Considerable evidence exists that aseptic loosening is initiated by wear particles that recruit macrophages and stimulate their production of pro-inflammatory cytokines. The cytokines primarily act indirectly by inducing production of RANKL, which stimulates osteoclast differentiation, osteolysis, and inflammatory bone loss. There is also considerable evidence that activation of macrophage Toll-like Receptors (TLRs) contributes to this cascade of events. It is however controversial whether bacterially-derived immunostimulatory molecules known as Pathogen-Associated Molecular Patterns (PAMPs) can contribute to aseptic loosening by stimulating their cognate TLRs on macrophages. Priming and subsequent activation of the NLRP3 inflammasome is essential for macrophage production of mature, active IL-1β in response to wear particles. We recently confirmed that wear particles can activate pre primed NLRP3 inflammasomes in the absence of PAMPs. Thus, activation of the NLRP3 inflammasome is the only macrophage-based event in the aseptic loosening cascade that we have found to date is independent of PAMPs. In contrast, priming of the NLRP3 inflammasome by wear particles requires PAMPs as well as their cognate TLRs. These results add to the growing body of evidence that bacterially-derived PAMPs can contribute to aseptic loosening.

Aseptic loosening is a major cause of revision surgeries and occurs when osteolysis is stimulated around the implant by pro-inflammatory cytokines including IL-1β. Production of active IL-1β in response to orthopedic wear particles depends on processing by the NLRP3 inflammasome which requires priming followed by activation. We found that pathogen associated molecular patterns (PAMPs) adherent to wear particles are necessary to prime the NLRP3 inflammasome. In contrast, in pre-primed macrophages, particles themselves are sufficient to activate the NLRP3 inflammasome and induce secretion of active IL-1β. Particles themselves also induce cell death, kinetically preceding the release of active IL-1β. Phagocytosis of particles is required to initiate both responses as the phagocytosis inhibitor cytochalasin blocks cell death and IL-1β release. Lysosome membrane destabilization is also critical as inhibition of lysosomal function with bafilomycin or chloroquine significantly abrogated the release of active IL-1β and cell death in response to wear particles. The pan-cathepsin inhibitors Ca-074-Me or K777 also inhibit cell death and IL-1β release indicating that cathepsin release from lysosomes is also a necessary step in the particle-induced response. Our results open the possibility of clinical intervention with lysosomal or cathepsin inhibitors to treat aseptic loosening as these drugs have better specificity and less in vivo toxicity than the phagocytosis inhibitors. Testing of these inhibitors in vivo in models of particle induced osteolysis is a key future direction.

Introduction:

Wear particles cause aseptic loosening by stimulating macrophages to produce inflammatory cytokines. Recent studies indicate that Toll-like receptor 2 (TLR2) and TLR4 mediate macrophage responses to the wear particles [1–3]. TLR2 and TLR4 uniquely activate MyD88-dependent signaling via an additional adapter protein known as TIRAP/Mal [4]. Del Vescovo et al reported that three single nucleotide polymorphisms (SNPs) within the TIRAP/Mal gene associate with aseptic loosening in THA patients [5]. The goal of the current study was therefore to determine whether TIRAP/Mal mediates responses to orthopaedic wear particles.

Total hip arthroplasty (THA) wear debris induced macrophage expression of pro-inflammatory cytokines has been associated with osteolysis both in vitro and in animal and human subjects. Interleukin-1 receptor antagonist (IL-1RA) is an anti-inflammatory cytokine which may limit bone destruction. Polymorphisms (SNPs) within the IL-1RN gene are associated with differences in susceptibility to infectious and inflammatory conditions and disorders of bone remodelling. This study investigated the association between the IL-1RA+2018T/C SNP (rs419598) and osteolysis after THA, and with mRNA and protein expression in an in-vitro wear debris-macrophage stimulation assay.

611 North European Caucasians who had received a cemented THA for primary osteoarthritis were genotyped for the IL-1RN+2018 SNP using Taqman methods. 62 subjects with a Charnley THA were selected from the genotyping population. Control subjects had no radiographic osteolysis and the osteolysis group had previously undergone revision surgery for aseptic loosening. Peripheral blood mononuclear cells were extracted and stimulated with endotoxin-stripped titanium particles (TiCL, endotoxin level 0 Eu/ml) and endotoxin-stripped particles with adherent LPS added back (TiAB, endotoxin level 140 Eu/ml); non-stimulated and LPS-stimulated cells were used as negative and positive controls. Cell lysate IL-1RA mRNA levels were assessed by rqRT-PCT following a 3-hour stimulation. Cell supernatant IL-1RA protein levels were assayed after 24 hours stimulation using a multiplex method.

The IL-1RN+2018C allele was underrepresented in patients with osteolysis after THA versus control THA subjects (chi-squared test 5.96, P=0.015). After correction for other risk factors for osteolysis, the adjusted odds ratio for osteolysis associated with carriage of the IL-1RN+2018C SNP was 0.69 (0.48 to 0.99, p=0.048). IL-1RA mRNA expression increased linearly with IL-1RN+2018C allele copy number in gene-dose dependent manner (ANOVA p=0.013). The IL-1RA+2018C allele did not significantly affect IL-1RA protein expression (ANOVA p> 0.05), however a similar trend towards increased levels with increased C allele copy number was observed.

Carriage of the IL-1RA+2018C allele is associated with both a decreased risk of osteolysis after THA and increased IL-1RA mRNA expression in-vitro. The mechanism for this functional effect remains unclear, however these findings support the importance of the IL-1RA in osteolysis and aseptic loosening.

Introduction: Immune responses in patients susceptible to aseptic loosening may differ to those without this susceptibility. We compared stimulated cytokine mRNA and protein expression in peripheral blood mononuclear cells (PBMC) in 34 subjects (M:F 16:18; mean age 75 years) with previous revision surgery for aseptic loosening versus 28 subjects (14:14; 75 years) with well-fixed implants after Charnley THA for osteoarthritis.

Methods: Extracted PBMCs were stimulated with endotoxin (LPS 200ng/mL), endotoxin-free titanium particles (Ti, endotoxin level =0 Eu/mL), or particles with adherent LPS (TiLPS, 140 Eu/mL). Cell lysate IL-1α, IL-1β, IL-1RA, IL-6, IL-10, IL-18, and TNF mRNA were assayed after 3 hours stimulation using standard rqRT-PCR techniques. Cell supernatant IL-1β, IL-1RA, IL-6 and TNF protein were assayed after 24 hours stimulation using a multiplex method.

Results: mRNA and protein levels in non-stimulated cells were lower in revision versus control subjects for all cytokines (p< 0.05 all analyses). mRNA expression relative to baseline was greater in revision subjects versus controls for all cytokines and all modes of stimulation (LPS, Ti, and TiLPS, p< 0.05 all analyses). LPS induced the greatest inflammatory cytokine response at both the mRNA and protein level in both groups, TiLPS particles induced a more attenuated response, and responses to Ti particles were weakest. In the control group endotoxin free particles showed a negative cytokine mRNA response for IL-1α, IL-1β, and IL-6 (p< 0.05), and reduced protein levels for IL-1β, IL-1RA, IL-6, and TNF versus non-stimulated cells (p< 0.05).

Discussion: Patients with a susceptibility to aseptic loosening have lower baseline but greater stimulated immune responses versus patients without loosening that may contribute to the pathogenesis of aseptic loosening.

Introduction and Aims: Accumulating evidence suggests that bacterially derived endotoxins may contribute to aseptic loosening. This study determined whether lipopolysaccharide (LPS), the classical endotoxin from Gram-negative bacteria, can be detected in periprosthetic tissue from patients with aseptic loosening. We utilised an assay that detects all forms of LPS and is unaffected by beta-glucan-like molecules.

Method: Periprosthetic tissue from revision total hip arthroplasty and synovia from primary total joint arthroplasty were homogenised in PBS in endotoxin-free conditions. Non-specific amidases in the homogenates were inactivated at 100 degrees C. LPS was measured using the Endospecy assay (Associate of Cap Cod). Multiple dilutions of the homogenates were assayed to maximise sensitivity, while avoiding assay inhibition assessed by spike recovery determinations. Results were corrected for colour and spike recovery. Assay results were considered positive if the absorbances were higher than the lowest standard and the LPS level was significantly greater (p< 0.05) than the PBS control. Statistical analysis was by ANOVA with Bonferroni-Dunn (Control) post-hoc tests.

Results: Samples from 13 patients have been studied to date. Multiple assays of four of these samples showed no detectable LPS while nine of these samples resulted in both positive and negative assays. This inter-assay variability prevents measurement of the concentration of LPS in the samples. Nonetheless, many of the samples contain detectable amount of LPS. Thus, six out of eight samples from revision THA patients with aseptic loosening had positive assays, as did two of four primary TJA patients. LPS was also detected in a sample from a revision control. These results demonstrate that samples from THA patients with aseptic loosening and from primary TJA contain detectable amounts of LPS derived from Gram-negative bacteria.

Conclusion: This conclusion is consistent with numerous studies, showing that human serum contains LPS derived from minor infections, gut flora, or dental procedures. It is likely that many of these samples also contain molecules derived from Gram-positive bacteria that have very similar biological effects as LPS. However, detection of these Gram-positive molecules await further improvements in assay specificity and sensitivity.