Investigation of the activation of a human serum complement protein, C3, by orthopedic prosthetic particulates

Abstract

Myriad molecular, cellular, and physiological processes underlie the inflammatory and osteolytic processes induced by particles of biomaterials resulting from the wear of implants such as total joint replacement prostheses. The objective this study was to investigate the role that the complement system may be playing in these phenomena. The aim was to evaluate the degree to which particles of selected orthopaedic materials—high density and ultrahigh molecular weight polyethylene, polymethylmethacrylate, and commercially pure titanium—cause the elevation of a key complement molecule, C3a, in an in vitro assay that directly measured the concentration of C3a. The results demonstrated that HDPE particles, at high concentration, are capable of causing the elevation of C3a in the in vitro assay. This finding is discussed in the context of other work and the mechanics of the complement system as it may affect the osteolytic process.

Introduction

Studies of osteolysis around joint replacement prostheses have generally focused on the response of phagocytes to particulate wear debris as the inciting cause [1]. Certain inflammatory mediators such as prostaglandin E2, (PGE2) [2], and interleukin-1 (IL-1) and IL-6 [3], released by macrophages—also referred to as histiocytes when they are tissue resident-phagocytes—as they phagocytose the debris, are known to be potent stimulators of bone resorption [4], [5]. Similar processes appear to explain the inflammatory response to other types of implants (e.g., temporomandibular disc replacement devices [6], [7], [8]). There are, however, other biological processes that could also be involved in wear particle-induced inflammation and osteolysis.

Exposure of blood to foreign surfaces such as those on wear particles, particularly the energetic surfaces of particles as they are being produced, can cleave certain molecules engaged in contact-activated host defense systems, including coagulation, fibrinolysis, and the complement system. The complement system [9] is composed of a group of more than 30 separate proteins present either in plasma, or as receptors on the surface of many cells, that have evolved to protect the host from invasion by foreign materials. Complement activation can occur by two distinct pathways, the classical and alternative pathways. The classical pathway is usually triggered by antigen–antibody complexes, whereas activation of the alternative pathway occurs spontaneously at a slow rate and is augmented by substances such as the polysaccharides of bacterial cell walls, endotoxins or artificial materials. Both pathways merge at the formation of an enzymatic complex, a C3 convertase, capable of cleaving the third complement component (C3), with generation of C3a and C3b. Surface-bound C3b, in conjunction with other complement factors, is responsible for cleaving C5 into C5a and C5b. The latter combines with C6, C7, C8, and C9 to form the multimolecular C5b-9 complex or terminal complement complex, which has cytolytic functions and can either react with the foreign surface or be inactivated in the fluid phase [10].

The smaller fragments C3a and C5a are anaphylatoxins that have multiple biological effects including the induction of the release of inflammatory mediators (including IL-1) from monocytes, basophils, and mast cells. C3b can facilitate aggregation of macrophages [11] and their adherence to other cells or particles bearing C3b on their surface and thus can play an important role in phagocytosis. C3b may also stimulate “frustrated phagocytosis” by phagocytes [12], a process which would result in degradative enzymes and high-energy oxygen species being secreted around the implant surface. Macrophage aggregation may lead to macrophage fusion and the formation of giant cells [13]. The accumulation of macrophages and giant cells in the periprosthetic tissues is one of the hallmarks of the particle-induced prosthesis failure, and the inflammatory products of monocytes and macrophages activated by complement molecules have been implicated in osteolysis following total joint arthroplasty (TJA). These striking similarities between the mechanisms implicated in osteolysis and the known biologic activities of C3a and C5a suggest that these inflammatory mediators may play a role in promoting osteolysis. A recent in vitro study [14] has demonstrated that polyethylene particles can in fact result in the elevation of levels of C3b, further prompting the current study, the objective of which was to evaluate the degree to which particles of selected orthopaedic materials—ultrahigh molecular weight polyethylene (UHMWPE), polymethylmethacrylate (PMMA), and commercially pure titanium (CPT)—cause the elevation of C3a in an in vitro assay that directly measured the concentration of C3a.

Physiologically significant complement activation that can propagate inflammatory reactions may not be detectable by immunoelectrophoretic techniques. The hemolytic assay for total complement (CH₅₀) is neither sensitive nor specific for any individual component proteins since deficiency in any of the components in the classical or terminal pathway can result in subnormal hemolysis. Measurement of individual components such as C3a (and to a certain extent other C3 activation products) is a more sensitive indicator of C3 activation than the other techniques. Sensitivity of radioimmunoassay for C3a is approximately 50 times greater than that of other techniques [15].