To assess this concern, 1.9 billion anti-CTXII particles (460 antibody molecules per particle) in 30 L of Buffer 1 were mixed with 300 L of either hyaluronidase-treated or native bovine synovial fluid, incubated with gentle mixing for 1 h, then divided into 25 L samples of approximately 150 million particles per sample. knee OA. CTXII could be magnetically captured from a MK-6913 rodent stifle without the need to aspirate fluid and showed 10 fold changes in CTXII levels from OA-affected bones relative to contralateral control bones. Combined, these data demonstrate the ability and level of sensitivity of magnetic capture for analysis of OA biomarkers in the rat. Keywords: Magnetic Nanoparticles, Biomarkers, Synovial Fluid, Osteoarthritis, Rodent Model Intro Osteoarthritis (OA) is typically diagnosed through individual reports of pain and disability and verified by changes in joint structure on radiographic scans. While pharmacologic and non-pharmacologic strategies have some ability to improve patient quality-of-life, current OA treatments are primarily palliative31. Disease-modifying and preventative strategies may be more successful if OA can be diagnosed earlier in the disease process10. Toward this end, several study initiatives have emphasized the development of early diagnostic tools for OA2,16. Regrettably, radiographic evidence of early-stage OA is definitely delicate, and imaging techniques that allow for more sensitive analysis of cartilage loss, such as magnetic resonance imaging (MRI), are cost-prohibitive for OA screening. As such, molecular biomarkers have drawn interest as potential OA diagnostics, and detection of OA biomarkers in urine PTPRR and serum has shown promise for diagnosing OA prior to MRI, ultrasound, or radiographic scans8,11,21. Since OA is definitely controlled by local catabolic and pro-inflammatory mediators7, the earliest molecular evidence of OA will likely be contained in synovial fluid3,25. Recent studies in horses confirm OA biomarker analysis in synovial fluid may have higher potential for early OA analysis than analysis of the same biomarkers in serum and urine4,17,27. As with MRI, synovial fluid analysis may be impractical for OA screening, but substantial opportunity exists to use synovial fluid analysis to monitor the effects of traumatic joint injury and to assess OA severity in preclinical studies. Evaluation of synovial fluid biomarkers in preclinical models, however, offers significant difficulties. Synovial fluid aspiration from small joints is non-trivial and MK-6913 technologically demanding due to a complex geometry and limited synovial fluid volume. For the rodent stifle (similar to the human being knee), synovial fluid aspiration is MK-6913 definitely practically impossible, and as such, biomarker analysis in rodent models is typically carried out using joint lavage (washing saline through the joint) or fluid wicking (pressing filter paper against the joint surface). Lavage is currently the most widely used technique to investigate joint-level biomarkers in rodent OA models. However, lavage fluids dilute biomarker and expose errors if fluids are not well-mixed1,12. For fluid wicking, proteins must also become eluted from filter paper, again resulting in biomarker dilution. As such, biomarker analysis following lavage and fluid wicking typically reflect biomarker concentration in the recovered sample; however, a secondary analyte, such as urea, or a known concentration of an internal standard within the lavage fluid can be used to estimate the dilution element and therefore biomarker concentrations within the joint13,23. However, biomarker concentrations in the joint are susceptible to joint effusion, and steps such as total biomarker in the joint or the rates of production of a biomarker are likely preferable for OA assessment26. Thus, fresh systems to collect OA biomarkers from small synovial fluid quantities may improve our ability to assess OA pathogenesis in small joints. Over the past decade, the use of magnetic nanoparticle systems offers improved dramatically in a variety of biomedical applications19. Magnetic MK-6913 nanoparticles are already in clinical use as MRI contrast providers and in biomedical laboratories for cell separation, drug and gene delivery, and remote activation of cell signaling pathways5,15,20. In this study, a new magnetic nanoparticle-based technology (termed magnetic capture) is explained for the deterimation of an OA biomarker in small quantities of synovial fluid and from a rat model of knee OA. Due to the wide use of the c-terminus telopeptide of type II collagen (CTXII)18 in OA study and the commercial availability of a highly specific, purified, HRP-labeled anti-CTXII monoclonal antibody, CTXII was selected for the development of magnetic capture. Using the CTXII antibody, we demonstrate the ability to assess CTXII in synovial fluid using magnetic capture. In so doing, the phases of magnetic capture are characterized and validated, and the ability of magnetic capture to accurately determine CTXII levels is shown for small quantities of bovine synovial fluid and in a rat monoiodoacetate (MIA) model of knee OA. Materials and Methods Magnetic Capture Concept Magnetic capture functions through the properties of superparamagnetic iron oxide nanoparticles (SPIONs) inlayed within a polymer. In the absence of an external magnetic field, SPIONs do not retain stable magnetization; however in a high-gradient magnetic field, SPIONs encounter a.
