Novel biological strategies for treatment of wear particle-induced periprosthetic osteolysis of orthopaedic implants for joint replacement

1. Introduction

Total joint replacement (TJR) of the lower extremity is a highly successful surgical procedure for alleviating pain, and improving ambulation and function for patients with end stage arthritis of the hip and knee. However, all TJR implants undergo wear of the bearing surfaces, producing particulates and other by-products of the different materials used in the surgical reconstruction [1]. These particulates, in sufficient amounts, may lead to the destruction of bone in the implant bed (periprosthetic osteolysis) jeopardizing the longevity of the implant. This may necessitate further surgery to replace worn parts, complex revision surgery to excise loose implants and re-implant new ones, or the fixation of pathologic fractures dues to bone degradation.

Debris from polymers such as polyethylene (PE) and polymethylmethacrylate (PMMA), metals and ceramics are capable of inciting an adverse tissue reaction, which is orchestrated by cells of the monocyte/macrophage lineage. Particles and their by-products are phagocytosed by macrophages and other local cells, which become activated [2–5]. This results in the release of pro-inflammatory substances such as cytokines, chemokines, prostanoids, degradative enzymes, reactive oxygen species and other factors [6–12]. This chronic inflammatory scenario leads to upregulation of pathways leading to bone destruction, and downregulation of bone formation [13–15]. Thus, the normal homoeostatic mechanisms that lead to continued successful coexistence between the implant and the surrounding musculoskeletal tissues is perturbed [16,17]. These events may lead to painful synovitis and impaired function including restriction of range of motion, instability and loosening of the implant, and pathologic fracture of the surrounding bone.

There are no pathognomonic laboratory tests to diagnose ongoing periprosthetic osteolysis. Surgeons rely on the clinical presentation and periodic serial radiographic imaging studies, which may demonstrate evidence of progressive wear of the bearing surfaces, osteolysis, loosening of the prosthesis or fracture [18,19]. In some cases, these events can be detected when the prosthesis is still salvageable, that is, still well fixed and asymptomatic, such as when early wear and minimal bone destruction have occurred. Although weight loss and activity restriction may theoretically slow down the wear rate, these options have proved to be impractical. To date, no biological approaches to curtailing the chronic inflammatory reaction associated with wear particles have proved successful clinically, owing to the plethora of inflammatory mediators that are upregulated by particles, redundancy of inflammatory pathways and potential adverse effects of systemic treatments [20,21].

Although the chronic inflammatory reaction to implant degradation products is primarily a local phenomenon, remote inflammatory and mesenchymal cells migrate to the anatomical location where the particles are generated [22–25]. This cellular trafficking occurs along chemokine or chemorepellent gradients produced by local cells exposed to the by-products of wear. Systemic treatments attempting to limit one pro-inflammatory cytokine or induce osteoclast apoptosis have been unsuccessful in mitigating osteolysis. In vitro and in vivo models that use some novel therapies to modulate particle-induced inflammation, for example gene therapy, risk potential adverse events [26].

Our group has approached the problem of osteolysis due to wear particles as a local biological phenomenon that could theoretically be modulated by local rather than systemic treatment. Three experimental approaches have been taken to potentially mitigate the adverse biological sequela of particle disease. These include (i) interfering with ongoing migration of monocyte/macrophages to the implant site by inhibiting the chemokine–receptor axis [23,27], (ii) altering the functional activities of local macrophages by converting pro-inflammatory M1 (classically activated pro-inflammatory) macrophages to an anti-inflammatory pro-tissue healing M2 phenotype [28–30], and (iii) modulating the production and release of pro-inflammatory chemokines, cytokines and other potentially harmful factors by inhibiting the key transcription factor nuclear factor kappa B (NF-κB) ([31–33]; figure 1).

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2. Interfering with ongoing migration of monocyte/macrophages to the implant site by modulating the chemokine–ligand–receptor system

In the process of periprosthetic osteolysis, the complex underlying network of chemokines is mainly driven by macrophages [34]. The production of polymer wear particles leads to a non-specific macrophage-mediated chronic inflammatory and foreign body reaction [35] in which both local and systemic macrophages are involved. This ultimately leads to dysregulation of bone formation and resorption favouring the latter. Local macrophages are activated by particles either by cell membrane contact through surface receptors, such as CD11b, CD14, toll-like receptors (TLRs), or through phagocytosis of wear debris. Macrophage activation takes place through different intracellular pathways: myeloid differentiation primary response gene 88 (MyD88) and p38 mitogen-activated protein kinase (MAP kinase) and others which in the end, activate nuclear factors, most importantly NF-κB. Transduction of nuclear factors induces release of pro-inflammatory cytokines (tumour necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, prostaglandin E (PGE)-2, macrophage colony-stimulating factor (M-CSF), receptor activator of NFκB ligand (RANKL), etc.) [36,37]. These factors act in an autocrine and paracrine way [38] to induce a series of biological events such as recruitment of more macrophages and osteoclast precursors, their differentiation [39] and further release of cytokines, some of which have chemotactic properties (chemokines) [40]. These chemokines belong to a large family of active biomolecules [41,42] that are directly dedicated to the migration of monocyte/macrophage lineage cells and other cells. Locally activated macrophages release monocyte chemoattractant protein-1 (MCP-1, also called CCL2) [34]. MCP-1 (human gene 17q11.2) belongs to the γ-chemokine subfamily (C–C chemokines) and is an immediate early stress-responsive factor [43]. MCP-1 is primarily involved in the systemic recruitment of pro-inflammatory cells (monocytes, neutrophils and lymphocytes) [44]. Once released in the bloodstream, MCP-1 binds its receptors (G-protein-coupled receptors) CCR2A and CCR2B (human gene ID 1231), with preference for CCR2B expressed by monocytes and activated natural killer lymphocyte (NK) cells [44–46]. With regards to bone, MCP-1 is also expressed by osteoblasts and osteoclasts. Huang et al. [47] have shown in vitro that when murine macrophages (RAW cells 264.7) were challenged with ultra-high molecular weight PE (UHMWPE) and PMMA particles, MCP-1 was released at fourfold higher levels than the constitutional level of secretion. Moreover, they showed that the conditioned media-induced chemotaxis of human macrophages and mesenchymal stem cells (MSCs) and that this chemotaxis was blocked with an MCP-1 neutralizing antibody. Similarly, when exposed to PMMA and titanium (Ti) particles, human fibroblasts released increased amounts of MCP-1 [48]. Nakashima et al. [27] produced similar findings using human monocytes/macrophages exposed to Ti-alloy and PMMA particles. High levels of MCP-1 as well as macrophage inflammatory protein-1α (MIP-1α, also called CCL3), another chemokine, were found after exposure to these particles. When MCP-1 and MIP-1α neutralizing antibodies were added to the media from cultures exposed to PMMA particles and Ti-alloy, the chemotactic activity of these chemokines was inhibited.

Recent studies have subsequently demonstrated in vivo systemic migration of macrophages to wear particles deposited at a remote site. Ren et al. [49] using in vivo bioluminescent imaging showed systemic trafficking of macrophages to PMMA particles injected into the mouse femoral shaft. Macrophages were also found to migrate to sites with UHMWPE injection: this has been shown using either a single bolus of UHMWPE particles in the femoral shaft [50] or using a continuous infusion of UHMWPE particles through a micro-diffusion pump [22,51,52], a more clinically relevant model. The next step was to link in vivo MCP-1 secretion and macrophage recruitment in the presence of particles. In a recent in vivo study [23], a single injection of MCP-1 was placed in the femoral shaft of mice and genetically modified reporter macrophages were remotely injected intravenously in the tail vein. The macrophages were genetically modified to express the bioluminescent optical reporter gene firefly luciferase (fluc) so that their migration could be tracked by bioluminescence at different time intervals. Signal intensity and subsequent immunohistochemistry confirmed the systemic recruitment of macrophages to the femur induced by local injection of MCP-1. Furthermore, after injection of the MCP-1 receptor antagonist (acting on the CCR2B receptor) associated with a continuous infusion of UHMWPE particles, the systemic migration of macrophages was dramatically reduced. This indicated that the MCP-1-CCR2 ligand–receptor axis is strongly involved in the particle-induced periprosthetic osteolysis. Bone mineral density demonstrated decreased bone deposition in the group receiving particles but no antagonist; this finding was reversed when either a MCP-1 receptor antagonist was given, or CCR−/− reporter cells were infused in the tail vein. Systemic trafficking of MSCs in the presence of UHMWPE particles was affected by the interruption of the MIP-1α-CCR1 ligand–receptor axis [53]. Taken together, these findings suggest that strategies that interfere with cell recruitment may provide a potential method for modulating the inflammatory reaction to orthopaedic implants and their by-products. Technologies, for example bioactive orthopaedic implant coatings, may have a role to play in increasing prosthesis longevity, by modulating cell trafficking to the bone–implant interface [54].

3. Targeting macrophage polarization as a means to mitigate wear particle-induced osteolysis

3.3. Modulation of macrophage polarization as a means to mitigate wear particle-induced osteolysis

As macrophage polarization has profound impact on expression of TLRs and other pattern-recognition receptors (PRRs), on the macrophages ability to produce pro- and anti-inflammatory cytokines and can impact the macrophage's capacity to support tissue healing, modulation of macrophage polarization and thus its activation state would seem as a viable strategy to mitigate wear particle-induced inflammation. Indeed, evidence is emerging that not only particles per se but also macrophage polarization plays an essential role in how macrophages respond to biomaterial particle challenge.

Trindade et al. [99] previously reported that IL-4 treatment reduced the production of TNF-α, IL-1β and GM-CSF from PMMA-particle-stimulated human peripheral blood monocyte-derived macrophages in a dose-dependent manner. Im & Han [100] demonstrated similar suppressive effect of IL-4 treatment on production of TNF-α and IL-6 from human peripheral blood monocytes stimulated with retrieved Ti6Al4V alloy wear particles.

More recently, Rao et al. [28] investigated the sequential modulation of macrophage phenotype in vitro using mouse bone marrow-derived macrophages and found that IL-4 administration even after PMMA particle challenge was sufficient to reduce particle-induced TNF-α production. This effect was more pronounced if particles were delivered together with LPS but reduction in TNF-α production was observed also in the PMMA-only group. Thus, sequential modulation of macrophage phenotype from an M1 phenotype to an M2 phenotype is a possible strategy to reduce particle-induced inflammation; in fact, some aspects of the M2 phenotype, including the production of IL-Ra, Ym1 and Arginase 1 were more evident if macrophages had first been activated into the M1 phenotype with LPS or possibly PMMA particles and then polarized into M2-phenotype with IL-4.

Pajarinen et al. [101] compared Ti particle responses of polarized human M0, M1 and M2 macrophages using genome-wide microarray expression profiling, qRT PCR and protein suspension array. In comparison to M0 macrophages, the overall chemotactic and inflammatory responses to Ti particles were greatly enhanced upon challenge of M1 macrophages but effectively suppressed when M2 macrophages were challenged. The authors concluded that the mode in which macrophages responded to particle stimulus is dependent on the polarization status of the macrophages and that induction of M2 polarization might be a means to limit particle-induced macrophage activation.

Antonios et al. [102] investigated the dynamics of macrophage polarization and PMMA particle stimulation using mouse bone marrow macrophages. IL-4 treatment reduced PMMA-particle-induced TNF-α production, as previously noted. The effect was most prominent if IL-4 was applied to the cells before, rather than concurrently with the PMMA stimulus; the production of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1Ra) was highest if macrophages had first passed from the M0 to the M1 state before being further polarized into an M2 phenotype.

In addition to these in vitro studies, some recent in vivo studies have investigated the effect of IL-4 treatment on wear particle-induced osteolysis. Using a mouse calvarial model of particle-induced osteolysis, Rao et al. observed that daily IL-4 injections to the subcutaneous bursa overlying the calvaria significantly reduced PE-particle-induced osteolysis as assessed by micro computed tomography (µCT) and histomorphometry [29]. The number of tartrate resistant acid phosphatase (TRAP) positive osteoclasts was reduced to the level of controls in the IL-4-treated group. In addition, prior IL-4 treatment reduced particle-induced TNF-α and RANKL production from calvarial samples cultured ex vivo. Using western blot analysis of calvarial proteins using iNOS as M1 marker and Ym1 as M2 marker, increased M1 to M2 ratio was observed in PE particle-treated group while in IL-4 treatment returned this ratio to that of negative controls. Thus, IL-4 treatment reduced particle-induced osteolysis by modulating macrophage activation from M1 towards M2-like macrophage phenotype.

Wang et al. [103] observed similar phenomenon in a mouse air pouch model of PE-particle-induced osteolysis; daily IL-4 or IL-13 injections reduced particle-induced bone collagen loss and the area of bone surface covered by TRAP-positive osteoclasts. Corresponding reduction in the production of RANKL and TRAP and increase in osteoprotegerin production was observed in IL-4-treated groups using both RT-PCR and ELISA. Interestingly, these effects were more pronounced if both IL-4 and IL-13 were administered rather than IL-4 or IL-13 alone. However, no analysis of the local macrophage phenotype was performed.

Taken together, current in vitro and in vivo data would seem to support the hypothesis that macrophage polarization is an essential factor that determines how macrophages respond to biomaterial particle challenge, and that local modulation of the macrophage phenotype appears to be an effective means to limit biomaterial wear particle-induced inflammation and subsequent osteolysis (figure 2).

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Prior observations note that not only are the inflammatory wear particle responses suppressed in M2 macrophages, but they are also aggravated in M1 macrophages. This phenomenon was first reported by Trindade et al., who showed that IFN-γ pre-treatment enhances the production of TNF-α and IL-6 from human monocytes stimulated with PMMA particles and later confirmed by Pajarinen et al. showing that the overall inflammatory and chemotactic response to particles was enhanced if macrophages had first been polarized into M1 phenotype [101,104]. The potential significance of these in vitro observations remains to be determined; however, they conjure some interesting hypotheses. For example, it is possible that chronic, low-grade inflammation in other anatomic locations caused by atherosclerosis, metabolic syndrome, periodontitis or other conditions might predetermine the systemic M1–M2 balance of macrophages and thus the mode that local macrophages react to wear particles. These other conditions may impact the susceptibility of an individual to develop aseptic osteolysis. Future studies will explore the correlation among individual patient characteristics, macrophage polarization and the reaction to orthopaedic implants and their by-products.

4. Suppression of wear particle-induced periprosthetic osteolysis via direct targeting NF-κB

Despite their essential role in wear particle-induced osteolysis, blocking individual pro-inflammatory cytokines, for example TNF-α, did not mitigate osteolysis in a clinical study [105]. The failure of such therapies may be due to the compensatory role of other wear particle-induced cytokines that can activate or be activated by NF-κB [106,107]. NF-κB signalling can be activated in macrophages and osteoclasts by exposure to wear particles [28], leading to the hypothesis that targeting NF-κB activity could be a potential strategy to mitigate osteolysis (figure 3).

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The core transcriptional component of the canonical NF-κB pathway is composed of RelA/p50 heterodimer, which is usually in an inactive state through association with IκB (inhibitor of κB) protein. Once the signal is activated, the IκB protein is phosphorylated by the IKK (IκB kinase) which is then degraded through ubiquitination. The released RelA/p50 dimers then translocate into the nucleus, bind to the response element and modulate the target gene expression. The strategy of direct NF-κB inhibition includes modulation of (i) IKKα/β kinase activity, (ii) IκB protein stability, and (iii) the transactivation of the heterodimer RelA/p50. IKKα/β is one of the most common targets for NF-κB inhibition [108]. Suppression of IKKα/β activity can be achieved by using IKKα/β-specific ATP analogues [109], expression of a dominant negative form [110] or blocking the IKKα/β interaction with IKKγ [111]. A previous study showed that inhibition of IKKα/β kinase activity can block PMMA-particle-induced osteolysis in a murine calvarial model [111]. Targeting IκB via prevention of protein degradation or over-expression of IκB protein is also an efficient strategy to modulate NF-κB activation [110,112].

The NF-κB heterodimer transactivation process includes nuclear translocation, specific DNA binding to the target sequence and enhancement of target gene transcription. Among these, suppression of the DNA binding ability via decoy oligodeoxynucleotide (ODN) is one of the most potent and specific ways to suppress NF-κB transactivation. Decoy ODNs are short synthesized duplex DNAs that mimic the transcription response element and can specifically suppress transcription factor activity via competitive binding with endogenous protein [113]. Although the low bioavailability and short half-life of decoy ODN may limit its clinical application, recent findings suggest that decoy ODNs can be improved by chemical modifications for example replacing the phosphodiester bond with a phosphothioate bond [113]. Application of decoy ODNs has been shown to prevent NF-κB transactivation of pro-inflammatory cytokine genes in primary cultured macrophage [114]. In addition, NF-κB ODN has also been used in several inflammatory disease animal models, including chronic obstructive pulmonary disease [115], vascular and cardiovascular disease [116–119], liver injury [120], atopic dermatitis [121] and periodontal disease [122].

To optimize the NF-κB inhibition strategy, the delivery route, timing and dosage, and potential toxicity should all be considered. As wear particle-induced osteolysis is generally an anatomically confined disease, local treatment could be an ideal way to prevent the undesired effect on the normal host immune system [123]. On the other hand, blocking NF-κB activation in a confined microenvironment may also suppress the protective effects on osteolysis. For example, the type I collagen in osteoprogenitor cells [124] and the IL-10 secreted by MSCs [125] are both targeted by NF-κB [124,126]. Recent studies have suggested that inhibition of NF-κB can enhance osteogenic differentiation in periodontal ligament stem cells or MSCs via suppression of β-catenin signalling [127–129]. Therefore, the overall beneficial effects among different cell types must be carefully evaluated.

Crosstalk and potential compensatory regulation between the NF-κB pathway and other transcription factors may be critical in order to optimize the therapeutic efficiency of NF-κB ODN. For example, suppression of NF-κB activity may unexpectedly enhance cytokine expression owing to enhanced AP-1 transactivation [130] or block the negative feedback regulator, TNF-α-induced protein 3 [131]. In addition, wear particles can activate NF-κB and nuclear factor interleukin-6 (NF-IL-6) transcriptional activity [31]; both of these transcription factors have essential roles in pro-inflammatory cytokine regulation. Thus, further preclinical studies are necessary to establish the efficacy and safety of local delivery of NF-κB ODN for the treatment of periprosthetic osteolysis.

5. Discussion

The by-products of wear of the bearing surfaces of current TJRs stimulate an inflammatory response that may result in chronic synovitis, periprosthetic osteolysis, implant loosening and pathologic fracture. These processes are initiated in otherwise well-functioning joint replacements. Although many advances have been made in bearing materials, implant technology and design, and surgical technique, revision surgeries due to implant wear are still commonplace. As joint replacements are being extended to younger, more active individuals who will potentially live for many decades, the wear problem will become even more complex in the future. Especially, troublesome is the patient with radiographic wear of their joint replacement and synovitis or early osteolysis, but the implant is not at the point of needing revision surgery. Are there potential interventions to modulate the inflammatory environment to extend the lifetime of the implant in an efficacious and safe manner?

Systemic treatments to date have been shown to be ineffective in humans, and may be associated with adverse effects on other body systems [21,105]. For example, systemic inhibition of specific cytokines such as TNF or IL-1 with biologics may result in blunted reactions to harmful agents such as bacteria and other infective agents, and might even interfere with the eradication of cancer cells. Bisphosphonates inhibit osteoclast-mediated bone degradation associated with particle disease, but may also lead to pathologic femoral fracture, mandibular lesions, impairment of fracture healing and other adverse events [132,133].

Our approach has been to work upstream in the particle-induced inflammatory cascade and use local treatments and interventions, if possible. This has involved interfering with macrophage migration from remote sites to the area of particle generation, changing the local macrophage polarization phenotype from a pro- to an anti-inflammatory one involved in repair and inhibiting the release of numerous pro-inflammatory chemokines, cytokines and other substances by interfering with the transcription factor NF-κB.

Interference with the MCP-1-CCR2 chemokine ligand–receptor systemic is novel and may have a direct clinical application. Surgical placement of a prosthesis evokes an inflammatory reaction initially that may eventually compromise the prosthesis–bone interface. Mitigating this early inflammatory reaction may provide a more robust bone–implant interface initially, which can function as a physical deterrent to particle and inflammatory mediator migration throughout the ‘effective joint space’ [134]. Potential therapies to accomplish this goal may be delivered as an injected or infused biologic, or a coating on the prosthesis or bone bed during initial prosthesis implantation. Using a layer-by-layer technique or other drug delivery platforms, the biologic may be released according to a desired profile for a time period of seconds to weeks. Further research is necessary to determine the appropriate time period and release profile.

The identification of different macrophage phenotypes is a recent discovery with great potential to modulate the processes involved in inflammation and its resolution [135]. Damage to adjacent normal ‘bystander’ cells and tissues is an unwanted by-product of the inflammatory cascade. Modulation of the intensity, timing and duration of events associated with acute inflammation, and avoidance of potentially persistent and injurious chronic inflammation could ostensibly alter our conceptual thinking regarding orthopaedic implants. With regards to the prosthesis interface, local delivery of IL-4 or -13 might be a sound therapeutic strategy to convert M1 pro-inflammatory macrophages into an M2 anti-inflammatory pro-tissue healing phenotype [28,29,136]. This could be performed with ‘minimally invasive’ techniques, for example local injection in the radiology suite; this treatment could potentially be repeated as long as the prosthesis is salvageable.

Once activated, macrophages and other cells in the prosthetic bed demonstrate upregulation of NFκB, the most important transcription factor with over-reaching control over pro-inflammatory factor synthesis and release, and osteoclastogenesis and function. Others have shown that inhibiting NF-κB activation by attenuating the assembly of the IKK complex (using a short peptide termed NEMO-binding domain peptide, that blocks binding of IKK2 and IKK1 to IKKg/NEMO) is effective in inhibiting osteoclastogenesis and PMMA-induced osteolysis in mice [111]. Our novel approach uses an NF-κB decoy ODN to interfere with the functionalization of NF-κB. This ODN can be locally injected, infused or coated onto a surface, and has been shown to be safe and effective in vitro. Ongoing in vivo studies will help substantiate whether this approach is translatable to the clinical situation.

The continuous production of wear particles is expected, whether the joint is natural or artificial. In general, the body's homoeostatic mechanisms can deal with this foreign material without the development of overwhelming local chronic inflammation, which may lead to bone loss and implant loosening. The novel strategies outlined above may have a potential role when periprosthetic inflammation is ongoing but the joint replacement is still functional and salvageable.

Funding statement

This work was supported by NIH grant nos. 2R01AR055650-05 and 1R01AR063717; the Ellenburg Chair in Surgery at Stanford University; the Stanford Medical Scholars Program, the Sigrid Jusélius Foundation, HUS evo grant, Orthopaedic Hospital of the Invalid Foundation, Finska Läkaresällskapet, Academy of Finland.