research into the cellular and molecular processes associated with periprosthetic osteolysis, no in vivo studies have elucidated whether remote macrophages are stimulated to migrate to wear particles, or whether these events are a local phenomenon only. If macrophage recruitment to particles is a systemic phenomenon then novel strategies to mitigate these events may be potential targets for treatment.

We hypothesized that exogenous reporter macrophages introduced from a distant site would migrate and concentrate to an area in which phagocytosable polymer particles have been implanted. To examine this hypothesis, we use a model of femoral intramedullary polymer particle placement [18] in nude mice, a murine macrophage cell line transfected with a bioluminescent reporter gene, and sequential non-invasive imaging in vivo using bioluminescence.

Section snippets

Animals and cells

8 to 11-week-old adult male nude mice (Charles River Laboratories, Inc., MA) were housed and fed in our Institution's Animal Facility. The murine macrophage cell line RAW264.7 was transfected with the lentiviral vector to express the bioluminescent optical reporter gene, firefly luciferase (fluc), and a fluorescence reporter gene, green fluorescent protein (gfp) [19].

Bone cement particles

Simplex® P bone cement (BC) powder (Howmedica Osteonics, Allendale, NJ) was used in the study. The BC powder is composed of 15%

Polarized light microscopy of frozen section of femora

To demonstrate the presence of the BC particles in the femoral medullary canal of experimental animals, femora of selected mice were harvested immediately after injection. Frozen sections were stained with hematoxylin and eosin (H&E). The adopted histological protocol utilized reagents that precluded particle disruption and dissolution during preparation of the slides. Polarized light microscopy was used to observe the birefringence of the BC particles. As shown in Fig. 1, bright white spots

Discussion

Wear particles, periprosthetic osteolysis and implant loosening jeopardize the longevity of joint replacements. Macrophages play an important role in these biological processes [8], [15], [17], [21], [22], [23], [24], [25]. Macrophages release proinflammatory cytokines, chemokines and other factors, and can differentiate into osteoclasts that resorb bone [8], [15], [17], [21], [22], [23], [24], [25], [26], [27]. The precursor cells for activated macrophages, foreign body giant cells and

Conclusion

This study employing the techniques of sequential in vivo imaging using bioluminescence and immunohistochemistry clearly demonstrates that exogenous reporter macrophages injected into the tail vein of nude mice are systemically recruited to a distant site, the femur that contains bone cement particles. Modulation of the signaling mechanisms that regulate systemic macrophage recruitment and homing may provide a new strategy for mitigating the chronic inflammatory response associated with wear

Acknowledgements

We gratefully acknowledge Dr. Gobalakrishnan Sundaresan who supplied the fluc and gfp expressing RAW264.7 macrophage cell line. This study was supported in part by Grants R21 AR053189 and R01 AR055650-01 from the National Institute of Health, the Ellenburg Chair in Surgery.

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