Phagocytosis of wear particles by perimplant macrophages results in cytokine release and osteoclast activation and osteolysis. Some investigators have proposed that this response may be mediated by adherent endotoxin. The aim of this study was to determine the role of endotoxin in modulating pro-inflammatory cytokine mRNA expression of macrophages when stimulated with titanium particles using relative quantitative real-time polymerase chain reaction (rqRT-PCR)

Human peripheral blood mononuclear cells were isolated from healthy subjects and plated in chamber slides. Three types of titanium particles were prepared; commercially pure titanium particles (cpTi), endotoxin stripped particles and endotoxin stripped particles with endotoxin (LPS) added back. Endotoxin levels of 450, 0 and 140 Eu/ml respectively were confirmed by high sensitivity Limulus Amebocyte Lysate assay. Macrophages were stimulated with particle concentrations of 0, 8.3, 83 and 830 particles per cell at time points 0 and 3 hours. LPS (200ng/ml) was used as a positive control. rqRT-PCR was performed using standard techniques.

Stimulation of human macrophages with cpTi demonstrated a significant dose dependent increase in TNFα, IL-1A, IL-1B and, IL-6. (Kruskal-Wallis p=0.01, p=0.017, p=0.001 and p=0.013 respectively). IL-18 mRNA levels were not increased (P> 0.05). The expression of mRNA following stimulation with the highest dose of titanium particles was similar to that following LPS stimulation. Endotoxin-free cpTi particles did not elicit any increase in mRNA expression above base line levels (P > 0.05, all cytokines). This lack of response was rescued in endotoxin-stripped particles with LPS added back. Particle dose dependent increases in cytokine mRNA levels were observed for TNFα, IL-1A, IL-1B and, IL-6 mRNA but not IL-18 (p=0.01, p=0.01, p=0.01, p=0.05 and p=0.> 0.05 respectively).

Our results show that adherent endotoxin plays a role in modulating particle induced pro-inflammatory cytokine mRNA expression in-vitro. Further study is required in evaluating the role of adherent endotoxin in vivo

Cytokine mediated activation of osteoclasts can lead to peri-implant osteolysis and aseptic loosening. The aim of this study was to determine the IL-1β and TNFα mRNA cytokine expression profile of human macrophages when stimulated with polyethylene particles using relative quantitative real-time polymerase chain reaction (rqRT-PCR).

Human peripheral blood monocytes or human monocytes from the cell line THP-1 were used in this study. rqRT-PCR conditions were optimized by stimulating human macrophages with 200ng/ml lipopolysaccharide (LPS). The median CV% value for duplicate measures was 12.6 (range 4.5–54). Stimulation assays were performed using unfractionated endotoxin-free commercial polyethylene particles (median size 7μm); or fractionated particles (size range 0.1–1.2μm). Human macrophages were stimulated with high dose unfractionated polyethylene particles at 0, 3500 or 10500 mm³/cell or with fractionated polyethylene particles at 0 and 100mm³/cell at time points 0 and 3 hours. Low dose unfractionated polyethylene stimulation was performed on THP-1 cells at 0, 50, 100, 1000 and 10000 mm³/cell. In all experiments LPS stimulation was used as a positive control. RNA was extracted and rqRT-PCR was performed using standard techniques

High dose unfractionated polyethylene stimulation did not result in a significant difference in cytokine mRNA levels between groups. Using fractionated polyethylene, a small increase in IL-1β mRNA was identified (21% versus maximal stimulation using LPS). Low dose unfractionated polyethylene stimulation of THP-1 cells demonstrated dose dependent decreases in TNFα and IL-1β mRNA expression that was not due to inhibition of RNA extraction or a decrease of cell viability.

Endotoxin-free polyethylene particles do not appear to be a major stimulus for IL-1β and TNFα mRNA production as measured by rqRT-PCR. We did observe a small positive effect on IL-1β mRNA expression using a fractionated polyethylene stimulus. However it remains unclear whether this effect is due to fractionation of particles into the submicron range or is due to introduction of endotoxin during the filtration process.

Aseptic loosening due to periprosthetic bone loss is a major cause of implant failure after total hip arthroplasty (THA). Interleukin 1-B (IL-1B) is thought to play a role in aseptic loosening by stimulating the activity of osteoclasts, the main bone resorbing cell type. A restriction fragment length polymorphism due to a C/T single base variation at +3954 in exon 5 of the IL-1B gene has been associated with differences in susceptibility to chronic periodontitis, a condition associated with bone loss. In this study we tested whether carriage of the C and T alleles at this site resulted in differential risk of aseptic loosening in 481 Caucasians (214 failed versus 267 radiologically intact implants) at 11.7± 4.1 years following primary cemented THA for osteoarthritis. Genomic DNA extracted from peripheral blood was genotyped using the Taqman 5′ nuclease method. Carriage rates were calculated and analysed using the 2 test.

In the intact implant group the frequency of the T allele was 0.253. The distribution of the C and T alleles was 147:105:15 (CC:CT:TT, respectively). In the failed implant group the frequency of the T allele was 0.241). The distribution of the C and T alleles was 124:77:13. The carriage rate of the T alleles in each group was 44.9% and 42.1%, respectively (odds-ratio P> 0.05). The genotype frequencies were in Hardy-Weinberg equilibrium for both intact and loose implant populations (Chi-squared P> 0.05).

Using the multivariate Cox proportional hazards model significant risk factors for loosening of both implant components included gender and age at THA (P< 0.05). However, carriage of the +3954 allele was not a significant independent risk factor for aseptic loosening (P> 0.05). Our data suggests that the IL-1B gene restriction fragment length polymorphism at +3954 does not influence the risk of aseptic loosening after THA.

Polyethylene wear particle-induced osteolysis is a major cause of implant failure after total hip arthroplasty (THA). Tumour necrosis factor (TNF) is a pro-inflammatory cytokine that is thought to play a pivotal role in this process. We have recently shown that carriage of the −238 ‘A’ allele in the TNF gene promoter is associated with a higher rate of osteolysis after THA versus carriage of the [more common] ‘G’ allele. The aim of this study was to determine the effect of this polymorphism on TNF gene transcriptional activation in response to polyethylene particle stimulation using a luciferase reporter gene assay.

A 691 bp fragment (−585 to +106) of the TNF gene was amplified by polymerase chain reaction and directionally cloned into the PGL3.basic vector (Promega, Madison, WI). Insert sequences were checked using an ABI 377 DNA sequencer (PE Applied Biosystems, Foster City, CA). RAW264.7 murine macrophage-like cells in rapid growth phase were transfected with plasmids containing either the TNF-238G allele or the TNF-238A allele. pTK-RL (Promega), that expresses the Renilla luciferase gene under the control of Herpes simplex virus minimal promoter, was used as a transfection control. The cells were then either left unstimulated or were induced using polyethylene particles generated from a hip simulator. Lipopolysaccharide (LPS) and LTA (Lipoteichoic acid) were used as positive controls. Luciferase reporter activity was measured after 4 hours (Dual luciferase assay, Promega Corp., Southampton, U.K.) and the relative firefly luciferase activity was calculated. Results were analysed using repeated measures ANOVA.

Polyethylene particle stimulation at concentrations of 0, 1, 15, and 30mg/mL resulted in relative luciferase activities (mean (SD)) of 21.4 (2.9), 36.2 (8.2), 45.9 (11.1), and 40.7 (5.1) for the −238A allele; and 19.7 (5.0), 26.4 (8.0), 35.9 (2.3), and 32.4 (2.4) for the −238G allele (ANOVA P=0.01). LPS and LTA stimulation also resulted in increased reporter activity for −238A versus −238G (ANOVA P=0.02 and P=0.04, respectively).

The promoter allele TNF-238A results in higher levels of transcriptional activation versus the TNF-238G allele in response to a clinically relevant stimulus, and provides functional evidence for the significance of this polymorphism in the development of osteolysis after THA.