Introduction: In attempting to unravel the complex cellular responses leading to prosthetic loosening investigators have been limited to studying gene expression of extracellular molecules about which most is known whereas new microarray technology allows simultaneous expression profiling of thousands of genes from a complex sample such as the membrane formed around loosened hip prostheses.

Methods: Two groups of 8 patients were recruited who have undergone primary total hip arthroplasty for osteoarthritis and subsequently developed either septic or aseptic loosening +/− osteolysis. The control group consisted of one group of 5 patients with the same initial diagnosis who had undergone identical procedures, developed no clinical or radiological signs of aseptic or septic loosening, but had come to revision surgery for other complications as defined by the Swedish Hip register: fracture without previous osteolysis, dislocation, technical error, implant fracture, polyethylene wear or pain. Periprosthetic membrane was harvested at the time of revision surgery and subjected to RNA extraction. cDNA was then synthesized and hybridised to a Human Genome u95 Genechip ® array which contains a complete set of known human genes. Data normalisation, data filtering and pattern identification was performed using Genechip®3.1 software (Affymetrix, Santa Clara, CA).

Results: This has revealed the involvement of a large number of genes coding for transcriptional regulators upstream from the extracellular and cell-cell signalling molecules already known to be involved in osteolysis and deep infection and which may ultimately control the responses to wear particles and bacterial challenge. Differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation and immune response was found. Most interestingly pathways for control of local bone resorption and inflammatory response have been shown to be highly activated.

Conclusions: The identification of these new pathogenetic mechanisms of total hip replacement failure make new indicators of disease susceptibility and prognosis plus new drug targets direct possibilities.

Introduction: Our group has previously reported on microarray gene expression profiling of failed aseptic and septic THRs. The data obtained from the Affymetrix DNA chips suggested a range of 21 differentially expressed genes between the tissue samples obtained from the control and study patients with failed aseptic THRs. The variation in expression that was demonstrated did not suggest that the basis of the local tissue reaction that occurs in aseptic loosening of THR is primarily inflammatory in nature. In order to validate these results we have performed quantitative real-time polymerase chain reaction (RT-PCR) to analyse the transcriptional levels of genes expression in the samples used in our original study and to formulate a hypothesis of how these candidate genes can be related to aseptic join loosening.

Methods: 3 control and 6 aseptic samples of peri-prosthetic membrane were subjected to RNA extraction. RNA quality analysis and quantification were performed. SYBRâ Green I real time quantitative PCR (RT qPCR) assays were designed using Primer Express [Applied Biosystems] and BLAST searching the resulting sequences. The comparative method for quantitation of gene expression levels, which utilizes arithmetic formulas to give the similar results to those achieved with standard curves, was utilised to validate the cDNA microarray data.

Results: We were able to devise successful quantitative real-time PCR for 15 of the 21 candidate genes plus the reference gene GAPDH. The genes coding for complement component C4B, Osteonectin , ATP2A2 (an ATPase linked to the regulation of adhesion, differentiation and proliferation in tissue that expresses this gene such as bone) and Phospholipase2A, were all found to be under-expressed whereas SLC2A5 (a solute carrier that can facilitate glucose/fructose transport)and NPC1 (intimately involved in cholesterol and glycolipid trafficking and inversely related to PLA2-mediated release of eicosanoids such as PGE2) were found to be over-expressed.

Conclusions: The data from our gene expression and RT-PCR studies have suggested novel pathways that may be intimately involved in the development of peri-prosthetic osteolysis and aseptic loosening that are distinctly different from the currently accepted theory of a proinflammatory cytokine cascade initiated by tissue reaction to particulate wear debris. These include possible alteration in both extra- and intracellular Ca2+ metabolism together with a possible effect upon extra-cellular matrix function. Altered lipid metabolism may also be evident and in particular decreased eicosanoid production. Intriguingly, the pattern of gene expression that is seen our studies would appear to be quite different than that seen in synovial inflammatory arthritidies such as rheumatoid and osteo-arthritis and suggests that previous studies that has used these pathological mechanisms as comparisons or controls may be flawed.

Aims: Tumour necrosis factor-alpha is a proinflammatory cytokine that has been implicated in the inflammatory response to bacterial infection and wear debris particles around loosened total hip replacements (THR). Individual TNF responses to such stimuli may be dictated by genetic variation and we have studied the effect of single nucleotide polymorphisms (SNPs) within the TNF gene.

Methods: We performed a case control study of 9 SNPs (−1031, −863, −857, −376, −308, −238, +489, +851 and +1304) for possible association with deep sepsis or aseptic loosening. All patients included in the study were Caucasian and had had a cemented Charnley THR. Cases consisted of 44 patients with early aseptic loosening and 30 patients with microbiological evidence at surgery of deep infection. Controls consisted of 85 THRs that were clinically asymptomatic for over 10 years and demonstrated no radiographic features of aseptic loosening. DNA was extracted from venous blood and genotyped by Snapshot assay.

Results: Genotype and allele frequencies for all SNPs were in Hardy-Weinberg equilibrium between THR controls and a random sample of UK Caucasians. A significant association was found for the -863 SNP and aseptic loosening (p< 0.05; OR=2.36; 95% CI: 0.976 – 5.71). A trend towards association was found between the -863A SNP and deep infection (p=0.80; OR=2.42; CI: 0.800 – 7.34).

Conclusions: Genetic polymorphism of TNF-alpha may play a significant role in THR aseptic loosening and possibly in deep infection. SNP markers may serve as predictors of implant survival and response to therapy such as anti-TNF treatment.

Purpose: In attempting to unravel the complex cellular responses leading to prosthetic loosening investigators have been limited to studying gene expression of extracellular molecules about which most is known whereas new microarray technology allows simultaneous expression profiling of thousands of genes from a complex sample such as the membrane formed around loosened hip prostheses.

Methods: Two groups of 8 patients were recruited who have undergone primary total hip arthroplasty for osteoarthritis and subsequently developed either septic or aseptic loosening +/− osteolysis. The control group consisted of one group of 5 patients with the same initial diagnosis who had undergone identical procedures, developed no clinical or radiological signs of aseptic or septic loosening, but had come to revision surgery for other complications as defined by the Swedish Hip register: fracture without previous osteolysis, dislocation, technical error, implant fracture, polyethylene wear or pain. Peri-prosthetic membrane was harvested at the time of revision surgery and subjected to RNA extraction. cDNA was then synthesized and hybridised to a Human Genome u95 Genechip ® array which contains a complete set of known human genes. Data normalisation, data filtering and pattern identification was performed using Genechip®3.1 software (Affymetrix, Santa Clara, CA).

Results: This has revealed the involvement of a large number of genes coding for transcriptional regulators upstream from the extracellular and cell-cell signalling molecules already known to be involved in osteolysis and deep infection and which may ultimately control the responses to wear particles and bacterial challenge. Differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation and immune response was found. Most interestingly pathways for control of local bone resorption and inflammatory response have been shown to be highly activated.

Conclusions: The identification of these new pathogenetic mechanisms of total hip replacement failure make new indicators of disease susceptibility and prognosis plus new drug targets direct possibilities.