Elsevier

Biomaterials

Volume 27, Issue 30, October 2006, Pages 5161-5169
Biomaterials

Association between UHMWPE particle-induced inflammatory osteoclastogenesis and expression of RANKL, VEGF, and Flt-1 in vivo

https://doi.org/10.1016/j.biomaterials.2006.04.004Get rights and content

Abstract

Wear debris-induced vascularized granulomatous periprosthetic tissue may augment the progress of prosthetic loosening, a major clinical problem after total joint replacement. The purpose of this study is to investigate the association of ultra-high-molecular-weight polyethylene (UHMWPE) particle-induced inflammatory osteoclastogenesis and expression of RANK/RANKL and VEGF/VEGF receptors (Flt-1 and Flk-1) using a mouse osteolysis model. UHMWPE particles were introduced into established air pouches on BALB/c mice, followed by implantation of calvaria bone from syngeneic littermates. Mice were injected with either recombinant VEGF or VEGF inhibitor (VEGF R2/Fc Chimera). Mice without drug treatment, as well as mice injected with saline alone were included. Each group contains 10 mice. Pouch tissues were harvested 2 weeks after bone implantation for histological and molecular analysis. UHMWPE stimulation significantly increased VEGF gene expression, and exerted a lower enhancement effect on the gene expression of Flt-1 and Flk-1. UHMWPE-stimulated VEGF production was markedly reduced by VEGF inhibitor treatment. Immunofluorescent staining indicated that pouch tissue macrophages were the main source of both VEGF and Flt-1 production. A positive association was observed between tissue inflammation and the levels of VEGF and Flt-1 gene transcripts. Both RANK and RANKL gene transcripts were significantly increased by UHMWPE stimulation, which was subsequently reduced by VEGF inhibitor treatment (p<0.05). VEGF treatment increased TRAP+ cells in pouches either with or without UHMWPE particle stimulation, and VEGF inhibitor treatment caused a significant reduction in the number of TRAP+ cells in UHMWPE-containing pouches. This study suggests that VEGF has a role in the regulation of RANK/RANKL-mediated osteoclastogenesis, and warrant future investigations to elucidate the role of VEGF signaling in the pathogenesis of prosthetic loosening.

Introduction

Osteolysis around knee and hip implants that subsequently leads to aseptic loosening has become a major clinical problem, and occurs in association with the formation of a vascularized granulomatous tissue in response to wear debris [1]. Since neovascularization is a hallmark of both bone remodeling and inflammation, the pro-angiogenetic status in periprosthetic tissue may augment the inflammatory response to wear debris [1], [2]. Recent studies have provided insights into molecular mechanisms that may be critical to this process. Vascular endothelial growth factor (VEGF), the most potent angiogenic growth factor, is produced by many cells including macrophages, lymphocytes, and osteoblasts [3], [4]. VEGF expression is regulated by growth factors, hormones, and cytokines [4], [5], [6], [7]. The receptors for VEGF belong to the tyrosine kinase receptor family, and two specific VEGF receptors have been identified to date; VEGF receptor-1 (VEGFR-1; fms-like tyrosine kinase, Flt-1) and VEGFR-2 (Flk-1/KDR) [8]. Current findings suggest that Flt-1 may play a critical role in the process of inflammation and osteoclastogenesis, based upon the observation that (i) Flt-1 is expressed on cells of the monocyte-macrophages lineage and is involved in macrophage activation [9]; (ii) Flt-1 signaling is a driving force in the recruitment of macrophages to inflamed tissues [10], [11], [12], [13], and (iii) Flt-1 is involved in both osteoclast formation (osteoclastogenesis) [14] and osteoclast bone resorptive activity [15]. Since VEGF signaling is involved in the process of osteoclastogenesis [14], [15], [16], receptor activator of nuclear factor kappa B ligand (RANKL) and its receptor RANK [17], [18] may act as a common mediator for inflammatory osteoclastogenesis [19]. RANKL and VEGF both regulate their activity through multiple signal transduction pathways, which have not yet been completely characterized. These two cytokines are known to be present in the local bone environment, and their corresponding receptors RANK (for RANKL) and Flt-1 (for VEGF) may be detected in osteoclasts and osteoclast precursor cells [14], [20], [15]. Both factors stimulate the recruitment of osteoclasts in a similar manner with respect to kinetics and signal transduction pathway [21], [22].

We have demonstrated that ultra-high-molecular-weight (UHMWPE) particles initiate a significantly higher RANK/RANKL gene expression in a murine model of tissue inflammation and osteoclastogenesis [23]. However, the possible interrelationship between VEGF and RANKL biological signaling during inflammatory osteoclastogenesis remains unclear. Using a mouse osteolysis model [24], we have recently demonstrated that stimulation by UHMWPE particles significantly increased VEGF expression, and immunohistology revealed that intense VEGF staining occurred predominantly on inflammatory cells around UHMWPE deposits. These findings suggest that VEGF signaling may be actively involved in the process of UHMWPE particle-induced inflammatory osteolysis. The purpose of this study was to test the hypothesis that amelioration of wear debris-induced inflammatory osteoclastogenesis by VEGF inhibitor treatment occurs through the down regulation of RANK, RANKL, and VEGF receptor genes in a mouse osteolysis model.

Section snippets

UHMWPE particles

UHMWPE particles (the generous gift of Dr. John Cuckler, University of Alabama, Birmingham, AL) were used to stimulate inflammatory reactions. The size and distribution of UHMWPE particles was evaluated with a Coulter particle counter and by scanning electron microscopy as described previously [25]. The UHMWPE particles had a mean particle diameter of 2.6 μm (range from <0.6 to 21 μm). The particles were washed in 70% ethanol solution to remove endotoxin and heat sterilized, and the absence of

Expression of VEGF and VEGF receptors in mouse pouch tissues

The gene expression of VEGF and VEGF receptors in the pouch tissues were determined by both immunostaining and RT-PCR. As shown in Fig. 1A, intense VEGF staining was observed in UHMWPE particle-stimulated pouches, compared with low expression in the PBS controls. VEGF staining was predominantly observed on the inflammatory cells surrounding UHMWPE deposits. VEGF treatment exerted little effect on the level of VEGF staining, either in the presence or absence of UHMWPE stimulation. However, VEGF

Discussion

This study indicates that UHMWPE particle stimulation significantly increases VEGF gene activity, but exerts a lesser influence on the levels of Flt-1 and Flk-1 gene activity in this model. UHMWPE particle-stimulated VEGF production was markedly reduced by VEGF inhibitor treatment. Pouch membrane macrophages were the predominant cells expressing both VEGF and Flt-1, and there was a positive association between tissue inflammation status and the levels of VEGF and Flt-1 gene activity. Both RANK

Conclusion

The present study shows that VEGF is actively involved in the regulation of RANK/RANKL gene expression, and exerts a regulatory effect on the development of UHMWPE particle-induced inflammatory osteoclastogenesis. These observations warrant future investigations to elucidate the possible interrelationship between these two cytokines.

Acknowledgments

The authors acknowledge the excellent technical assistance of Ms. Lois Mayton. Striker Company has provided support for this project.

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