Synovial tissue‐derived extracellular vesicles induce chondrocyte inflammation and degradation via NF‐κB signalling pathway: An in vitro study

Abstract Osteoarthritis (OA) is a whole‐joint disease characterized by synovial inflammation and cartilage degeneration. However, the relationship between synovial inflammation and cartilage degeneration remains unclear. The modified Hulth's method was adopted to establish a knee OA (KOA) rabbit model. Synovial tissue was collected after 8 weeks, and synovial tissue‐derived extracellular vesicles (ST‐EVs) were extracted by filtration combined with size exclusion chromatography (SECF), followed by identification through transmission electron microscopy (TEM), nanoparticle tracer analysis (NTA) and Western blot (WB). The collagenase digestion method was used to extract normal rabbit chondrocytes, which were then treated with the SF‐EVs to observe the effect and mechanism of SF‐EVs on chondrocytes. The morphology, particle size and labelled protein marker detection confirmed that SECF successfully extract ST‐EVs. The ST‐EVs in the KOA state significantly inhibited chondrocyte proliferation and promoted chondrocytes apoptosis. Moreover, the ST‐EVs also promoted the expression of pro‐inflammatory cytokines (IL‐1β, IL‐6, TNF‐α and COX‐2) and cartilage degradation‐related enzymes (MMP13, MMP9 and ADAMTS5) in the chondrocytes. Mechanistically, the ST‐EVs significantly promoted the activation of NF‐κB signalling pathway in chondrocytes. Inhibition the activation of the NF‐κB signalling pathway significantly rescued the expression of inflammatory cytokines and cartilage degradation‐related enzymes in the ST‐EVs–induced chondrocytes. In conclusion, the ST‐EVs promote chondrocytes inflammation and degradation by activating the NF‐κB signalling pathway, providing novel insights into the occurrence and development of OA.


| INTRODUC TI ON
Osteoarthritis (OA) is a common complex disease that manifests as pain, swelling and limited mobility. 1,2 Epidemiological studies have shown that OA affects more than 10% of the elderly, 3 and 140 million people worldwide suffer from OA. 1 To date, the pathogenesis of OA is not completely understood. 4,5 Cartilage degeneration has long been regarded to play a vital role in the pathogenesis of OA. [6][7][8][9] However, intensive research suggests that inflammation in the synovial tissue may play a pioneering role in the early stage of OA. Based on a case-control study, Atukorala et.al suggested that synovitis is a precursor of radiographic OA and increases the risk of cartilage loss. 10 Wang et al. reported that synovial tissue may cause pathological changes in cartilage via the innate immune system to aggravate the progression of OA. 11 However, how the inflammation of synovial tissue aggravates OA cartilage degeneration and how the signal between synovium and cartilage is transmitted remains unclear.
Extracellular vesicles (EVs), a type of microvesicle with a diameter of about 30-150 nm, a round or elliptical cup holder-like structure, have a double-layer phospholipid membrane structure, and play an important role in exchanging information between cells and tissues. 12 EVs are widely present in cell medium supernatants and bodily fluids. 12 Kato extracted exosomes from IL-1β-induced fibroblast-like synoviocytes (FLS) supernatants, and then added FLS-exosomes to chondrocytes. The results showed that FLS-exosomes significantly promoted the expression of matrix metalloproteinase- 13 (MMP13) and decreased the expression of collagen II, thereby accelerating the cartilage degeneration. 13 These results suggested that synovial tissue inflammation may regulate cartilage degeneration by secreting EVs.
Several studies have investigated the extraction and mechanism of EVs in cell supernatants and body fluids; however, only a few articles have studied the extraction and mechanism of tissue-derived EVs.
In our previous study, 14 for the first time, we extracted and identified synovial tissue-derived EVs (ST-EVs), and found that the background of ST-EVs extracted by the size exclusion + ultrafiltration method (SECF) was the cleanest, without the expression of EVs negative markers, and recommended for the follow-up study of the function and mechanism of ST-EVs. However, the effect and mechanism of ST-EVs on OA cartilage are still unclear. Therefore, in this study, we used the modified Hulth's method to establish an OA rabbit model, which was used to extract ST-EVs after 8 weeks, and then, the ST-EVs were added to normal chondrocytes to observe their effect on OA state in cartilage and to study the mechanism underlying this effect.

| Establishment of OA rabbit model
Eight male rabbits (6 months, 2.5 ± 0.5 kg) were purchased from Beijing Keyu Animal Breeding Center (production license: SCXK (Jing)-2018-0010), and then raised in separate cages in the Medical Animal Experiment Center of the Chinese Academy of Chinese Medical Sciences. The experimental protocols were approved by the Animal Care and Use Committee of China Academy of Chinese Medical Sciences. All rabbits are fed under the same breeding conditions, temperature: 21 ± 3℃, humidity: 55 ± 5%, dark/light cycle: 12/12 h. After one week of adaptive feeding, the modified Hulth's method was used to establish a moderate KOA rabbit model as previously described. 11 Briefly, after anaesthesia, a 1 cm incision was made on the inside of the right knee joint, transected the medial collateral ligament (MCL) and then resection of the anterior cruciate ligament and medial meniscus. Penicillin was continuously injected for 3 days after the operation to prevent infection, and the rabbits were driven to simulate exercise for 30 min every day after modelling. After 8 weeks, the synovial tissue of the right knee was taken and extracted EVs by SECF; the cartilage of the right knee was taken for morphological detection, and the cartilage of left knee was taken and then digested into chondrocytes with collagenase. activating the NF-κB signalling pathway, providing novel insights into the occurrence and development of OA.

K E Y W O R D S
cartilage degeneration, extracellular vesicles, NF-κB signalling pathway, osteoarthritis, synovial inflammation 2.3 | Collection and treatment of synovial tissue specimens As previously described, 14 the obtained synovial tissue is immediately placed in PBS containing 1% antibiotic mixture, and transferred to the ultra-clean table as soon as possible. After washed 3 times with PBS containing 1% antibiotic mixture, the synovial tissue was cut into pieces of 1 × 1 × 1 mm 3 , added the culture medium (containing 10% exosome free foetal bovine serum and 1% antibiotic mixture), and the culture supernatant was collected after 24 h. The collected medium was filtered through a 0.22 μm filter and SECF was used to extracted the ST-EVs, all procedures were performed in strict accordance with the kit instructions.

| Chondrocytes isolation
Primary normal rabbit chondrocytes were isolated from articular cartilage of left knee joint by collagenase digestion as previously described. 15 In a nutshell, after washing with PBS containing 1% antibiotic mixture, the articular cartilage was cut into small pieces, and then digested with type II collagenase for 6 h. The filtered supernatant was centrifuged for 5 min, and the pellet was washed twice with ice-cold PBS and resuspended in DMEM F12 with 10% FBS and 1% antibiotic mixture.

| EVs characterization and uptake of EVs
As previously described, 14 transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were performed to detect the morphology, particle size and concentration of EVs. For uptake of EVs, ST-EVs were labelled with PKH67 (Sigma-Aldrich) according to the manufacturer's protocol. Simply put, 1 × 10 7 ST-EVs were suspended in 1 ml PBS mixed with PKH67 dye, incubated at 4°C for 5 min, and then stopped the labelling reaction by the addition of BSA. The labelled EVs were incubated with primary normal rabbit chondrocytes for 4 h, and then visualized with fluorescence microscope.

| Western Blot (WB)
Chondrocytes were lysed RIPA supplemented with phenylmethanesulfonylfluoride (PMSF), and the protein concentration was quantified by the BCA protein assay kit. Extracted proteins were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis; then, transferred to PVDF membrane as previously described. 14,15 After incubating 5% non-fat dried milk at room temperature for 1 h, add primary antibody and incubate overnight at 4℃. After four washes in TBST, membranes were incubated with secondary antibodies for 1 h at room temperature. Then, the membranes were washed with TBST for 5 min × 8 times. Finally, ECL was added for the visualized of proteins according to the manufacturer's protocol.

| Real-time quantitative polymerase chain reaction analysis
Total RNA was extracted from chondrocytes using TRIzol reagent according to the manufacturer's instructions. The concentration of RNA samples was determined using the NanoDrop spectrophotometer, and then, the quality of RNA samples was checked by the 260 nm/280 nm ratios were checked. Complementary DNA was prepared using a commercially available kit under the following conditions: 15 min at 50°C and 5 s at 85°C. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using a thermocycler and SYBR Premix Ex Taq II Kit under the following conditions: 10 min at 95°C, followed by 40 cycles at 95°C for 15 s and at 60°C for 1 min. The expression of GAPDH was used to normalize data, and the data were analysed using the 2 −ΔΔCT method. The RT-qPCR primers are listed in Table 1.

| Flow cytometry
The chondrocytes of all groups were harvested. The cells were de-

| Histological analysis
The femoral cartilage samples and synovial tissue were fixed in 4% paraformaldehyde, and then decalcified using the EDTA (Sigma-Aldrich, US) microwave method. The cartilage samples were sectioned after conventional paraffin-embedding. Put the cartilage sections in xylene Ⅰ and Ⅱ for 5 min each for dewaxing, and then debenzene step by step. Afterwards, haematoxylin and eosin (HE) staining was performed to reveal the cartilage morphology.

| Enzyme-linked immunosorbent assay
Briefly, chondrocyte supernatants were collected, and then, the proteins of the chondrocyte supernatants were extracted with RIPA and PMSF buffer. BCA protein assay kit was used to quantified the concentration of proteins. Afterwards, commercially available enzyme-linked immunosorbent assay (ELISA) kit was used to detecte the expression of (IL-1β, IL-6, TNF-a and COX2) according to the manufacturer's protocol.

| Statistical analyses
In general, data obtained from this research were presented as the mean ± standard deviation (SD). All analyses were performed using GraphPad Prism 7.0 software. The statistical differences among groups were analysed by t test or one-way ANOVA. p < 0.05 was considered statistically significant.

| Isolation and identification of ST-EVs
To evaluate the effect and mechanism of ST-EVs on OA cartilage, the modified Hulth's method was used to establish an OA rabbit model, to extract ST-EVs after 8 weeks of modelling, and then interfere with normal chondrocytes ( Figure 1A). NTA results showed that the particle size of ST-EVs was mainly distributed in the range of 30-150 nm, and the main peak was approximately 100-120 nm, which is consistent with the particle size of exosomes ( Figure 1B). Furthermore, the exosomal positive markers CD63 and flotillin-1 were highly expressed in ST-EVs, while the negative marker-calnexin was expressed at significantly lower level ( Figure 1C). The TEM results showed that ST-EVs had circular and elliptical disc-shaped and cup holder-shaped vesicles, about 30-150 nm in size, consistent with the morphological characteristics of EVs ( Figure 1D).

| OA model identification and EVs uptake by chondrocytes
To verify the effect of ST-EVs in the KOA state on cartilage, we employed the modified Hulth's method to establish the OA rabbit model.

| ST-EVs inhibit chondrocytes proliferation and promote apoptosis
To detect the effect of ST-EVs on the cell viability of chondrocytes, the CCK-8 assay was used to detect the cell viability of chondrocytes.
As shown in Figure

| ST-EVs promote the inflammation and degeneration of normal cartilage via NF-κB signalling pathway
The They found that among patients whose X-ray examination revealed KOA, the higher the Kellgren-Lawrence grade, the higher the probability of synovitis; the higher the synovitis score, the higher the degree of cartilage damage. 21 Atukorala et al. 10  In summary, our findings indicate that synovial inflammation can significantly promote cartilage damage and chondrocyte inflammation and degradation via activating the NF-κB signalling pathway, which provides novel insight into the occurrence and development of OA ( Figure 6).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.