Analysis of Human Knee Joint Fluid Using Metabonomic, Genomic and Tribology Techniques

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Description

Swollen joints represent a diagnostic challenge. Possible diagnoses include: osteoarthritis, gout, rheumatological disease or infection. Diagnostic methods are limited to microscopy, microbiology and trace analysis. These methods can be slow, time consuming, expensive and often inconclusive. Osteoar...

Swollen joints represent a diagnostic challenge. Possible diagnoses include: osteoarthritis, gout, rheumatological disease or infection. Diagnostic methods are limited to microscopy, microbiology and trace analysis. These methods can be slow, time consuming, expensive and often inconclusive. Osteoarthritis varies in the severity of any inflammatory response and subsequent symptoms. To date no molecular link has been established to explain this ambiguity. Previous studies have identified metabolites of the inflammatory response but failed to investigate any correlation with symptoms or response to targeted injections. Microbial infections of joints are destructive. It typically takes 2-3 days to identify a causative organism and confirm diagnosis. Recent studies suggest low-level bacterial colonisation within joints. The investigators aim to analyse the joint fluid for any bacterial colonisation using enrichment and 16S Polymerase Chain Reaction (PCR) then correlate the results to the metabolic profile. In addition the investigators aim to seek metabolites indicative of the presence of a biofilm which, if present severely limits treatment options. Metabonomics is the quantitative measurement of the dynamic multiparametric response of a living system to pathophysiological stimuli or genetic modification. This provides a global metabolic profile despite a complex biological sample and has proven useful in many fields. Our hypotheses are: Each pathology will have a unique metabolic profile Inflammatory osteoarthritis will contain a different metabolic profile and potentially biomarkers to treatment and/or response Specifically the presence and detection of organism sub-type and biofilm biomarkers These profiles can be characterised by analysis of blood, urine and joint fluid This data can be used to generate diagnostic tests that will aid management of a swollen joint. There will be a correlation between metabolic profile and lubricant properties of the joint fluid, that can be utilised in future engineering strategies to manage osteoarthritis. 1. To analyse joint fluid from osteoarthritic joints to identify potential biomarkers of the disease and further understanding of the cartilage destruction pathways 2. To assess population variances in joint fluid composition using metabonomics and whether this composition correlates to arthropathy of joints 3. To study the lubrication and wear properties of the joint fluid and understand how the metabolic profile influences lubrication and subsequently joint wear. 4. To study joint fluid using metabonomics to identify biomarkers of bacterial colonisation in tandem with microbial 16S Polymerase Chain Reaction and genomic sequencing. Project Timeline: Phases of the project are to be as follows: Full research, Site and Ethical Approval: Provisional ethics is already granted. No obstacles to research and site approval are expected and this should be achieved shortly afterwards. (success = ethical approval) Expected June 2015 Sample Collection: The investigators aim to recruit 150 patients to the study capturing a conservative estimate of 10 per week. All orthopaedic patients will be targeted. The majority are expected to be recruited from the elective theatres. (success = 150 pts recruited) June - September 2015 (onwards) Metabonomic Analysis Method Development: Establishing Nuclear Magnetic Resonance (NMR) methodology for metabolic profiling of synovial fluid as a sample matrix. Method development of Liquid Chromatography Mass Spectroscopy (LCMS) untargeted and targeted methodology for synovial fluid (success = achieving robust, sensitive methodology) October 2015 Joint Fluid Tribology: Analysis of the lubricating properties of the joint fluid (success = 150 samples assayed) October - December 2015 Metabonomic Analysis: In order to yield the maximum results the investigators aim to analyse using NMR screening with untargeted and targeted LCMS analysis. With approximately 150 patients and thus 450 body fluid samples the investigators are estimating an analysis time of 6 months (success = metabolic profiling and targeted analysis) November - April 2016 Microbial Genetic Analysis: PCR and sequencing of the microbial genetic content of the joint fluid (success = completion in 150 samples) April - June 2016 Collation of results, Analysis and Reporting: Preparation of results for submission to a journal with an impact factor >10. (success = editorial acceptance of manuscript) July - October 2016

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