Preparation of SC-derived MSCs, primary articular chondrocytes and synovial cells
The subcutaneous adipose tissue was obtained by liposuction from young healthy donors. The methods and guidelines were approved by the People’s Liberation Army No. 85 Hospital, Shanghai, P.R. China (review serial number NO.2013/18) [34]. All donors provided written informed permission. All operation was conducted in accordance with the regular procedures and Declaration of Helsinki.
SC-derived MSCs was isolated in accordance with the earlier reported reference [25]. After washed with phosphate-buffered solution (PBS) (Gibco, Rockford, IL, USA), 500 mg of adipose tissue cut into 1-mm3 pieces was digested for 45 min with 0.1% collagenase I (Gibco, USA). Then, we added equivalent volume of complete culture medium (DMEM-F12 culture medium (Gibco, USA), 10% FBS, 1% Penicillin–Streptomycin) and centrifuged at 1000 rpm for 10 min. cells were cultured at a 1 × 106/ml density in complete culture medium with 10 ng/ml bFGF (Stem Cell, USA) after being resuspended in PBS and filtered using a 40 μm cell strainer. When adhering cells reach 80–90% confluence, the cells should be passaged in a 1:3–1:4 ratio. The primary SC-derived MSCs before passage 7 were used.
The surface markers of MSCsSC were identified according to the experiment design of the Human MSC Analysis Kit (BD Biosciences) and by using flow cytometry (FACSCalibur, BD, NJ, USA). Data was assessed with Flow Jo V10 software.
The primary articular chondrocytes was isolated according to the earlier research [35]. The cartilage samples from newborn Sprague-Dawly (SD) rats was rinsed in sterile PBS contained penicillin–streptomycin and then sliced. The matrix was digested overnight in high-glucose DMEM (Gibco, USA) with 0.2% type II collagenase (Gibco; 17101-015) and 1% P/S to extract cartilage. The cell solution was filtered through a 70 μm cell strainer; and the collected primary chondrocytes were centrifuged at 400 g for 5 min before resuspending in high-glucose DMEM supplemented with 10% FBS and 1% P/S. The medium was exchanged every next day. The primary chondrocytes before passage 4 were used. C28/I2 cells, which are the normal human chondrocytes, were cultured in DMEM (Gibco, USA) with 1% P/S and 10% FBS (Gibco, USA) in a 5% CO2 environment at 37 °C.
The primary synovial cells was isolated as described previously [36]. The synovial tissue samples from 8-week old SD rats was rinsed in sterile PBS contained P/S and then sliced. The matrix was digested in RPMI medium (Gibco, USA) with collagenase (2 mg/ml) (Sigma, C5138, St. Louis, MO) and 1% P/S at 37 °C for 1 h. The cell solution was filtered through a 70 μm cell strainer; and the collected primary synovial cells were centrifuged at 400g for 5 min before resuspending in RPMI medium supplemented with 10% FBS and 1% P/S. The medium was exchanged every next day.
Isolation and identification of Exos derived from MSCsSC
To obtain the MSCsSC derived exosomes, when MSCsSC on the 2nd passage neared 80–90% confluence, the DMEM-F12 culture medium added with 10% exosome-depleted serum and 10 ng/ml bFGF were replaced for 24 h, after that the cell supernatant was all collected. The cell supernatant was pre-centrifuged in 300g for 10 min, 2000g for 20 min, 10,000g for 30 min to eliminate dead cells, apoptotic bodies and cell debris. Then, after 0.22 μm filtration, the whole supernatant was moved to ultracentrifuge tube (Beckman, #355618, USA) and ultracentrifuged in 100,000g for 70 min twice to separate exosomes. The pellets were then resuspended in 100 μl PBS per tube. All operations were performed at 4 °C and the generated exosomes were stored at − 80 °C or used instantly.
The Nanoparticle Tracking Analysis (NTA) was used to measure exosomes concentration, the distribution of particle size and purity (NanoSight300, Malvern Instruments Ltd, UK). 1 ml PBS was used to dilute the samples with the appropriate concentration before being put into the sample cubicle. The particles were then monitored and illuminated via Brownian motion of laser light. Each sample was performed three times, and the procedure was documented. The Stokes–Einstein equation was used to calculate the concentration of particles, the distribution of size and scatter intensity.
The transmission electron microscopic (TEM) technique was employed to identify the ultrastructure of the exosomes. Hitachi HT7800 electron microscope (HT-7800, Hitachi, Japan) was used to capture images of MSCsSC-Exos.
Plasmid construction and transfection
The pEGFP-C1-RVG-Lamp2b expressing vector was kindly provided by Prof. Matthew J. A. Wood’s lab from University of Oxford [37]. The DNA sequence encoding a glycosylation motif (GNSTM) [38], CAP Peptide (DWRVIIPPRPSA) [39], and a glycine-serine spacer were synthesized and subcloned to replace the RVG fragment in the plasmid vector, then was refined as plasmid CAP-Lamp2b. Meanwhile, the other two plasmid encoding Lamp2b and CAP-EGFP-Lamp2b, were constructed respectively, according to the previous report [31]. The above three plasmids encoding the lamp2b constructs were transfected into the SC-derived MSCs using Lipofectamine 3000 transfection reagent (Invitrogen, L3000008, USA), when adhering cells reach 50–60% confluence. The corresponding exosomes were isolated according the previous method in 2.2.
In vivo studies
Ethics statement
The animal experiments were approved by Ethical Committee of Laboratory Animals Research Center, Tongji University. The approval number was TJAA07622701. All experimental procedures on procedures on animals were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health.
Rats OA model and grouping
Six-week-old male SD rats about 180–200 g were purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). All rats were maintained on a 12-h light cycle in the animal facility of the Animal Unit of Tongji University. When the rats were eight weeks old, they were subjected to destabilization of the medial meniscus (DMM) and anterior cruciate ligament transection (ACLT) surgery to induce OA, as previously described [40]. Specifically, the DMM + ACLT surgery was performed by surgical sectioning of the medial meniscotibial ligament and the sham operation was performed by incision of the cutaneous and muscular planes at baseline.
In the first animal section (Fig. 2B), all rats underwent DMM + ACLT surgery or sham surgery were randomly divided into three groups: (1) sham group; (2) PBS group; (3) PBS-ExosSC group. Three weeks after operation, rats were given multiple intra-articular injections of 50 μl PBS or 50 μl ExosSC (2 × 1010 particles/ml) during the following 6 weeks (once a week). After 8 weeks of operation, anaesthesia of the rats was maintained with isoflurane and the samples were subjected to pathological analysis.
In the second animal section (Fig. 6A), all rats underwent DMM + ACLT surgery or sham surgery were randomly divided into four groups: (1) sham; (2) PBS + antagomir-NC group; (3) PBS-ExosSC + antagomir-NC group; (33) PBS-ExosSC + antagomir-199-3p group. The rats were pre-injected with 50 μl antagomir-NC or antagomir-199-3p for 3 weeks (twice a week), 2 week after surgery; and then the rats were given multiple intra-articular injections of 50 μl PBS or 50 μl ExosSC (2 × 1010 particles/ml) during the following 6 weeks (once a week). After 11 weeks of operation, anaesthesia of the rats was maintained with isoflurane and the samples were subjected to pathological analysis.
Mice OA model and grouping
Seven-week-old male C57BL/6 J mice about 18–22 g were purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). All mice were maintained on a 12-h light cycle in the animal facility of the Animal Unit of Tongji University. When the rats were nine weeks old, they were subjected to DMM to induce OA, as previously described [40]. The sham group were operated was by incision of the cutaneous and muscular planes at baseline. All mice underwent DMM surgery or sham surgery were randomly divided into five groups: (1) sham group; (2) OA + PBS group; (3) OA + ExosSC group, (4) OA + ExosSC/mir-199a-3p group; (5) OA + CAP-ExosSC/mir-199a-3p group. Six weeks after operation, mice were given multiple intra-articular injections of 10 μl PBS or 10 μl ExosSC (1 × 1010 particles/ml) during the following 4 weeks (once a week). After 10 weeks of operation, anaesthesia of the mice was maintained with isoflurane and the samples were subjected to pathological analysis.
The tissues preparation, safranin O/fast green staining trials and immunohistochemical (IHC) trials
The testing animals were killed and the entire knee joints were preserved in 4% paraformaldehyde for 24 h before being decalcified in 0.5 M EDTA at pH 7.4 for 40 days (rats)/7 days (mice) and then being embedded in paraffin. Sections of 5 μm thickness were cut for Safranin O/Fast Green staining and immunohistochemical analysis.
The Osteoarthritis Research Society International (OARSI)-modified Mankin criteria were used to grade the cartilage deterioration in Safranin-O/Fast Green-stained specimens [41]. Two independent, blindfolded graders evaluated each section, and the statistical analysis was based on the average score.
For IHC analysis, the trial sections were incubated overnight at 4 °C with primary antibodies specific for COL2A1 (Proteintech, 28459-1-AP, China), MMP13 (Proteintech, 18165-1-AP). The DAB chromogen kit (Servicebio, G1212, Wuhan, China) was used to visualize the staining of sections. The number of positive antigen-stained chondrocytes and the corresponding total number of chondrocytes was calculated, through using Image-Pro Plus 6.0 software (Media Cybernetics, USA). The percentage of positive antigen-stained chondrocytes in different sections and the relative fold changes to the sham group were compared.
The tissue immunofluorescence trial
Following decalcification, the joint tissues were rinsed three times with 1 × PBS for 5 min before submerging in 30% sucrose for 12-16 h at 4 °C until the tissues sank. Transmit the tissues to an OCT-containing cryomold (Sakura, 4583, USA), and preserve at − 80 °C. For tissue immunofluorescence, 5 μm thick sections were cut using a freezing microtome (Leica, CM1950, Germany).
The specimen was rinsed three times in chilled PBS for 5 min each before being blocked with blocking buffer [1X PBS (BI, 02-024-1ACS)/5% normal serum (Jackson lab, 005-000-121, USA)/0.3% Triton™X-100 (Sangon Biotech, A110694, Shanghai, China)] for 60 min at room temperature. After incubating the specimen with the appropriate antibodies overnight at 4 °C, they were washed three times in PBS for 5 min each. Then specimen was treated for 1 h with matching fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer protected from light, and rinsed three times in PBS for 5 min each. After staining with DAPI (Sigma, 32670), each specimen was rinsed three times for 5 min in PBS. Photographs of the representative images were taken with a confocal microscope laser scanning (Leica, TSC SP8). The mouse anti-COL2A1 (Invitrogen, MA5-12789), mouse anti-MMP13 (Invitrogen, MA5-14238), LC3B-antibody (CST, 43566, Danavers, MA), mTOR-antibody (CST, 2983) was employed as e primary antibodies. For secondary reactions, Alexa Fluor 488 goat anti-mouse IgG secondary antibody (Invitrogen, A32723) and Alexa Fluor 594 goat anti-rabbit IgG secondary antibody (Invitrogen, A32754) were used.
Transmission electron microscopy (TEM) assay for animal cartilages
After decalcification, the tissues were subjected to transmission electron microscopy (TEM) assays. The cartilage samples were fixed in 2.5% glutaraldehyde for at least 4 h at room temperature, rinsed with 0.1 M phosphate buffer four times for 15 min each. Then the samples were post-fixed with 1% osmic acid for 1–2 h at room temperature, dehydrated using an ascending acetone, embedded in Epon 812. The samples were sliced into 70-nm-thick sections and were photographed with the JEOL JEM-1230 electron microscope.
Sequencing of tissues RNA
Cartilages specimens of Control group (OA + PBS) and MSCsSC-Exos treatment group (OA + ExosSC) rats were gathered (n = 3/group). The total RNA was then extracted as described above. LC Bio Technology CO., Ltd (Hangzhou, China) sequenced the RNA libraries using the illumina Novaseq™ 6000 platform. The bioinformatic analysis, including differentially expressed genes (DEGs), KEGG enrichment, GSEA enrichment was carried out using the OmicStudio tools at https://www.omicstudio.cn/tool. The heatmap was drawn based on the R (https://www.r-project.org/) on the OmicStudio platform (https://www.omicstudio.cn/tool).
In vitro studies
The co-culture assay
The rat chondrocytes were pretreated with IL-1β for 24 h and resuspended at a density of 1 × 105 cells/well (2 ml) and then seeded in the lower chambers (6-well migration chambers, 0.4 μm pore membrane, 83.3930.041, SARSTEDT, Germany). The upper chambers were seeded with rat synovial cells of 1 × 105 cells/well in 6-well plates filled with DMEM/F12 medium supplemented with 10% FBS, and incubated at 37 °C with 5% CO2 and a humidified incubator. Subsequently, the exosomes (1 × 109 particles/ml) were added in the upper cell culture medium. After 48–72 h incubation, the lower and upper cells were harvested for mRNA and protein analysis, IF and autophagic flux detection.
Cell viability assay
The rat chondrocytes were pre-seeded at a density of 4000 cells/well in 96-well plates and cultured overnight. After the administration of MHY1485 (MedChemExpress, Shanghai, China), cell proliferation ability of the rat chondrocytes was evaluated by cell counting kit-8 (CCK-8, Beyotime, China). For different treatments of different doses or times, the OD450 values of cells were measured using microplate reader (SpectraMax M5, Molecular Devices, SanJose, USA).
The cell immunofluorescence trial
The chondrocytes staining was performed following a standard protocol. Briefly, chondrocytes were rinsed three times in cool PBS for 5 min each, fixed in paraformaldehyde (4%) or methanol for 20 min, and then blocked with Blocking Buffer for 60 min at room temperature. The chondrocytes were then incubated with the indicated antibodies overnight at 4 °C, and rinsed three times in PBS for 5 min each. Then chondrocytes were incubated with corresponding fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1 h protected from light, and rinsed three times in PBS for 5 min each. The samples were stained with DAPI (Sigma, 32670), and then rinsed three times in PBS for 5 min each. Representative images were photographed using a laser scanning confocal microscope (Leica, TSC SP8). The primary antibodies used were mouse anti-COL2A1 (Invitrogen, MA5-12789), mouse anti-MMP13 (Invitrogen, MA5-14238). And the Alexa Fluor 488 goat anti-mouse IgG secondary antibody (Invitrogen, A32723) were used as for secondary reactions. Four random fields of each group were captured and used for statistical analysis.
Western blot analysis
The cartilage and chondrocytes were treated with the RIPA lysis buffer (Epizyme, PC101, China) added with a protease inhibitor cocktail (Epizyme, GRF101, China) and a phosphatase inhibitor cocktail (Epizyme, GRF102). The protein contents were calculated using a BCA protein assay kit (Takara, T9300A), and the samples was instantly boiled for 10 min with the addition of loading buffer. An equal quantity of protein extracts (20 μg) was placed onto a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel for electrophoresis and transmitted to a PVDF membrane. Following that, the PVDF membrane was sequentially incubated with primary and secondary antibodies. Finally, the enhanced chemiluminescence (BI, 20-500-120) was used to react with secondary antibodies and the images were acquired. The mTOR-antibody (CST, 2983), P-p70S6 (CST, 9234), Calnexin-antibody (Abcam, ab133615, USA), CD9-antibody (Abcam, ab263019), CD81-antibody (Abcam, ab109201), TSG101-antibody (Abcam, ab125011), Alix-antibody (CST, 92880), Lamp2b-antibody (Abcam, ab18529), anti-rabbit IgG, HRP-linked Antibody (CST, 7074) were used as the antibodies.
Autophagic flux analysis
The adenoviral vector carrying RFP-GFP-LC3 (HB-AP2100001) were purchased from HANBIO. This construct fluorescence depends on the difference in pH between the acidic autolysosome and the neutral autophagosome, and the exhibited red/green (yellow) or red fluorescence makes it possible to monitor progression of autophagic flux. To analyze the autophagic flux in rat chondrocytes, the cells were planted on cover slips that had been retained in 24-well plates were treated with: induced with IL-1β, or co-culture with MSCsSC-Exos for 48 h, or MHY1485, up to the need of the trial. Subsequently the cells were infected with the RFP-GFP-LC3 adenovirus for 24 h. Finally cultured cells on the cover slips were washed in cool PBS and fixed in 4% PFA, stained the nuclear with DAPI for immunostaining and detected for the images using a laser scanning confocal microscope (Leica, TSC SP8). For each condition, at least 4 RFP-GFP-LC3-transfected images were subjected to fluorescence analysis, and the percentage of transfected cells showing puncta RFP-GFP-LC3 were used to indicate the accumulation of autophagosomes.
MiR-199-3p loading by electroporation
The miR-199-3p mimic was loaded inside exosomes derived MSCsSC by electroporation. Briefly, exosomes at a total protein concentration of 10 μg and 500 nmol miR-199-3p mimic were mixed in 400 μl of electroporation buffer. After electroporation at 250 V and 125 μF in a 4 mm electroporation cuvette using a Gene Pulser Xcell™ system (Bio-Rad Laboratories, CA), the mixture was incubated at 37 °C for 30 min to ensure the recovery of the exosome membrane. The exosomes were ultracentrifugated for 70 min at 100,000 ×g and 4 °C to remove the unloaded miR-199-3p. Then the pellets were resuspended in PBS, and the generated exosomes were stored at − 80 °C or used instantly. The Cy3-labeled miR-199-3p was used to measure the loading efficiency of miR-199-3p, which was quantified using a fluorimeter with excitation at 532 nm and emission at 580 nm (SpectraMax M5, Molecular Devices, USA). The loading efficiency of miR-199-3p was determined by calculating the encapsulated miR-199-3p over total initial miR-199-3p.
RNA extraction, reverse transcription, and quantitative real-time (RT)-polymerase chain reaction (PCR)
Total RNA was isolated from tissues or cultured cells using Trizol reagent (Invitrogen, 15596-026). HiScript® III 1st Strand cDNA Synthesis Kit was used to synthesize the first-strand cDNA (Vazyme, R312-02, China). Real-Time PCR was used on a light cycler (Roche, Basel, Switzerland) to evaluate the mRNAs expression using ChamQ™ Universal SYBR® qPCR Master Mix (Vazyme, Q711-03) and Actin as an endogenous control. Furthermore, miRNA-specific real-time qPCR was performed in accordance with the manufacturer instructions (Vazyme, Q711-03), and U6 small nuclear RNA (snRNA) was employed as a control to determine miRNAs. Primer sequences are shown in Table 1 (Additional file 1).
Dual-luciferase reporter assay
The bioinformatics web software miRDB was employed to predict target genes and verify if miR-199a-3p and mTOR have binding sites. The wild-type mTOR dual-luciferase reporter vector (WT mTOR) and mutant mTOR dual-luciferase reporter vector (MUT mTOR) was constructed respectively, and then co-transfected into C28/I2 cells with miR-199a-3p mimic and the negative control. A miRNA control was employed as a negative control. To determine whether miR-199a-3p contributes to the effect of ExosSC on OA model, C28/I2 cells were pretreated with ExosSC for 24 h, then were transfected with the antagomir-199a-3p and the reporter plasmids. The activity of luciferase in C28/I2 cells was measured 48 h after transfection, and reporter tests were carried out according to the manufacturer’s instructions of a dual luciferase activity detection kit (Promega, E1910, USA). The activity of renilla luciferase was normalised to that of firefly luciferase and represrnted as % of the control.
Sequencing of MSCsSC-Exos derived MiRNA
Total RNAs of MSCsSC-Exos were isolated and used for miRNA sequencing. The miRNA Library was built and sequenced at LC Bio Technology CO.,Ltd (Hangzhou, China), and was collected by the Illumina HiSeq 2500 platform.
Statistical analysis
All experiments were conducted in duplicate or triplicate and were monitored by separate observers. Student’s t-test was employed to compare two groups, whereas the one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test were used to compare three groups. GraphPad Prism 8.0 was used for all statistical analyses (GraphPad Software Inc., La Jolla, CA, USA). The data are shown as the mean ± standard error (SEM), and p < 0.05 regarded statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, ns not significant.
Add Comment