Morphological and Mechanical Characterization of Extracellular Vesicles and Parent Human Synoviocytes under Physiological and Inflammatory Conditions

Abstract

The morphology of fibroblast-like synoviocytes (FLS) issued from the synovial fluid (SF) of patients suffering from osteoarthritis (OA), rheumatoid arthritis (RA), or from healthy subjects (H), as well as the ultrastructure and mechanical properties of the FLS-secreted extracellular vesicles (EV), were analyzed by confocal microscopy, transmission electron microscopy, atomic force microscopy, and tribological tests. EV released under healthy conditions were constituted of several lipid bilayers surrounding a viscous inner core. This "gel-in" vesicular structure ensured high mechanical resistance of single vesicles and good tribological properties of the lubricant. RA, and to a lesser extent OA, synovial vesicles had altered morphology, corresponding to a "gel-out" situation with vesicles surrounded by a viscous gel, poor mechanical resistance, and poor lubricating qualities. When subjected to inflammatory conditions, healthy cells developed phenotypes similar to that of RA samples, which reinforces the importance of inflammatory processes in the loss of lubricating properties of SF.

Keywords: extracellular vesicles; inflammation; osteoarthritis; rhumatoid arthritis; synovial fluid; synoviocytes.

Figure 1

Selection of the most appropriate conditions for the structural and mechanical characterization. (A) Cells (top) and supernatants (bottom) confocal micrographs at 5, 10, 20, and 30 days of cell culture (X63 immersion oil microscope objective) after passage 6. (B) Cells (top) and supernatants (bottom) micrographs from passage 6 to 9 each after 20 days. Labeling of the different components was done by lipophilic fluorescent label “DiI” (λex~549 nm). (C) PLT concentration (µg/mL) in the supernatant and (D) number of vesicles per image as a function of cell culture day. MC, medium of culture. Bars represent mean ± SD. *** p < 0.0001, and NS non-significant.

Figure 2

Structural characterization of FLS in the absence (A) or presence (B) of an inflammatory environment (IL-17: 50 ng/mL, TNF-α: 1 ng/mL). Cells at 20 days of culture after passage P6 were labeled with lipid fluorescent label “DiI” (λex~549 nm) in all cases. Differences in cells morphology in term in terms of shape, spread and number of pseudopodia were observed by confocal microscopy (a–f). Synoviocyte area (µm²) (C) and elongation (D) in the absence or presence of inflammatory environment. Bars represent mean ± SEM. *** p < 0.0001, ** p < 0.001 * p < 0.01 and NS non-significant.

Figure 3

Structural characterization and comparison of supernatants between healthy (H) and pathological (OA and RA) in the absence (A) or presence (B) of inflammatory environment (IL-17: 50 ng/mL, TNF-α: 1 ng/mL). Differences in vesicular structures were observed by confocal microscopy (a–c) and transmission electron microscopy (TEM) (d–f) (20 days of cell culture after cell passage 7). Arrows: Phospholipid multilayers. (C) Fluorescence intensity of the image. (D) PLT concentration (µg/mL) in the supernatant. Bars represent mean ± SEM; *** p < 0.0001, ** p < 0.001 and NS non-significant.

Figure 4

Tribological behavior of vesicle supernatant (H, OA, and RA) after wear and friction tests. (A) The principle of functioning of the bio-tribometer apparatus showing the ex vivo contact model. (B) Wear: micrographs showing the wear of the lipid interfaces deposited at on the surfaces in contact using white light microscopy (a) unworn interface, an even image in the viewing octagon of the microscope (b) interface degradations, lighter clusters in the viewing octagon of the microscope. (C–H) Micrographs of the lipid interfaces wear for vesicle supernatant (H, OA, and RA) on absence and presence of inflammatory environment. (I) Coefficient of friction as a function of pathologies in the absence (white bars) or presence (green) of inflammatory environment (IL-17: 50 ng/mL, TNF-α: 1 ng/mL). Error bars represent SD values calculated on at least 3 independent measurements. * p < 0.01.

Figure 5

Characterization of vesicles from supernatants of healthy and pathological (OA and RA) FLS in the absence or presence of inflammatory environment (IL-17: 50 ng/mL, TNF-α: 1 ng/mL). Vesicle sectional curves and height topographies under standard (A) and inflammatory conditions (B). Differences in the height topographies (a–c) and sectional curves (d–f) depending on culture conditions were observed by Atomic Force Microscopy. Average vesicle area (C) and average height (D) as a function of pathology and inflammatory environment compared to control and represented by mean ± SEM and with ANOVA test *** p < 0.0001, ** p < 0.001 * p < 0.01 and NS non-significant.

Figure 6

Mechanical characterization of vesicles in supernatant by Atomic Force Microscopy in healthy (H) and pathological conditions (OA and RA) in the absence or presence of inflammatory environment (IL-17: 50 ng/mL, TNF-α: 1 ng/mL). Vesicle intrinsic rigidity as a function of pathology and inflammatory environment compared to control and represented by mean ± SEM. * p < 0.01 and *** p < 0.001.

Figure 7

AFM settings for indentation tests performed on synovial EV. Vesicles were fixed on a glass slide through biotin–streptavidin coupling and were tested in HEPES buffer using a cantilever with a spherical tip (a). Insets: a zoom of the tip and a cartoon of the tip interacting with a vesicle. Vesicle height topographies (b) and sectional curves (c).