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.