Ultrastructural Analysis of Healthy Synovial Fluids in Three Mammalian Species

Matei, Constantin; Boulocher, C.; Boulé, Christelle; Schramme, Michael; Viguier, É.; Roger, Thierry; Berthier, Yves; Trunfio-Sfarghiu, Ana-Maria; Blanchin, M. G.

doi:10.1017/s1431927614000415

Cited by 13 publications

(21 citation statements)

References 31 publications

(52 reference statements)

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“…As these hydrophobic interactions occur at different rates, a wide range of vesicle sizes is formed [47]. The size range of 0.1 to 50 μm in diameter (Figure 4) for the vesicles synthesized in this study overlaps with the range of 0.1 μm to a few hundred nanometers of the vesicles found naturally in synovial fluid [48], but it also indicates the presence of much larger vesicles. However, this is the range prior to tribological testing, and, as pointed out above, the larger vesicles are expected to divide into smaller vesicles under shear and compressive stresses.…”

Section: Discussionmentioning

confidence: 58%

Phospholipid Vesicles in Media for Tribological Studies against Live Cartilage

et al. 2018

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Introduction Pre-clinical testing of hemiarthroplasty devices requires that the tribological conditions present in vivo with live cartilage be closely duplicated. A current limitation in the tribological testing of live cartilage involves the use of cell-culture media as lubricant. Study Aim to develop and test a new hyaluronan-phospholipid based medium (HA–phospholipid medium) that combines the rheological and frictional properties of synovial fluid with the nourishing properties of culture media to keep cells alive. Materials and Methods The HA–phospholipid medium consisted of culture medium with added phospholipid dipalmitoylphosphatidylcholine (0.3 mg/mL), and hyaluronic acid (2.42 mg/mL). A standard cell culture medium was used as the control. The rheology of each medium was determined using a flat plate configuration. Bovine calf cartilage was used to assess cell viability and friction in each medium. For friction measurements, a cobalt-chrome alloy ball was articulated against cartilage disks immersed in medium. Results Lipid vesicles 0.1 to 50 μm in diameter were identified in the HA–phospholipid medium. Cartilage cell viability was significantly higher in the HA–phospholipid medium (62% ± 8%, 95% CI) than in control medium (49.5% ± 5%) (p = 0.009). The HA–phospholipid medium exhibited strong shear-thinning behavior, similar to synovial fluid, with viscosities ~100-fold higher at 10 s−1 and 5-fold higher at 20,000 s−1 than the approximately Newtonian control medium. The HA–phospholipid medium also yielded 20% lower friction values than the control medium after one hour of testing. Conclusions The rheological and friction results indicate that the HA–phospholipid medium is superior to the control cell culture medium in emulating the shear thinning and lubricative properties of natural synovial fluid, making it more clinically relevant for in vitro wear and friction testing with live cartilage.

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Section: Discussionmentioning

confidence: 58%

Phospholipid Vesicles in Media for Tribological Studies against Live Cartilage

et al. 2018

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“…The present study connects to the recent findings from cell cultures that HA‐EVs originate from the tips of microvilli or bleb directly from the plasma membrane, and suggests that this process also occurs in vivo. HA‐coating could have been also formed via CD44 or HAS after EV secretion into the synovial fluid, or HA could be contained within EVs by envelopment of HA . High HA concentrations and active EV shedding are both observed in conditions, such as inflammation, diabetes and cancer .…”

Section: Discussionmentioning

confidence: 99%

First in vivo detection and characterization of hyaluronan‐coated extracellular vesicles in human synovial fluid

Mustonen

Nieminen

Joukainen

et al. 2016

Journal Orthopaedic Research

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Extracellular vesicles (EVs) function in intercellular signaling by transporting different membrane and cytosolic molecules, including hyaluronan (HA) and its synthesis machinery. As both EVs and HA are abundant in synovial fluid, we hypothesized that HA synthesized in synovial membrane would be carried on the surface of EVs. Synovial fluid (n = 15) and membrane samples (n = 5) were obtained from knee surgery patients. HA concentrations were analyzed in synovial fluid and HA and its synthesis machinery were examined with histochemical stainings in synovial membrane. To assess the size distribution of EVs in synovial fluid and to visualize HA on EVs, nanoparticle tracking analysis (NTA), confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) were utilized. The average HA concentration in synovial fluid was 2.0 ± 0.21 mg/ml without significant differences between the patients with trauma/diagnostic arthroscopy and primary or post-traumatic osteoarthritis. Positive stainings of HA synthases (HAS1-3), HA and its receptor CD44 in synovial cells indicated active HA secretion in synovial membrane. According to NTA, EVs were abundant in synovial fluid and their main populations were ≤300 nm in diameter after differential centrifugation. There were no significant differences in the EV counts between the patients with primary or post-traumatic osteoarthritis. TEM verified that HA-positive particles detected by CLSM were lipid membrane vesicles surrounded by a HA coat. Our results provide the first in vivo evidence that human synovial fluid contains HA-positive EVs, one source of which presumably is the long HAS-positive protrusions of synovial fibroblasts. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1960-1968, 2016.

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“…1. The protocol was based on earlier protocols used for EV isolation from SF as summarized in Table I, (2–19) but was more comprehensive than most of these, as it was made to meet the recently defined minimal requirements for functional EV studies (31). The technical specifications of ultracentrifugation steps (centrifugation speeds, rotor types, etc.)…”

Section: Methodsmentioning

confidence: 99%

“…In this context, EVs derived from synovial fluid (SF) are currently being investigated to unravel their role in these processes and their biomarker potential for joint disease. Several studies have examined EVs isolated from SF (2–19). In most of these studies EVs have only been isolated from human SF obtained from inflamed joints.…”

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Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Boere

Lest

Libregts

et al. 2016

J of Extracellular Vesicle

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Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) – a prominent extracellular matrix component – it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20–200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.

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Ultrastructural Analysis of Healthy Synovial Fluids in Three Mammalian Species

Cited by 13 publications

References 31 publications

Phospholipid Vesicles in Media for Tribological Studies against Live Cartilage

Phospholipid Vesicles in Media for Tribological Studies against Live Cartilage

First in vivo detection and characterization of hyaluronan‐coated extracellular vesicles in human synovial fluid

Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

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