Anne-Mari Mustonen1,2, Janne Capra3, Kirsi Rilla4, Petri Lehenkari5,6, Sanna Oikari4, Tommi Kääriäinen7, Antti Joukainen7, Heikki Kröger7, Tommi Paakkonen4, Johanna Matilainen4, Petteri Nieminen4,8. 1. Faculty of Health Sciences, School of Medicine, Institute of Biomedicine, University of Eastern Finland, P.O. Box 1627, FI-70211, Kuopio, Finland. anne-mari.mustonen@uef.fi. 2. Faculty of Science and Forestry, Department of Environmental and Biological Sciences, University of Eastern Finland, P.O. Box 111, FI-80101, Joensuu, Finland. anne-mari.mustonen@uef.fi. 3. Faculty of Health Sciences, School of Medicine, Institute of Biomedicine, Cell and Tissue Imaging Unit, University of Eastern Finland, P.O. Box 1627, FI-70211, Kuopio, Finland. 4. Faculty of Health Sciences, School of Medicine, Institute of Biomedicine, University of Eastern Finland, P.O. Box 1627, FI-70211, Kuopio, Finland. 5. Faculty of Medicine, Cancer and Translational Medicine Research Unit, University of Oulu, P.O. Box 5000, FI-90014, Oulu, Finland. 6. Department of Surgery and Medical Research Center, Oulu University Hospital, P.O. Box 21, FI-90029, Oulu, OYS, Finland. 7. Department of Orthopaedics, Traumatology and Hand Surgery, Kuopio University Hospital, P.O. Box 100, FI-70029, Kuopio, KYS, Finland. 8. Faculty of Science and Forestry, Department of Environmental and Biological Sciences, University of Eastern Finland, P.O. Box 111, FI-80101, Joensuu, Finland.
Abstract
BACKGROUND: Hyaluronic acid (HA) is the major extracellular matrix glycosaminoglycan with a reduced synovial fluid (SF) concentration in arthropathies. Cell-derived extracellular vesicles (EV) have also been proposed to contribute to pathogenesis in joint diseases. It has recently been shown that human SF contains HA-coated EV (HA-EV), but their concentration and function in joint pathologies remain unknown. METHODS: The aim of the present study was to develop an applicable method based on confocal laser scanning microscopy (CLSM) and image analysis for the quantification of EV, HA-particles, and HA-EV in the SF of the human knee joint. Samples were collected during total knee replacement surgery from patients with end-stage rheumatoid arthritis (RA, n = 8) and osteoarthritis (OA, n = 8), or during diagnostic/therapeutic arthroscopy unrelated to OA/RA (control, n = 7). To characterize and quantify EV, HA-particles, and HA-EV, SF was double-stained with plasma membrane and HA probes and visualized by CLSM. Comparisons between the patient groups were performed with the Kruskal-Wallis analysis of variance. RESULTS: The size distribution of EV and HA-particles was mostly similar in the study groups. Approximately 66% of EV fluorescence was co-localized with HA verifying that a significant proportion of EV carry HA. The study groups were clearly separated by the discriminant analysis based on the CLSM data. The intensities of EV and HA-particle fluorescences were lower in the RA than in the control and OA groups. CONCLUSIONS: CLSM analysis offers a useful tool to assess HA-EV in SF samples. The altered EV and HA intensities in the RA SF could have possible implications for diagnostics and therapy.
BACKGROUND:Hyaluronic acid (HA) is the major extracellular matrix glycosaminoglycan with a reduced synovial fluid (SF) concentration in arthropathies. Cell-derived extracellular vesicles (EV) have also been proposed to contribute to pathogenesis in joint diseases. It has recently been shown that human SF contains HA-coated EV (HA-EV), but their concentration and function in joint pathologies remain unknown. METHODS: The aim of the present study was to develop an applicable method based on confocal laser scanning microscopy (CLSM) and image analysis for the quantification of EV, HA-particles, and HA-EV in the SF of the human knee joint. Samples were collected during total knee replacement surgery from patients with end-stage rheumatoid arthritis (RA, n = 8) and osteoarthritis (OA, n = 8), or during diagnostic/therapeutic arthroscopy unrelated to OA/RA (control, n = 7). To characterize and quantify EV, HA-particles, and HA-EV, SF was double-stained with plasma membrane and HA probes and visualized by CLSM. Comparisons between the patient groups were performed with the Kruskal-Wallis analysis of variance. RESULTS: The size distribution of EV and HA-particles was mostly similar in the study groups. Approximately 66% of EV fluorescence was co-localized with HA verifying that a significant proportion of EV carry HA. The study groups were clearly separated by the discriminant analysis based on the CLSM data. The intensities of EV and HA-particle fluorescences were lower in the RA than in the control and OA groups. CONCLUSIONS: CLSM analysis offers a useful tool to assess HA-EV in SF samples. The altered EV and HA intensities in the RA SF could have possible implications for diagnostics and therapy.
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