2.1 Main instruments and reagents
Transmission electron microscope H-7650 was purchased from Hitachi (Japan); Nanoparticle tracking analyzer was purchased from PMX (Germany); 0.22um filter was purchased from Millipore Corporation (USA); Size exclusion chromatography and filtration kit from Echo Biotech (China) Purchase; BCA protein concentration detection kit, cryostat centrifuge were purchased from Thermo Scientific (USA); anti-CD9, CD63, Flotillin-1 and calnexin primary antibodies were purchased from CST (Cambridge, Massachusetts, USA); Hyaluronidase and polyethylene glycol (PEG6000) were purchased from Sigma (USA).
2.2 Acquisition of joint effusion
According to the diagnostic criteria developed by the American College of Rheumatology,  24 patients with primary KOA were collected from the outpatient clinic of the The Third Affiliated Hospital of Beijing University of Chinese Medicine from August 2019 to December 2019. All patients were randomly divided into 3 groups, PEG group, UC group and SECF group, 8 cases in each group. This study was approved by the Ethics Committee of the The Third Affiliated Hospital of Beijing University of Chinese Medicine, and all patients signed an informed consent form.
After sterilization, the knee joint was punctured along the lower patellar edge with a puncture needle in the outpatient operating room to extract joint effusion. The joint effusion sample was centrifuged at 3000 rpm and 4°C for 10 minutes to remove cell debris. In order to clarify the sample, the sample was treated with 2ug/ml hyaluronidase for 30min, then centrifuged at 10000×g for 20min, the supernatant was taken through a 0.22um filter, and then stored at -80°C.
2.3 Exosome isolation
2.3.1 PEG group The first step is to configure a concentration of 16% PEG6000 solution (16g PEG6000, 5.844g NaCl dissolved in 100mL ultrapure water), after filtering through a 0.22um filter, to be used. Mix the above solution with the thawed sample at a 1:1 ratio (to make the final PEG concentration 8%), and incubate overnight at 4°C. The next day at 12 000×g, centrifuge at 4°C for 30 min, discard the supernatant, add 100ul PBS to resuspend, and store at -80°C.
2.3.2 UC group, the samples were thawed at room temperature, vortexed and mixed, balanced, and placed in a 100 000×g centrifuge. After ultracentrifugation at 4° C. for 2 h, the supernatant was discarded, resuspended in PBS and placed in 100 000×g again. In a centrifuge, ultracentrifuge at 4℃ for 2h, discard the supernatant, add 100ul PBS to resuspend, and store at -80℃ until use.
2.3.3 SECF group The procedures used strictly follow the kit instructions. The resulting exosomes were resuspended in 100ul PBS, and then placed at −80°C and kept for use.
2.4 Characterization of exosomes
2.4.1 Transmission electron microscopy (TEM)
Take 10μL of the sample re-suspension solution on the sample-loaded copper mesh and let it stand at room temperature for 5 min. Use a filter paper to suck the liquid from the side. Add 10ul of 4% phosphotungstic acid solution onto the copper mesh. The filter paper absorbs the negative dye solution, after baking under the incandescent lamp, observe under the transmission electron microscope and image under 80KV.
2.4.2 Nanoparticle tracer analysis (NTA)
Nanometer tracing technology was used to detect the size and concentration distribution of vesicles. According to the corresponding operation process, set the parameters, the measurement time is 60s, the 500ul resuspended sample is appropriately diluted, and the temperature is monitored throughout the measurement process. The samples were measured three times and analyzed using NanoSight NS300 NTA software.
2.5 Western Blot(WB)
After quantification of BCA protein, protein immunoblotting (WB) was used to detect the expression of the corresponding protein. The protein was separated by 12% SDS-PAGE gel electrophoresis. The electrophoresis environment was (80V, 30min; 120V, 60min); The protein was transferred to the PVDF membrane at 250mA constant flow ,5% non-fat milk was blocked at room temperature for 2 hours, and then added to the primary antibody and incubated at 4°C overnight, and TBST was washed 4 times for 5 min each time,added with corresponding secondary antibodies and incubated for 1 h at room temperature, and TBST was washed 4 times for 5 min each time. The ECL luminescence kit was developed, and the Bio-Rad fluorescence scanner scanned the image.
2.6 Statistical analyses
The data of this study are expressed as mean ± standard deviation (SD). GraphPad Prism 7.0 software was used for statistical analysis, and t-test or one-way analysis of variance was used for comparison between groups. P<0.05 is considered significant.