Distinct gene expression of receptor activator of nuclear factor-κB and rank ligand in the inflammatory response to variant morphologies of UHMWPE particles

Abstract

Recent studies have examined the role of wear debris-induced bone resorption in the aseptic loosening of orthopedic prostheses. Research has shown that inflammation depends not only on the amount of particulate debris, but also the shape and size of the accumulated wear particles. Our previous studies have demonstrated that variant shapes of ultra-high molecular weight polyethylene (UHMWPE) particles induce diverse cellular and apoptotic responses in a murine inflammation model. Since enhanced osteoclastogenesis is recognized as a hallmark of bone loss in prosthetic loosening, we have now investigated the gene expression of receptor activator of nuclear factor-κB (RANK) and receptor activator of nuclear factor-κB ligand (RANKL) during the inflammatory response to different shapes of UHMWPE particles. Two shapes of UHMWPE particles (globular or elongated) were implanted in established air pouches on BALB/c mice, and pouches harvested 7 days after stimulation with UHMWPE particles. Gene levels of RANK, RANKL, TNFα, IL-1β, and cathepsin K (CK) were quantified by real time RT-PCR, and TRAP staining of pouch membrane was used to evaluate osteoclastogenesis. We found that (i) elongated particles generated significantly higher RANK and RANKL gene expression than globular particles in pouch tissue; (ii) elongated particles provoked significantly higher IL-1β and TNFα gene expression; (iii) a positive association was found between tissue inflammation status and the gene level of RANK/RANKL; and (iv) elongated particles stimulated significantly higher CK gene expression in comparison with globular particles. Histology revealed that clusters of TRAP+ cells were located in regions in contact with elongated particles. Overall, these data suggest that the morphology of wear debris may be a critical factor in the pathogenesis of prosthetic loosening.

Introduction

Aseptic loosening is associated with the generation of wear debris particles that become embedded in peri-prosthetic tissue in direct apposition to bone [1], [2]. Since ultra high molecular weight polyethylene (UHMWPE) has been widely used in orthopaedic prostheses, and UHMWPE components removed at revision surgery usually show evidence of wear [3], [4], [5], many investigators have focused on the role of UHMWPE debris in the pathogenesis of aseptic loosening. Previous studies have indicated that UHMWPE and other wear debris generated from the prosthesis stimulates phagocytosis by macrophages and the subsequent release of pro-inflammatory cytokines, resulting in local inflammation at the contact zone between tissue and wear debris deposit [6], [7], [8], [9], [10], [11].

Enhanced osteoclastogenesis mediated by the inflammatory cytokines is an important contributor to the development of aseptic loosening [12]. Two important regulators of osteoclastogenesis have been recently identified [13], [14]. These are the receptor activator of nuclear factor-κB (RANK) and the receptor activator of nuclear factor-κB ligand (RANKL). RANKL, also known as ODF/TRANCE/OPGL, is a TNF-related cytokine that is produced by marrow stromal cells and osteoblasts [14]. RANK, a member of TNF receptor super family, is present on osteoclasts and their precursors and initiates osteoclastogenesis after ligation with RANKL or agonistic anti-RANK antibodies [14]. An emerging concept is that RANK may act as a common mediator for inflammatory osteoclastogenesis. Li et al. [15] reported that osteoclastogenesis was not observed in the RANK gene knock out (RANK^−/−) mice following IL-1 treatment, shown by the complete absence of TRAP⁺ and cathepsin K⁺ (CK) osteoclasts in calvaria. In addition, the failure of RANK^−/− mice to mount a significant osteoclastic response in the presence of experimental inflammatory arthritis further supports the concept that RANK plays a gate-keeping role in controlling osteoclastogenesis [16]. In contrast, mice lacking RANKL have defects in osteoclastogenesis that leads to severe osteopetrosis [16]. However, no studies have addressed the physical properties of wear debris that influence the gene activation of these important mediators of bone loss.

Previous studies have demonstrated that both uncemented and cemented prostheses generate wear debris with similar globular morphology with occasional elongated, fibrillar or flak-like particles [17], [18]. Minimal research has been conducted to differentiate the tissue response to the various shaped wear debris. We have used a mouse air pouch model to study the physical properties of orthopaedic wear debris under controlled experimental conditions [19]. Using this model, we have demonstrated that different morphologies of UHMWPE particles can elicit diverse cellular and apoptotic responses [20]. Using the same model, the aim of the present investigation was to address whether gene activation of RANK and RANKL varies in response to the different morphologies of UHMWPE particles, and determine whether an association exists between RANK/RANKL gene expression and tissue inflammation and osteoclastogenesis.